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THE ROLE OF LACTATE MEASUREMENT
IN THE PREDICTION OF
FETAL HYPOXIC-ISCHAEMIC BRAIN INJURY
DURING LABOUR
Craig Edward Pennell
MBBS(Hons) FRANZCOG
A thesis submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy
School of Women’s and Infants’ Health
Faculty of Medicine and Dentistry
The University of Western Australia
June 2003
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This thesis is dedicated to my darling wife Nicola,
for without her my achievements would not be possible.
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Science
To the physician particularly, a scientific discipline is an incalculable gift,
which leavens his whole life, giving exactness to habits of thought and
tempering the mind with that judicious faculty of distrust which can alone,
amid the uncertainties of practice, make him wise unto salvation.
For perdition inevitably awaits the mind of the practitioner
who has never had the full inoculation with the leaven,
who has never grasped clearly the relations of science to his art,
and who knows nothing and perhaps cares less, for the limitations of either.
Sir William Osler, 1894
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ABSTRACT
In this thesis the role of lactate measurement has been evaluated in intrapartum assessment
of fetal wellbeing. Specifically, I have addressed the question of whether fetal lactate
measurement is better than the assessment of fetal heart rate patterns or the measurement of
pH at predicting fetal brain injury after intrapartum asphyxia. Using an ovine model of
repeated umbilical cord occlusion designed to mimic events which may occur during human
labour, I have shown that the measurement of fetal lactate levels after repeated cord
occlusion is significantly associated with the severity of brain injury after the asphyxial insult.
No significant associations were identified with fetal pH measurements or with the duration
of decelerative or compound fetal heart rate patterns; however, this is the first study to
describe an association between the duration of both increased fetal heart rate variability and
fetal heart rate overshoot with the severity of subsequent brain injury. Although no
significant association was identified between fetal arterial pressure measured between
umbilical cord occlusions and the grade of brain injury, the studies performed in this thesis
are the first to show a strong correlation between the duration of specific arterial pressure
responses during cord occlusions and the grade of brain injury, accounting for approximately
90% of the variability seen in the severity of injury.
The mechanism responsible for the improved ability of lactate measurement to predict fetal
brain injury is unknown. It may be because fetal lactate levels are a more stable marker of
anaerobic metabolism of glucose than fetal pH levels, which are influenced by both
increasing levels of carbon dioxide and anaerobic metabolism of amino-acids and fatty acids.
In addition fetal pH levels can be rapidly normalised through placental exchange of carbon
dioxide whereas fetal lactate levels are slow to normalise across the placenta as they rely on
facilitated diffusion.
In this thesis I have provided evidence for two additional potential mechanisms explaining
why fetal lactate levels may be better predictors of fetal brain injury than fetal pH assessment.
First, evidence has been provided which suggests that fetal lactate levels are a more reliable
marker of cardiovascular instability during umbilical cord occlusion than other markers of
fetal acid-base status, with high sensitivity, specificity and accuracy. The second potential
mechanism may involve the association between fetal lactate levels and oxygen free radical
activity in the fetal brain. There is a growing body of evidence suggesting that oxygen free
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radical activity in the fetal brain is critically involved in the mechanism and timing of fetal
hypoxic-ischaemic brain injury. In this thesis I have provided evidence which suggests that
fetal lactate levels are significantly better than other markers of fetal acid-base status at
predicting increases in oxygen free radical activity in the blood perfusing the fetal brain.
In human labour the fetal scalp is the window for biochemical assessment of fetal wellbeing.
In this thesis I have thoroughly investigated the relationship between fetal scalp blood lactate
levels and central blood lactate levels, considering both the effects of fetal asphyxia on this
relationship and also the effects of the adverse intrauterine environment associated with fetal
growth restriction. The relationship between fetal scalp blood and carotid arterial lactate
levels was influenced by asphyxia but not by fetal growth restriction. In contrast, the
accuracy of fetal scalp pH assessment was impaired in growth restricted fetuses with scalp
pH levels significantly underestimating the severity of changes in central blood pH. Despite
the complex relationships between fetal scalp blood lactate levels and central blood lactate
levels, scalp lactate levels were significantly better at predicting lipid peroxidation in the fetal
brain than central markers of fetal acid-base status. In addition, fetal growth restriction did
not impair the ability of fetal scalp blood lactate levels to predict cardiovascular instability
during cord occlusion.
One of the major limitations with the current clinical technique for biochemical assessment
of fetal wellbeing during labour is the restricted availability of the expensive equipment
required to perform acid-base measurement on micro volume blood samples, thus limiting
the technique to major obstetric centres. In this thesis I have assessed the accuracy and
precision of a handheld lactate meter which is commercially available in Australia, costing
approximately AUS$900 with analysis of each 15µL sample costing $1, and found it to be
both accurate and precise in lactate measurement in human blood micro samples across the
gestational age range of 26 to 42 weeks.
Together the results of this combined clinical and laboratory study suggests that the
intrapartum assessment of fetal lactate levels may be a superior method of fetal assessment,
warranting consideration for widespread clinical practice.
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DECLARATION
I, Craig Edward Pennell, declare that this thesis is less than 100,000 words in length,
exclusive of references. Except where due acknowledgement has been made, this thesis
comprises original work that I have performed. To the best of my knowledge and belief, this
thesis contains no material that has been previously written or published by any other person,
except where due reference is given in the text. No part of this thesis has been submitted to
any institution for the award of any other degree or diploma.
Craig Pennell
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ACKNOWLEDGEMENTS
I take this opportunity to express my sincere gratitude to all those who, throughout my
doctoral candidature, have contributed to its completion.
I would like to begin by acknowledging my wife Nicola, who has supported me tirelessly
throughout my doctoral candidature which has been performed simultaneously with my
specialty training in obstetrics and gynaecology and my subspecialty training in maternal-fetal
medicine. Few women are capable of caring for a young family whilst providing practical,
psychological and emotional support during the thousands of hours which have dissolved
into this thesis.
This thesis began when I was a first year obstetric and gynaecology registrar in Sydney in
1997. I performed the human studies and many of the animal studies presented in this thesis
whilst I was completing my specialty training in obstetrics and gynaecology in Sydney during
1997 to 2001. I would like to sincerely thank Dr Henry Murray, my supervisor in Sydney for
encouraging me to begin the studies presented in this thesis. I would also like to thank him
for teaching me the principles of ovine surgery and for providing the practical and financial
support for the studies which I performed whilst in Sydney. In addition, I would like to
thank him for his assistance during the final stages of preparation of this thesis.
In January 2001, my family and I moved to Perth so I could accept the Forrest Fellowship in
Fetal Medicine to undertake subspecialty training in maternal-fetal medicine. It was in Perth
that I completed the animal studies and the final preparation of this thesis. I would sincerely
like to thank Professor John Newnham and Dr Timothy Moss, my supervisors at The
University of Western Australia, for allowing me to perform the last year of ovine studies
presented in this thesis and, more importantly, for their scientific guidance during the
preparation of this manuscript. Professor John Newnham has instilled in me the principles
of thorough scientific evaluation and taught me the importance of taking small steps
forwards during scientific investigation. In addition, he has been both a friend and mentor
during my time in Perth and his enthusiasm, encouragement, support and patience have
acted to shape my approach to scientific research. Dr Timothy Moss has provided both
practical and technical assistance during the final series of animal studies performed in this
thesis. More importantly, he has shaped the way that I present my research, both written and
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oral, and he has provided hours of invaluable support, guidance and friendship during the
preparation of this manuscript.
I would like to thank Mrs Anita Turner, Dr Heather Coughtrey, Dr John Smyth, Dr Ping
Yan, Dr Ilias Nitsos and Mr Adrian Jonker for their assistance in performing the animal
studies presented in this thesis. I would particularly like to thank Mrs Anita Turner for
teaching me the techniques required to chronically instrument fetuses and for her presence
and support during the many early mornings, late nights and weekends that were invested in
the animal studies in this thesis.
Dr Dorota Doherty, from the Women and Infants Research Foundation Biostatistics and
Research Design Unit, Perth, has provided invaluable assistance both during my postgraduate
statistics training at The University of Western Australia and during the preparation of my
thesis. Thank you for your time and your critical appraisal of the data and the analyses
presented in this thesis.
I take this opportunity to thank Dr Thomas Ng from the Institute of Chemical Pathology
and Medical Research, Westmead Hospital, The University of Sydney, for his assistance in
performing the histopathological assessment of the fetal brains in this thesis. In addition, I
would like to thank Dr CC (Ronald) Wang from the Chinese University of Hong Kong for
performing the measurements of lipid peroxidation presented in this thesis and Miss Vittoria
Lazzaro from the Renal Laboratory at King George the Fifth Hospital, Sydney, for
performing the catecholamine assays presented in this thesis.
Finally I would like to take this opportunity to thank my father Roy and my late mother
Wendy for instilling in me the belief that anything is possible if you try hard enough.
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SPECIAL ACKNOWLEDGEMENT
I wish to give special acknowledgment to Mr Andrew and Mrs Nicola Forrest who provided,
through donation, the financial resources to support my research fellowship. This fellowship
position is known as the Forrest Fellowship in Fetal Medicine and I was the inaugural
recipient of this award to undertake subspecialty training in maternal-fetal medicine.
The Forrest family has descended from a distinguished list of prominent Western Australians
acclaimed for pioneering the early development of this State. This pioneering tradition lives
on through Andrew and Nicola Forrest in the context of the modern mining industry. It has
been their wish to support advancement through research in fetal medicine which represents
the pioneering edge of contemporary science, with a promise to improve lives of people
through interventions at early times in life.
It is my hope this thesis has contributed to their vision.
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MANUSCRIPTS, ABSTRACTS AND AWARDS ARISING FROM
THIS THESIS
Manuscripts
PENNELL, C.E., TRACY, M.B. (1999). A new method for rapid measurement of lactate in fetal and neonatal blood. Aust N Z J Obstet Gynaecol 39, 227-33. ROGERS, M.S., MURRAY, H.G., WANG, C.C., PENNELL, C.E., TURNER, A., YAN, P., PANG, C.C., CHANG, A.M. (2001). Oxidative stress in the fetal lamb brain following intermittent umbilical cord occlusion: a path analysis. British Journal of Obstetrics and Gynaecology 108, 1283-90.
Published Abstracts
PENNELL, C.E., TRACY, M.B. A new method for rapid measurement of lactate in fetal and neonatal blood. Canberra Centre for Perinatal Care Fetal Medicine Meeting. Canberra, Australia. September, 1997. PENNELL, C.E. The role of intrapartum lactate analysis in fetal distress. Taking Midwifery to the Future. Sydney, Australia. March, 1998 PENNELL, C.E., MURRAY, H.G., TURNER, A., PING, Y. Fetal lactic acid changes during cord occlusion in the healthy ovine fetus. Twelfth Annual Workshop on Fetal and Neonatal Physiology. Alice Springs, Australia. March, 1998. PENNELL, C.E., MURRAY, H.G., TURNER, A., PING, Y. Fetal lactic acid changes during cord occlusion in the unhealthy ovine fetus. Twelfth Annual Workshop on Fetal and Neonatal Physiology. Alice Springs, Australia. March, 1998 PENNELL, C.E., MURRAY, H.G., TURNER, A., PING, Y. Fetal lactic acid changes during cord occlusion in the healthy and unhealthy ovine fetus. Royal Australian College of Obstetricians and Gynaecologists Annual Scientific Meeting. Canberra, Australia. April, 1998. PENNELL, C.E., TURNER, A., PING, Y., WANG, C.C., ROGERS, M.S., MURRAY, H.G. Lipid peroxidation and lactic acid production during cord occlusion in the ovine fetus. Fetal and Neonatal Physiological Society 26th Annual Meeting. Vlieland, Netherlands. September, 1999. PENNELL, C.E., TURNER, A., PING, Y., MURRAY, H.G. Central and peripheral lactic acid changes during cord occlusion in healthy and compromised ovine fetuses. Fetal and Neonatal Physiological Society 26th Annual Meeting. Vlieland, Netherlands. September, 1999.
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PENNELL, C.E., TURNER, A., PING, Y., WANG, C.C., ROGERS, M.S., MURRAY, H.G. Lipid peroxidation and lactic acid production during cord occlusion in the ovine fetus. Perinatal Society of Australia and New Zealand 4th Annual Congress. Brisbane, Australia. March, 2000. PENNELL, C.E., TURNER, A., PING, Y., MURRAY, H.G. Central and peripheral lactic acid changes during cord occlusion in healthy and compromised ovine fetuses. Perinatal Society of Australia and New Zealand 4th Annual Congress. Brisbane, Australia. March, 2000. PENNELL, C.E., NG, T., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., MURRAY, H.G. Predictors of physiologic and histologic responses to repeated cord occlusion in the near term ovine fetus. Fetal and Neonatal Physiologic Society 28th Annual Meeting. Auckland, New Zealand. August, 2001. PENNELL, C.E., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., DOHERTY, D.A., MURRAY, H.G., NEWNHAM, J.P. Fetal growth restriction produces discordance between peripheral and central acid base measurements in the ovine fetus. Perinatal Society of Australia and New Zealand 6th Annual Congress. Christchurch, New Zealand. March, 2002. PENNELL, C.E., NG, T., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., MURRAY, H.G. Predictors of physiologic and histologic responses to repeated cord occlusion in the near term ovine fetus. Perinatal Society of Australia and New Zealand 6th Annual Congress. Christchurch, New Zealand. March, 2002. PENNELL, C.E., TRACY, M.B., DOHERTY, D.A., MURRAY, H.G. Fetal scalp lactate for monitoring fetal wellbeing in labour. Perinatal Society of Australia and New Zealand 6th Annual Congress. Christchurch, New Zealand. March, 2002. PENNELL, C.E., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., DOHERTY, D.A., MURRAY, H.G., NEWNHAM, J.P. The effects of growth restriction on the fetal heart rate patterns and blood pressure responses to repeated cord occlusion in the ovine fetus. Perinatal Society of Australia and New Zealand 6th Annual Congress. Christchurch, New Zealand. March, 2002. SMYTH, J.P., PENNELL, C.E., COUGHTREY, H., TURNER, A.J., PING, Y., MURRAY, H.G., NEWNHAM, J.P. The effect of intrauterine growth restriction on lactate response during neonatal resuscitation following an asphyxial insult. Perinatal Society of Australia and New Zealand 6th Annual Congress. Christchurch, New Zealand. March, 2002. SMYTH, J.P., PENNELL, C.E., COUGHTREY, H., TURNER, A.J., PING, Y., MURRAY, H.G., NEWNHAM, J.P. The effect of intrauterine growth restriction on lactate response during neonatal resuscitation following an asphyxial insult. Society for Gynecologic Investigation 49th Annual Meeting. Los Angeles, California, United States of America. March, 2002. PENNELL, C.E., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., DOHERTY, D.A., MURRAY, H.G., NEWNHAM, J.P. Fetal growth restriction produces discordance between peripheral and central acid base measurements in the ovine fetus. Society for Gynecologic Investigation 49th Annual Meeting. Los Angeles, California, United States of America. March, 2002.
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PENNELL, C.E., SMYTH, J.P., TURNER, A.J., COUGHTREY, H., YAN, P., DOHERTY, D.A., MURRAY, H.G., NEWNHAM, J.P. The effect of umbilico-placental embolisation on the fetal response to acute asphyxia. Federation of Placenta Associations 8th Meeting, Melbourne, Australia. October, 2002.
Awards
Perinatal Society of Australia and New Zealand New Investigator Award in the discipline of obstetrics. Perinatal Society of Australia and New Zealand 4th Annual Congress. Brisbane, Australia. March, 2000. Society for Gynecologic Investigation President’s Presenter Award and Abstract Number One in the Opening Plenary Session; Society for Gynecologic Investigation 49th Annual Meeting. Los Angeles, California, United States of America. March, 2002.
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ABBREVIATIONS
AACB Australasian Association of Clinical Biochemists AAP American Academy of Pediatrics ACOG American College of Obstetricians and Gynecologists ACTH Adrenocorticotropic hormone AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA Analysis of variance ATP Adenosine triphosphate BP Blood Pressure BPM Beats per minute C Control Ca2+ Calcium CI Confidence intervals CO2 Carbon dioxide CTG Cardiotocography CV Coefficient of Variation DNA Deoxyribonucleic acid E Embolised EFM Electronic fetal monitoring ECG Electrocardiogram EEG Electroencephalogram FADH Flavin adenine dinucleotide FBS Fetal blood sample FHR Fetal heart rate H:- Hydride ion H+ Hydrogen ion HIE Hypoxic-ischaemic encephalopathy IL-1β Interleukin-1beta IM Intramuscular IQR Inter quartile range IR Ischaemia-reperfusion IV Intravenous kg Kilogram LTV Long term variation MAC Minimal alveolar concentration MAP Mean arterial pressure MDA Malondialdehyde mmHg Millimetres of mercury mmol Millimole MMR Mean minute R-R interval MRI Magnetic resonance imaging MRS Magnetic resonance spectroscopy msec Millisecond n Number N Normal fetuses NADH Nicotine adenine dinucleotide NE Neonatal encephalopathy ng Nanograms NIRS Near infrared spectroscopy NMDA N-methyl-D-aspartate NNT Number needed to treat NO Nitric oxide NS Not significant O2 Oxygen OFR Oxygen free radical OHP Organic hydroperoxide
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OR Odds Ratio PAF Platelet activating factor PB Passing Bablok PCO2 Partial pressure of carbon dioxide pg picograms PGE2 Prostaglandin E2 PO2 Partial pressure of oxygen PSANZ Perinatal Society of Australia and New Zealand PVL Periventricular leukomalacia RANZCOG Royal Australian and New Zealand College of Obstetricians and Gynaecologists RCOG Royal College of Obstetricians and Gynaecologists RCPA Royal College of Pathologists RCT Randomised clinical trial RIR Repetitive ischaemia-reperfusion ROC Receiver Operator Curve RR Relative risk SA Spontaneously acidaemic fetuses SC Subcutaneous SD Standard deviation SEM Standard error of the mean SOGC Society of Obstetricians and Gynaecologists of Canada SSS Superior sagittal sinus STV Short term variation TNF-α Tumour necrosis factor alpha UCO Umbilical cord occlusion XDH Xanthine dehydrogenase XO Xanthine oxidase
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TABLE OF CONTENTS
ABSTRACT IV DECLARATION VI ACKNOWLEDGEMENTS VII SPECIAL ACKNOWLEDGEMENT IX MANUSCRIPTS, ABSTRACTS AND AWARDS ARISING FROM THIS THESIS X
Manuscripts x Published Abstracts x
ABBREVIATIONS XIII TABLE OF CONTENTS XV TABLE OF FIGURES XXI TABLE OF TABLES XXIII CHAPTER 1: LITERATURE REVIEW AND THESIS OUTLINE 1
1.1 Introduction 1 1.1.1 The clinical problem of birth asphyxia.......................................................................................1 1.1.2 Chapter overview............................................................................................................................2
1.2 Birth asphyxia –aetiology and implications 3 1.2.1 Exploration of definitions.............................................................................................................3
1.2.1.1 Fetal distress........................................................................................................................3 1.2.1.2 Birth asphyxia and fetal asphyxia....................................................................................4 1.2.1.3 Neonatal encephalopathy.................................................................................................6 1.2.1.4 Hypoxic-ischaemic encephalopathy...............................................................................6 1.2.1.5 Cerebral palsy......................................................................................................................7
1.2.2 Historical perspective of birth asphyxia.....................................................................................8 1.2.3 Current view of fetal brain injury during labour ......................................................................9 1.2.4 Conclusion .................................................................................................................................... 15
1.3 Mechanisms of brain injury 16 1.3.1 The fetal response to asphyxia.................................................................................................. 16 1.3.2 Pattern and frequency of brain injury after asphyxia ........................................................... 19
1.3.2.1 Cerebral ischaemia models............................................................................................ 20 1.3.2.2 Global asphyxia models................................................................................................. 21 1.3.2.3 Partial asphyxia models.................................................................................................. 25 1.3.2.4 Summary of pattern and frequency of brain injury after asphyxia ....................... 27
1.3.3 Molecular mechanisms of perinatal brain injury ................................................................... 28 1.3.3.1 Biphasic energy failure ................................................................................................... 30 1.3.3.2 Pathogenesis of neuronal injury................................................................................... 30 1.3.3.3 The role of inflammatory mechanisms ...................................................................... 35
1.3.4 Summary of mechanisms of brain injury................................................................................ 37 1.4 Current techniques for intrapartum fetal surveillance 38
1.4.1 What are we aiming to prevent with intrapartum monitoring?.......................................... 38 1.4.1.1 Perinatal death, cerebral palsy and neurodevelopmental disability....................... 38 1.4.1.2 Neonatal encephalopathy.............................................................................................. 39 1.4.1.3 Neonatal seizures ............................................................................................................ 40
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1.4.1.4 Severe metabolic acidosis .............................................................................................. 40 1.4.1.5 Summary........................................................................................................................... 42
1.4.2 Current monitoring techniques and their limitations........................................................... 43 1.4.2.1 Fetal heart rate monitoring ........................................................................................... 44
1.4.2.1.1 Historical perspective.......................................................................................... 44 1.4.2.1.2 Fetal heart rate monitoring and adverse clinical outcomes ......................... 45 1.4.2.1.3 Limitations of fetal heart rate monitoring....................................................... 48
1.4.2.2 Fetal scalp blood gas analysis ....................................................................................... 50 1.4.2.2.1 Physiologic basis for fetal scalp blood gas analysis ....................................... 51 1.4.2.2.2 Fetal blood sampling and adverse clinical outcomes.................................... 54 1.4.2.2.3 Limitations of fetal blood sampling ................................................................. 55 1.4.2.2.4 Scalp stimulation as an alternative to fetal blood sampling......................... 57
1.4.2.3 Umbilical cord blood gas analysis................................................................................ 58 1.4.2.3.1 Physiological basis................................................................................................ 58 1.4.2.3.2 Prediction of adverse outcomes........................................................................ 58 1.4.2.3.3 Limitations of umbilical cord blood gas analysis........................................... 59
1.4.3 Summary........................................................................................................................................ 59 1.5 Lactate and fetal surveillance 61
1.5.1 Lactate physiology ....................................................................................................................... 61 1.5.1.1 Lactate production.......................................................................................................... 61
1.5.1.1.1 Hypoxic conditions ............................................................................................. 61 1.5.1.1.2 Non-hypoxic conditions..................................................................................... 62
1.5.1.2 Fetal lactate levels during pregnancy and delivery ................................................... 63 1.5.1.3 Negative effects of lactate ............................................................................................. 64 1.5.1.4 Positive effects of lactate............................................................................................... 64
1.5.2 Fetal scalp lactate ......................................................................................................................... 65 1.5.3 Umbilical cord lactate levels at delivery .................................................................................. 68 1.5.4 Lactate levels in the newborn.................................................................................................... 68
1.6 Thesis outline 69 1.6.1 Aim ................................................................................................................................................. 69 1.6.2 Studies performed ....................................................................................................................... 69
1.6.2.1 Evaluation of a new method for rapid assessment of lactate in fetal and neonatal blood................................................................................................................. 69
1.6.2.2 The relationship between neuropathologic damage and biochemical and physiologic markers of asphyxia induced by graded cord occlusion in chronically instrumented ovine fetuses. ..................................................................... 69
1.6.2.3 The relationship between fetal scalp lactate levels, central lactate levels and oxygen free radical activity in the fetal brain during asphyxia induced by graded cord occlusion in the ovine fetus ................................................................... 70
1.6.2.4 Fetal lactate responses in growth restricted ovine fetuses during asphyxia induced by graded cord occlusion............................................................................... 70
CHAPTER 2: EVALUATION OF A NEW METHOD FOR RAPID ASSESSMENT OF LACTATE IN FETAL AND NEONATAL BLOOD 72
2.1 Introduction 72 2.1.1 Aim ................................................................................................................................................. 74
2.2 Methods 75 2.2.1 Measuring principle ..................................................................................................................... 75
2.2.1.1 Accutrend® meter.......................................................................................................... 75 2.2.1.2 Reference method........................................................................................................... 75
2.2.2 Assessment of precision............................................................................................................. 75 2.2.3 Assessment of accuracy.............................................................................................................. 79
2.2.3.1 Neonatal plasma samples .............................................................................................. 79 2.2.3.2 Umbilical cord blood samples...................................................................................... 79 2.2.3.3 Neonatal arterial blood samples................................................................................... 79 2.2.3.4 Effect of temperature on accuracy.............................................................................. 79
2.2.4 Statistical methods ....................................................................................................................... 80 2.3 Results 82
2.3.1 Analysis of precision ................................................................................................................... 82
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2.3.2 Analysis of accuracy using neonatal plasma samples ........................................................... 83 2.3.3 Analysis of accuracy using umbilical cord blood samples................................................... 83 2.3.4 Analysis of accuracy using neonatal arterial samples............................................................ 85 2.3.5 Effect of temperature on accuracy........................................................................................... 89
2.4 Discussion 91 2.4.1 Reference method precision and accuracy ............................................................................. 91 2.4.2 Accutrend® handheld lactate meter precision and accuracy ............................................. 91 2.4.3 Temperature and lactate measurement ................................................................................... 92 2.4.4 Haematocrit and lactate measurement .................................................................................... 94 2.4.5 Lactate measurement bias.......................................................................................................... 95
2.5 Conclusion 96 CHAPTER 3: THE RELATIONSHIP BETWEEN NEUROPATHOLOGIC DAMAGE AND BIOCHEMICAL AND PHYSIOLOGIC MARKERS OF ASPHYXIA INDUCED BY GRADED CORD OCCLUSION IN CHRONICALLY CANNULATED OVINE FETUSES 97
3.1 Introduction 97 3.1.1 Aim ............................................................................................................................................... 100 3.1.2 Hypothesis .................................................................................................................................. 100
3.2 Methods 101 3.2.1 Surgical procedure ..................................................................................................................... 101 3.2.2 Cord occlusion protocol .......................................................................................................... 103 3.2.3 Fetal heart rate assessment ...................................................................................................... 103 3.2.4 Fetal arterial pressure assessment........................................................................................... 104 3.2.5 Umbilical blood flow ................................................................................................................ 104 3.2.6 Biochemical assessment ........................................................................................................... 105 3.2.7 Delivery and neuropathologic examination ......................................................................... 105 3.2.8 Statistical methods ..................................................................................................................... 106
3.3 Results 108 3.3.1 Biochemical responses to repeated cord occlusion ............................................................ 108
3.3.1.1 Fetal arterial blood gas and lactate responses during umbilical cord occlusion......................................................................................................................... 108
3.3.1.2 Fetal glucose, cortisol and catecholamine responses during umbilical cord occlusion......................................................................................................................... 110
3.3.1.3 Fetal haemoglobin response to experimental protocol......................................... 110 3.3.1.4 Biochemical prediction of fetal response to umbilical cord occlusion .............. 113
3.3.2 Fetal heart rate responses to repeated cord occlusion ....................................................... 113 3.3.2.1 Baseline FHR response to repeated cord occlusion.............................................. 113 3.3.2.2 Fetal heart rate variability response to repeated cord occlusion ......................... 113 3.3.2.3 Fetal heart rate patterns during repeated cord occlusion ..................................... 117
3.3.2.3.1 Variable fetal heart rate decelerations ............................................................ 117 3.3.2.3.2 Overshoot after variable fetal heart rate decelerations............................... 117 3.3.2.3.3 Late components to variable fetal heart rate decelerations ....................... 117 3.3.2.3.4 Increased fetal heart rate variability between cord occlusions.................. 123 3.3.2.3.5 Complex variable fetal heart rate decelerations ........................................... 123
3.3.3 Fetal arterial pressure responses to repeated cord occlusion............................................ 123 3.3.3.1 Fetal arterial pressure between cord occlusions ..................................................... 123 3.3.3.2 Fetal arterial pressure during cord occlusion .......................................................... 125
3.3.4 Neuropathologic findings after repeated cord occlusion .................................................. 125 3.3.5 Relationship between markers of fetal well-being during repeated cord occlusion
and fetal neuropathologic damage ......................................................................................... 127 3.3.5.1 Correlation of biochemical markers with fetal neuropathologic damage ......... 127 3.3.5.2 Correlation of fetal heart rate patterns with fetal neuropathologic damage..... 131 3.3.5.3 Correlation of fetal arterial pressure with fetal neuropathologic damage ......... 132
3.3.5.3.1 Fetal arterial pressure between the umbilical cord occlusions.................. 132 3.3.5.3.2 Fetal arterial pressure during the final three occlusions............................. 132 3.3.5.3.3 Duration of fetal arterial pressure responses during umbilical cord
occlusion .............................................................................................................. 133
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3.3.6 Prediction of fetal arterial pressure responses during cord occlusion ............................ 133 3.3.6.1 Prediction of arterial pressure responses during umbilical cord occlusion
from fetal heart rate patterns ...................................................................................... 134 3.3.6.2 Prediction of arterial pressure responses during umbilical cord occlusion
from fetal biochemical measures ............................................................................... 134 3.4 Discussion 138
3.4.1 Cord occlusion model............................................................................................................... 138 3.4.2 Histopathological changes induced by cord occlusion model ......................................... 138 3.4.3 Fetal acid-base status and neuropathologic damage........................................................... 139 3.4.4 Fetal lactate and neuropathologic damage ........................................................................... 142 3.4.5 Fetal glucose and neuropathologic damage ......................................................................... 144 3.4.6 Fetal heart rate patterns and neuropathologic damage...................................................... 145
3.4.6.1 Increased fetal heart variability and neuropathologic damage............................. 145 3.4.6.2 Fetal heart rate overshoot and neuropathologic damage ..................................... 146 3.4.6.3 Other fetal heart rate patterns and neuropathologic damage .............................. 148
3.4.7 Fetal arterial pressure and neuropathologic damage .......................................................... 149 3.4.8 Prediction of rate of response to repeated umbilical cord occlusion ............................. 151 3.4.9 Integration of results into clinical practice ........................................................................... 152 3.4.10 Future studies ............................................................................................................................. 153
3.5 Conclusion 155 CHAPTER 4: THE RELATIONSHIPS BETWEEN FETAL SCALP LACTATE LEVELS, CENTRAL LACTATE LEVELS AND OXYGEN FREE RADICAL ACTIVITY IN THE FETAL BRAIN DURING ASPHYXIA INDUCED BY GRADED CORD OCCLUSION IN THE OVINE FETUS 156
4.1 Introduction 156 4.1.1 Development of an experimental model .............................................................................. 157 4.1.2 The relationship between fetal scalp lactate levels and central lactate levels................. 158 4.1.3 The relationship between fetal scalp lactate levels and fetal brain injury....................... 160
4.1.3.1 Oxygen free radical activity and fetal brain injury.................................................. 160 4.1.4 Aim ............................................................................................................................................... 163 4.1.5 Hypothesis .................................................................................................................................. 164
4.2 Methods 165 4.2.1 Surgical Procedure..................................................................................................................... 165 4.2.2 Cord occlusion protocol .......................................................................................................... 166 4.2.3 Fetal heart rate and arterial pressure assessment................................................................. 167 4.2.4 Umbilical blood flow ................................................................................................................ 167 4.2.5 Biochemical assessment ........................................................................................................... 167 4.2.6 Study group classification......................................................................................................... 168 4.2.7 Statistical methods ..................................................................................................................... 169
4.3 Results 171 4.3.1 Baseline fetal biochemical status prior to the experimental phase .................................. 171 4.3.2 Investigation of the validity of the acutely catheterised model for the assessment
of fetal scalp blood samples..................................................................................................... 172 4.3.2.1 Fetal acid-base status and lactate responses ............................................................ 172 4.3.2.2 Fetal endocrine responses ........................................................................................... 172 4.3.2.3 Fetal heart rate responses............................................................................................ 175 4.3.2.4 Fetal arterial pressure responses ................................................................................ 175
4.3.3 Biochemical responses of normal (non-acidaemic) and spontaneously acidaemic fetuses to repeated umbilical cord occlusion ....................................................................... 178
4.3.3.1 Controls .......................................................................................................................... 178 4.3.3.2 Fetal acid-base status and lactate responses ............................................................ 178 4.3.3.3 Fetal endocrine responses ........................................................................................... 178
4.3.4 The relationship between fetal scalp blood lactate levels and central lactate levels..... 181 4.3.4.1 Fetal scalp blood lactate levels and carotid arterial lactate levels ........................ 181 4.3.4.2 Fetal scalp blood lactate levels and jugular venous lactate levels ........................ 186 4.3.4.3 Fetal carotid artery and jugular vein blood lactate levels ...................................... 186
4.3.5 The relationship between fetal scalp blood lactate and lipid peroxidation in the fetal brain..................................................................................................................................... 187
xix
4.3.5.1 Controls .......................................................................................................................... 187 4.3.5.2 Normal and spontaneously acidaemic fetuses ........................................................ 187 4.3.5.3 Prediction of the lipid peroxidation in fetal brain perfusate ................................ 190
4.4 Discussion 194 4.4.1 Comparison of the acute and chronic instrumented models ........................................... 194
4.4.1.1 Selection of maternal anaesthesia .............................................................................. 195 4.4.1.2 Similarities and differences between the acute and chronically
instrumented models.................................................................................................... 199 4.4.1.2.1 Control fetuses ................................................................................................... 199 4.4.1.2.2 Fetuses subjected to repeated cord occlusion.............................................. 199
4.4.1.3 What is the optimum model for assessing fetal scalp blood during asphyxia? ......................................................................................................................... 201
4.4.2 The relationship between fetal scalp blood, carotid arterial and jugular venous blood lactate levels..................................................................................................................... 202
4.4.2.1 Scalp lactate and central lactate levels in control and normal fetuses during asphyxia........................................................................................................................... 202
4.4.2.2 Information gained from spontaneously acidaemic fetuses................................. 206 4.4.3 Fetal scalp blood lactate and lipid peroxidation in the fetal brain ................................... 208
4.4.3.1 Markers of lipid peroxidation..................................................................................... 208 4.4.3.2 Patterns of lipid peroxidation during asphyxia ....................................................... 209 4.4.3.3 The relationship between fetal scalp lactate and lipid peroxidation in the
fetal brain........................................................................................................................ 212 4.4.4 Future studies ............................................................................................................................. 213
4.5 Conclusion 215 CHAPTER 5: FETAL LACTATE RESPONSES IN THE GROWTH RESTRICTED OVINE FETUS DURING ASPHYXIA INDUCED BY GRADED CORD OCCLUSION 217
5.1 Introduction 217 5.1.1 Aim ............................................................................................................................................... 220 5.1.2 Hypothesis .................................................................................................................................. 220
5.2 Methods 221 5.2.1 First surgical preparation.......................................................................................................... 221 5.2.2 Embolisation protocol.............................................................................................................. 223 5.2.3 Second surgical preparation..................................................................................................... 223 5.2.4 Cord occlusion protocol .......................................................................................................... 224 5.2.5 Fetal heart rate, arterial pressure and umbilical blood flow assessment......................... 225 5.2.6 Biochemical methods................................................................................................................ 225 5.2.7 Statistical methods ..................................................................................................................... 225
5.3 Results 228 5.3.1 Physiologic responses of the growth restricted fetus during umbilical cord
occlusion...................................................................................................................................... 228 5.3.1.1 Umbilicoplacental embolisation................................................................................. 228 5.3.1.2 Fetal acid-base responses to repeated cord occlusion........................................... 230 5.3.1.3 Fetal arterial lactate response to repeated cord occlusion .................................... 233 5.3.1.4 Fetal endocrine responses to repeated cord occlusion ......................................... 233 5.3.1.5 Fetal heart rate responses to repeated cord occlusion .......................................... 236
5.3.1.5.1 Baseline fetal heart rate response to repeated cord occlusion .................. 236 5.3.1.5.2 Fetal heart rate variability response to repeated cord occlusion .............. 236 5.3.1.5.3 Fetal heart rate patterns during repeated cord occlusion........................... 236
5.3.1.6 Fetal arterial pressure responses to repeated cord occlusion............................... 239 5.3.1.6.1 Fetal arterial pressure between cord occlusions .......................................... 239 5.3.1.6.2 Fetal arterial pressure during cord occlusions.............................................. 243
5.3.1.7 Physiologic associations with fetal heart rate patterns in embolised and non-embolised fetuses ................................................................................................. 243
5.3.1.7.1 Fetal heart rate accelerations............................................................................ 248 5.3.1.7.2 Late component to decelerations ................................................................... 249 5.3.1.7.3 Severe variable decelerations. .......................................................................... 249 5.3.1.7.4 Increased fetal heart rate variability................................................................ 250
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5.3.2 The effect of umbilicoplacental embolisation on the relationship between fetal scalp blood levels and central levels of lactate and pH...................................................... 251
5.3.2.1 Fetal scalp blood lactate levels ................................................................................... 251 5.3.2.1.1 Fetal scalp blood lactate levels and carotid arterial lactate levels ............. 251 5.3.2.1.2 Fetal scalp blood lactate levels and jugular venous lactate levels ............. 255 5.3.2.1.3 Fetal carotid artery and jugular venous blood lactate levels...................... 255
5.3.2.2 Fetal scalp blood pH levels ......................................................................................... 258 5.3.2.2.1 Fetal scalp blood pH levels and carotid arterial pH levels ........................ 258 5.3.2.2.2 Fetal scalp blood pH levels and jugular venous blood pH levels ............ 262 5.3.2.2.3 Fetal carotid artery and jugular venous blood pH levels ........................... 262
5.3.3 Prediction of fetal arterial pressure changes during cord occlusion................................ 263 5.3.3.1 Prediction of fetal arterial pressure responses during cord occlusion from
fetal heart rate patterns ................................................................................................ 263 5.3.3.2 Prediction of fetal arterial pressure responses during cord occlusion from
fetal scalp blood biochemistry.................................................................................... 265 5.3.4 Summary of similarities and differences in fetal responses to asphyxia in
embolised and non-embolised fetuses .................................................................................. 267 5.4 Discussion 268
5.4.1 Stability of fetal lactate responses during asphyxia ............................................................. 268 5.4.1.1 Arterial lactate responses............................................................................................. 269 5.4.1.2 Fetal scalp blood lactate responses............................................................................ 272 5.4.1.3 Lactate and the prediction of arterial pressure responses during cord
occlusion......................................................................................................................... 273 5.4.2 Variation in cardiovascular and endocrine responses during asphyxia .......................... 274
5.4.2.1 Fetal heart rate patterns ............................................................................................... 274 5.4.2.1.1 Fetal heart rate variability ................................................................................. 275 5.4.2.1.2 Fetal heart rate decelerations ........................................................................... 277 5.4.2.1.3 Fetal heart rate accelerations and overshoot ................................................ 278
5.4.2.2 Fetal arterial pressure responses ................................................................................ 279 5.4.2.3 Fetal endocrine responses ........................................................................................... 280
5.4.3 Influence of the experimental model of fetal scalp blood biochemistry........................ 281 5.4.4 Integration of results into clinical practice ........................................................................... 282 5.4.5 Future studies ............................................................................................................................. 283
5.5 Conclusion 285 CHAPTER 6: CLINICAL APPLICATION: IS THERE EVIDENCE FOR CHANGE? 286 REFERENCES 295
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TABLE OF FIGURES
Number Page 1.1 Fetal pathophysiology after an asphyxial exposure ................................................................................ 29 1.2 Hypoxia-ischaemia induced energy depletion and cascade to neuronal death ................................. 33 1.3 Pathways of hypoxia-mediated neuronal cell death................................................................................ 34 1.4 Fetal acidosis with abnormal fetal heart rate patterns............................................................................ 52 1.5 Fetal aerobic and anaerobic metabolism................................................................................................... 53 2.1 Accutrend® handheld lactate meter.......................................................................................................... 76 2.2 Design of lactate test strip for the Accutrend® handheld lactate meter............................................ 76 2.3 Reaction principles used for lactate measurement by the Accutrend® and ABL625®................. 77 2.4 Analysis technique for the Accutrend® handheld lactate meter ......................................................... 78 2.5 Accuracy of the Accutrend® lactate meter for measurement of lactate in neonatal plasma......... 84 2.6 Accuracy of the Accutrend® lactate meter for measurement of lactate in umbilical arterial
blood ................................................................................................................................................................ 86 2.7 The effect of haematocrit on accuracy of the Accutrend lactate meter ............................................. 87 2.8 Accuracy of the Accutrend® lactate meter in neonatal arterial blood ............................................... 88 2.9 The effect of temperature on the accuracy of the Accutrend® lactate meter .................................. 90 3.1 Experimental timeline and cord occlusion protocol ............................................................................ 102 3.2 Fetal arterial gas tensions, pH, base excess, lactate, and glucose over the seven day study
period ............................................................................................................................................................. 109 3.3 Fetal cortisol, noradrenaline, adrenaline and dopamine concentrations over the seven day
study period .................................................................................................................................................. 111 3.4 Fetal haemoglobin responses over seven day study period................................................................ 112 3.5 Biochemical prediction of response to repeated umbilical cord occlusion ..................................... 114 3.6 Baseline fetal heart rate during the experimental phase....................................................................... 115 3.7 Fetal heart rate variability during the experimental phase................................................................... 116 3.8 Variable fetal heart rate decelerations...................................................................................................... 118 3.9 Overshoot after fetal heart rate decelerations........................................................................................ 119 3.10 Late components to fetal heart rate decelerations ................................................................................ 120 3.11 Increased FHR variability between cord occlusions ............................................................................ 121 3.12 Complex variable fetal heart rate decelerations..................................................................................... 122 3.13 Fetal arterial pressure responses between repeated cord occlusion .................................................. 124 3.14 Fetal arterial pressure responses during repeated cord occlusion ..................................................... 126 3.15 Range of histopathologic damage to fetal brains .................................................................................. 129 3.16 Receiver operator curves for biochemical prediction of blood pressure patterns during
cord occlusion .............................................................................................................................................. 137 4.1 Adverse effects of oxidative stress on neuronal lipids, protein and, deoxyribonucleic acid........ 161 4.2 Comparison of arterial gas tensions, pH, base excess and lactate levels in acute and
chronically catheterized ovine fetuses during repeated umbilical cord occlusion.......................... 174 4.3 Comparison of endocrine responses in acute and chronically catheterized ovine fetuses
during repeated umbilical cord occlusion............................................................................................... 173 4.4 Comparison of FHR and FHR variability responses in acute and chronically instrumented
fetuses during repeated umbilical cord occlusion ................................................................................. 176 4.5 Comparison of inter-occlusion fetal blood pressure responses in acute and chronically
instrumented fetuses during repeated cord occlusion.......................................................................... 177 4.6 Fetal arterial gas tensions, pH, base excess and lactate in normal and spontaneously
acidaemic fetuses during experimental phase ........................................................................................ 179 4.7 Fetal endocrine responses to repeated cord occlusion in normal and spontaneously
acidaemic fetuses.......................................................................................................................................... 180 4.8 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
lactate levels in controls.............................................................................................................................. 182 4.9 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
lactate levels in normal fetuses during repeated cord occlusion ........................................................ 183
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4.10 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood lactate levels in spontaneously acidaemic fetuses during repeated cord occlusion ........................ 184
4.11 Comparison of three lactate sampling sites in control, normal and spontaneously acidaemic fetuses.......................................................................................................................................... 185
4.12 Lipid peroxidation in carotid arterial and jugular venous blood of control fetuses....................... 188 4.13 Lipid peroxidation in carotid arterial and jugular venous blood of fetuses that experienced
umbilical cord occlusion............................................................................................................................. 189 4.14 Range of carotid arterial pH and scalp blood lactate associated with lipid peroxidation in
jugular venous blood................................................................................................................................... 191 4.15 Receiver operator curves for the prediction of OHP production in jugular venous blood ........ 192 4.16 Control of jugular venous and scalp capillary blood content............................................................. 204 4.17 Mechanism of oxygen free radical production with umbilical cord occlusion ............................... 210 5.1 Outline of experimental protocol............................................................................................................. 222 5.2 Fetal acid-base responses during umbilicoplacental embolisation .................................................... 229 5.3 Fetal arterial lactate responses during repeated umbilical cord occlusion ...................................... 231 5.4 Fetal arterial lactate responses during repeated umbilical cord occlusion ...................................... 232 5.5 Fetal endocrine responses during umbilical cord occlusion ............................................................... 234 5.6 Fetal ACTH and cortisol responses during umbilical cord occlusion.............................................. 235 5.7 Fetal heart rate responses during umbilical cord occlusion ................................................................ 237 5.8 Fetal heart rate responses in two groups of fetuses exposed to repeated cord occlusion............ 238 5.9 Fetal heart rate decelerative patterns during umbilical cord occlusion............................................. 240 5.10 Fetal systolic pressure responses between umbilical cord occlusions .............................................. 241 5.11 Fetal diastolic pressure responses between umbilical cord occlusions............................................. 242 5.12 Fetal arterial pressure responses during umbilical cord occlusions................................................... 244 5.13 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
lactate levels in control fetuses.................................................................................................................. 252 5.14 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
lactate levels in occluded fetuses .............................................................................................................. 253 5.15 Comparison of three lactate sampling sites in control and occluded non-embolised and
embolised fetuses......................................................................................................................................... 254 5.16 Lactate and oxygen differences across the fetal cerebral circulation during cord occlusion ....... 257 5.17 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
pH levels in control fetuses ....................................................................................................................... 259 5.18 The relationship between fetal scalp blood, carotid arterial blood and jugular vein blood
pH levels in occluded fetuses.................................................................................................................... 260 5.19 Comparison of three pH sampling sites in control and occluded non-embolised and
embolised fetuses......................................................................................................................................... 261 5.20 Receiver operator curves for the prediction of any blood pressure instability during cord
occlusion in non-embolised and embolised fetuses ..................................................................... 266
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TABLE OF TABLES
Number Page 1.1 Classification of intrapartum fetal asphyxia ................................................................................................5 1.2 Criteria to define an acute intrapartum hypoxic event........................................................................... 11 1.3 Cardiovascular responses to an asphyxial exposure ............................................................................... 17 1.4 Four patterns of perinatal brain damage................................................................................................... 20 1.5 Brain damage after cerebral ischaemia ...................................................................................................... 21 1.6 Brain damage after global asphyxia............................................................................................................ 22 1.7 Brain damage after partial fetal asphyxia .................................................................................................. 25 1.8 Important adverse clinical outcomes......................................................................................................... 38 1.9 Short term neonatal complications of severe metabolic acidosis at birth.......................................... 41 1.10 Scalp pH and the prediction of neonatal outcome................................................................................. 55 1.11 Normal ranges for scalp lactate in first stage of labour ......................................................................... 66 1.12 Scalp lactate and the prediction of neonatal outcome ........................................................................... 68 2.1 Precision of the Accutrend® Reflectance Photometer ......................................................................... 82 2.2 Precision of the Radiometer ABL 625® blood-gas machine............................................................... 82 2.3 Characteristics of 160 cord samples utilized in accuracy analysis........................................................ 83 2.4 Normal reference range for umbilical arterial lactate after labour....................................................... 95 3.1 Outcome after term neonatal encephalopathy ........................................................................................ 99 3.2 Grading of histopathologic changes in fetal brains .............................................................................. 106 3.3 Histopathologic damage in right side and left side of fetal brain ...................................................... 128 3.4 Spearman’s Rho for fetal biochemical markers and the grade of fetal brain injury....................... 130 3.5 Spearman’s Rho for the duration of FHR patterns and the grade of fetal brain injury................ 131 3.6 Spearman’s Rho for the duration of compound FHR patterns and the grade of fetal
brain injury .................................................................................................................................................... 131 3.7 Spearman’s Rho for arterial pressure at the completion of the cord occlusion protocol
and the grade of fetal brain injury ............................................................................................................ 132 3.8 Spearman’s Rho for the duration of arterial pressure instability during UCO and the
grade of fetal brain injury ........................................................................................................................... 133 3.9 Fetal heart rate prediction of arterial pressure responses during cord occlusion........................... 134 3.10 Biochemical prediction of arterial pressure responses during cord occlusion................................ 135 3.11 Accuracy of biochemical prediction of arterial pressure responses during cord occlusion ......... 136 4.1 Baseline fetal biochemical condition prior experimental phase......................................................... 171 4.2 Accuracy of biochemical prediction of OHP production in brain perfusate ................................. 193 4.3 Comparison of maternal anaesthesia techniques between experimental models........................... 195 5.1 Effect of 23 days of umbilicoplacental embolisation on fetal biochemical and endocrine
status............................................................................................................................................................... 230 5.2 Univariate associations of FHR patterns with blood gas, blood pressure and
catecholamine variables .............................................................................................................................. 245 5.3 Multivariate associations of FHR patterns considering all blood gas variables and fetal
arterial pressure variables simultaneously ............................................................................................... 246 5.4 Multivariate associations of FHR patterns considering all blood gas, fetal blood pressure
and catecholamine variables simultaneously .......................................................................................... 247 5.5 Receiver operator curve analysis for the prediction of FHR patterns from fetal
physiologic parameters............................................................................................................... 248 5.6 Summary of differences between superior sagittal sinus blood and jugular
venous blood ............................................................................................................................... 256 5.7 Fetal heart rate prediction of arterial pressure responses during cord occlusion ........... 263 5.8 Odds Ratios of abnormal FHR pattern if fetal arterial pressure response is
present........................................................................................................................................... 264 5.9 Biochemical prediction of arterial pressure responses during cord occlusion ................ 265 5.10 Accuracy of biochemical prediction of arterial pressure responses during UCO .......... 267 5.11 Summary of differences and similarities in physiologic responses to asphyxia in
embolised and non-embolised fetuses .................................................................................... 267
1
C h a p t e r 1
LITERATURE REVIEW AND THESIS OUTLINE
At the outset do not be worried about the big question – Truth.
No human being is constituted to know the truth, the whole truth, and nothing but the truth;
And even the best of men must be content with fragments, with partial glimpses, never the full fruition.
Sir William Osler (1892)
1.1 Introduction
1.1.1 The clinical problem of birth asphyxia
Worldwide there are approximately 130 million births annually. Of these infants, it has been
estimated that four million babies suffer from birth asphyxia, one million die and a similar
number develop sequelae including cerebral palsy, seizures, or developmental delay (WHO,
1991, 1995). The incidence of birth asphyxia is higher in developing than in so-called
developed countries yet in the latter there are still between two and six neonates per
thousand who exhibit hypoxic-ischaemic encephalopathy (Levene et al., 1985). This amounts
to between 8,000 and 25,000 infants each year in both the United States of America and the
European Union and between 500 and 1,500 infants each year in Australia. Birth asphyxia
therefore represents an important, potentially preventable condition that has major personal,
social and financial implications for both families and the community worldwide.
The ultimate fear of every obstetrician, midwife and parent is a poor neonatal outcome. Fetal
distress is a widely used but poorly defined term (Steer, 1982; Parer & Livingston, 1990). Its
use commonly indicates anxiety about the fetal condition which then leads to a caesarean
section or instrumental delivery. The aim of intrapartum fetal surveillance is to more
accurately diagnose fetal distress to reduce the incidence of birth asphyxia.
The three techniques that are currently widely used for the assessment of fetal wellbeing in
labour are electronic fetal heart rate monitoring, scalp blood pH measurement and umbilical
cord blood gas analysis. Fetal heart rate (FHR) monitoring was introduced into clinical use
and patient management more than three decades ago. It continues to be the predominant
2
method for intrapartum fetal surveillance despite questions about its efficacy and outcomes
associated with its use (Freeman, 2002). It is generally accepted that both continuous
electronic FHR monitoring and intensive intermittent auscultatory monitoring are better than
non-intensive auscultatory monitoring in the prevention of intrapartum fetal death.
Nonetheless, despite three decades of immense changes in obstetric management, including
the widespread use of fetal surveillance tests and increased caesarean deliveries for fetal
indications, the incidence of cerebral palsy has remained constant (Scheller & Nelson, 1994;
MacLennan, 1999).
The principal biochemical markers of perinatal asphyxia are intrapartum fetal scalp acid-base
assessment and the assessment of umbilical cord acid-base status at delivery. Both these
techniques have a sound physiologic basis and umbilical cord acid-base assessment at delivery
provides the most sensitive reflection of birth asphyxia based on current published literature
(MacLennan, 1999). Unfortunately the use of these traditional markers of fetal acid-base
status is limited because they reflect fetal metabolic adjustments that are not directly related
to fetal damage unless the values are extreme (Gilstrap et al., 1989; Goldaber et al., 1991;
Winkler et al., 1991). Even a pH of less than 7.0 is poorly predictive of neurological deficit
because a severely acidotic infant will usually have a normal outcome (Winkler et al., 1991;
Socol et al., 1994; Yudkin et al., 1994).
In order to reduce the burden of birth asphyxia on families, health care systems and the
community, new techniques that are inexpensive and widely available are required to allow
recognition of early intrapartum asphyxia so that timely obstetric intervention can avoid
asphyxia-induced brain damage or death. Furthermore, new techniques are required to
identify which fetuses exposed to an asphyxial insult have acute brain injury so that
appropriate cases can be selected for therapies designed to limit the extension of this injury
that can occur after birth (Edwards & Azzopardi, 2000).
1.1.2 Chapter overview
In this chapter I will review birth asphyxia, its aetiology and its implications for the newborn
and child. This will be followed by a review of the animal studies of fetal asphyxia addressing
the fetal response to asphyxia, the pattern and frequency of brain injury after asphyxial
exposure, and the molecular mechanisms of brain injury. The third part of this chapter will
review our current markers of fetal wellbeing during labour with particular emphasis on their
ability to predict important outcomes. The final part of this chapter will review the role of
lactate measurement during intrapartum surveillance and outline the studies that I have
performed to expand knowledge in this field.
3
1.2 Birth asphyxia –aetiology and implications
1.2.1 Exploration of definitions
There are a number of important definitions that should be established at this point as they
have specific meanings and should not be interchanged.
1.2.1.1 Fetal distress
Fetal distress is a widely used but poorly defined term (Steer, 1982; Parer & Livingston,
1990). It is often prefixed by the term acute or chronic and it is commonly used as an
indication for caesarean section or as a justification for instrumental delivery. Some authors
have defined it based on FHR patterns such as “late decelerations”, “severe variable
decelerations”, or “tachycardia with total loss of short-term variability” (Haverkamp et al.,
1979; Haesslein & Niswander, 1980). Others believe that fetal acidosis or depressed Apgar
scores are necessary to define fetal distress (Sykes et al., 1982). Some authors suggest that the
presence of meconium-stained amniotic fluid and any abnormalities in the fetal heart rate
define fetal distress warranting delivery (Steer, 1999).
In 1893, Von Winckel described the criteria for fetal distress that were to remain essentially
unchanged until the arrival of fetal scalp blood sampling for pH measurement and electronic
heart rate monitoring (Von Winckel, 1893). These criteria included tachycardia (FHR more
than 160bpm), bradycardia (FHR less than 100bpm), irregular fetal heart rate, passage of
meconium and gross alteration of fetal movement. Few studies either challenged or
supported the validity of these auscultative and clinical criteria for fetal distress. It was not
until 1968, when the results of the Collaborative Project commissioned by the National
Institute of Neurologic Diseases and Blindness were published, that these criteria came under
serious scrutiny (Benson et al., 1968). This study reviewed the benefits of FHR monitoring in
the management of intrapartum fetal distress in 24,683 deliveries and concluded that there
was no reliable indicator of fetal distress in terms of fetal heart rate save in extreme degree
(Benson et al., 1968).
Parer et al. (1990) attempted to define fetal distress in terms of the fetal physiologic
adaptations that occur with decreased oxygenation. They defined fetal distress as persistent
fetal asphyxia that if not corrected or circumvented, will result in decompensation of the
physiologic responses (primarily redistribution of blood flow to preserve oxygenation of vital
organs) and cause permanent central nervous system damage or death. This definition,
4
although physiologically accurate, is difficult to apply clinically. The first half of the
definition (persistent fetal asphyxia) may possibly be determined from continuous FHR
monitoring through the identification of late, variable or prolonged decelerations; however,
the second half of the definition implies identifying fetal decompensation which is more
difficult (Quilligan, 1990). Some authors have proposed that this can be done through the
assessment of FHR variability and say that as long as variability is present, there is no need to
intervene (Parer & Livingston, 1990). Others have contended that if one waits until
variability is lost it may be too late (Goodlin, 1990; Freeman, 2002).
A more appropriate terminology for describing fetal condition during labour is “reassuring”
and “non-reassuring” fetal status as clinical signs often poorly predict a compromised fetus.
This terminology has been endorsed by the Perinatal Society of Australia and New Zealand
(PSANZ, 1995; MacLennan, 1999), the American College of Obstetricians and
Gynecologists (ACOG, 1998) and the Society of Obstetricians and Gynaecologists of Canada
(SOGC, 1996b) and it is stated by these three professional bodies that the continued use of
the term fetal distress may encourage incorrect assumptions or inappropriate management.
1.2.1.2 Birth asphyxia and fetal asphyxia
Asphyxia can literally be translated from the Greek as meaning absence of pulse. It is more
usually applied to situations where there has been anoxia or hypoxia. A task force set up by
the World Federation of Neurology defined asphyxia as a condition of impaired gas exchange
leading, if it persists, to progressive hypoxaemia and hypercapnia (Bax & Nelson, 1993). This
is a good definition of asphyxia, however, in labour asphyxia may occur intermittently.
Therefore, for fetal asphyxia during labour, a more appropriate definition is “a condition of
impaired blood gas exchange leading to progressive fetal hypoxaemia and hypercapnia with a
significant metabolic acidosis” (Low, 1998; MacLennan, 1999).
In the clinical context, Thorp et al. (1990) suggested that birth asphyxia should be defined to
include not only acidaemia (pH less than two standard deviations below the mean; <7.10) but
also neonatal depression and evidence of hypoxic end-organ damage such as early neonatal
seizures and renal or cardiac dysfunction (Thorp, 1990).
5
The American Academy of Pediatrics and the American College of Obstetrics and
Gynecology (AAP & ACOG, 1996) recommended that the diagnosis of birth asphyxia
should be based on the finding of three of the four following criteria:
1. Umbilical arterial pH<7.00
2. Apgar score <4 for more than 5 minutes
3. Multiple organ damage
4. Hypoxic-ischaemic encephalopathy
It is clear that the diagnosis of birth asphyxia can only be made retrospectively as it is the
sequence of symptoms and signs and how the brain and other organs react over time that
indicates the diagnosis (Nelson & Emery, 1993).
A classification system for fetal asphyxia has been developed to aid in the prediction of long
term outcome for the child (Low, 1998). This classification, summarised in Table 1.1,
includes metabolic acidosis, neonatal encephalopathy and multiple organ system dysfunction.
Table 1.1 Classification of intrapartum fetal asphyxia
Asphyxia* Metabolic Acidosis at Delivery
Encephalopathy Cardiovascular,
Respiratory and Renal Complications
Mild Moderate Severe Minor Moderate/Severe
Mild + +/- +/-
Moderate + + +/-
Severe + + + *Umbilical artery base deficit ≥12mmol/l, +/- present in some but not all fetuses (Adapted from Low, 1998) Although our current understanding of the physiologic mechanisms of intrapartum fetal
asphyxial brain injury has suggested a strong association with multiple organ system injury,
severe fetal brain injury can occur in response to severe acute insults in the absence of
multiple organ system dysfunction (Phelan et al., 1998). This finding was illustrated by Phelan
et al. (1998) who reported a series of cases of hypoxic-ischaemic encephalopathy in the
6
neonatal period, all due to acute catastrophic events, in infants who did not have multiple
organ system dysfunction but went on to have permanent neurologic injury. The acute
insults responsible for the encephalopathy included uterine rupture (43%), prolonged FHR
deceleration (36%), fetal exsanguination (7%), cord prolapse (7%), and maternal cardio-
pulmonary arrest (7%). These acute injuries, associated with a prolonged FHR deceleration,
may be linked to a severe decrease in cardiac output and hypotension that cause vulnerable
portions of the fetal brain to be injured before other organs.
1.2.1.3 Neonatal encephalopathy
Neonatal encephalopathy (NE) is a clinically defined syndrome of disturbed neurological
function in the infant at or near term during the first week after birth, manifest as difficulty
with initiating and maintaining respiration, depression of tone and reflexes, altered level of
consciousness, and often seizures (Leviton & Nelson, 1992).
In addition to asphyxia and hypoxic-ischaemic injury, a diverse group of disorders can result
in this syndrome, many of which begin antenatally. These disorders include maternal and
fetal infections; maternal thyroid and nutritional deficiencies; placental disorders; and genetic
diseases including Prader-Willi and Angelman’s syndromes, Rett’s syndrome, myotonic
dystrophy, non-ketotic hyperglycinaemia, thrombophilic disorders, brain malformations, and
a family history of epilepsy (Smith et al., 1996; Grether & Nelson, 1997; Badawi et al., 1998a;
Ellis et al., 2000; Impey et al., 2001; Richer et al., 2001; Geerdink et al., 2002; Johnston, 2003).
1.2.1.4 Hypoxic-ischaemic encephalopathy
Hypoxic-ischaemic encephalopathy (HIE) is a subtype of neonatal encephalopathy where the
origin of the condition is thought to be hypoxia and ischaemia.
The condition was first described in 1976 by Sarnat and Sarnat who described a syndrome of
abnormal neonatal behaviour that followed asphyxia. Hypoxic-ischaemic encephalopathy is
traditionally sub-classified into mild, moderate and severe (or Stage I, II or III) using a
classification system relying on the assessment of consciousness, neuromuscular control,
complex neonatal reflexes, autonomic function and seizures (Sarnat & Sarnat, 1976). Briefly,
mild encephalopathy is characterised by hyper-alertness, hyper-reflexia, dilated pupils,
tachycardia, and the absence of seizures. Moderate encephalopathy includes the presence of
lethargy, hyper-reflexia, miosis, bradycardia, seizures, hypotonia and weak suck and Moro
responses. Severe encephalopathy is characterised by stupor, flaccidity, small to mid-position
pupils which react poorly to light, decreased stretch reflexes, hypothermia and absent Moro
and suck responses (Sarnat & Sarnat, 1976; Finer et al., 1981). Various attempts have been
7
made to improve on these descriptions(Levene et al., 1985; Amiel-Tison & Ellison, 1986), but
the original Sarnat grading appears robust and clear in its categorisation.
There is debate in the literature regarding whether NE or HIE is the most appropriate term
to describe encephalopathy in the neonate. Multiple causes of brain injury have been
identified in both premature and term infants which include hypoxia-ischaemia,
haemorrhage, infection, and metabolic derangement. From this list, hypoxia-ischaemia is
recognised to predominate (Volpe, 2001b). The argument for using the term NE rather
than HIE is that hypoxia and ischaemia have often not been proven and have been assumed
from a variety of clinical markers that do not accurately reflect hypoxia or ischaemia of either
acute or chronic origin (Leviton & Nelson, 1992). This argument is supported by several
epidemiological studies which have described over 75% of cases of NE as having no clinical
signs of intrapartum hypoxia (Nelson & Leviton, 1991; Adamson et al., 1995; Badawi et al.,
1998b, a). In contrast a number of recent studies suggest that hypoxia-ischaemia, rather than
antenatal events, is the major cause of NE (Evans et al., 2001; Cowan et al., 2003). This
debate will be further discussed in section 1.2.3.
In this thesis, when referring to encephalopathy, I will use the term neonatal encephalopathy
unless citing a reference that specifically reports hypoxic-ischaemic encephalopathy.
1.2.1.5 Cerebral palsy
Cerebral palsy is an umbrella term covering a group of non-progressive, but often changing
motor impairment syndromes secondary to lesions or abnormalities of the brain arising in the
early stages of its development (Mutch et al., 1992; Stanley et al., 2000). There are a number
of different types of cerebral palsy. Spastic quadriplegia and, less commonly, dyskinetic
cerebral palsy are the only two subtypes of cerebral palsy associated with acute hypoxic
intrapartum events (Stanley et al., 1993; Rosenbloom, 1994; MacLennan, 1999). Spastic
quadriplegia is not specific to intrapartum hypoxia (Stanley et al., 2000). Only 24% of a
population base series of children with moderate or severe spastic quadriplegia were thought
to have been affected by intrapartum events (Stanley et al., 1993). Hemiplegic cerebral palsy,
spastic diplegia and ataxia have not been associated with acute intrapartum hypoxia
(MacLennan, 1999; Stanley et al., 2000).
8
1.2.2 Historical perspective of birth asphyxia
Fetal asphyxia was first described by the ancient Greeks who described severe depression at
birth as ‘asphyxia neonatorum’ meaning without pulse. This description was subdivided into
‘asphyxia livida’ meaning asphyxia with cyanosis, and ‘asphyxia pallida’ which was asphyxia
with shock and vasoconstriction of the periphery (Claye, 1963).
The first description of an association between abnormal neurologic outcome in children and
complications during birth was made by the English surgeon William John Little during the
19th century (Little, 1862). In a series of nine lectures beginning in 1843 entitled a "Course of
Lectures on Deformity of the Human Frame," Little presented the first medical descriptions
of cerebral palsy. He described cerebral palsy as "a peculiar distortion, never previously
described, that affects newborn children with spasmodic tetanus-like rigidity and distortion of
the limbs" (Accardo, 1989; Schifrin & Longo, 2000). By the end of the 19th century, the
disorder was commonly known as "Little's Disease". William Little began correlating the
relationship of Little's Disease to both premature and complicated births. By 1861, after
studying the disorder for 20 years, Little had tabulated over 200 cases of Little's Disease. In a
thesis before the Obstetrical Society of London, Little proposed that the causes of this
previously overlooked disorder included asphyxia at birth, premature birth, and direct
mechanical injury (Little, 1862; Schifrin & Longo, 2000). Little further noted that the
susceptibility of the fetal nervous system to damage depended on its stage of development.
Consistent pathological features associated with birth asphyxia have been recognised since
the 19th century. Anton described status marmoratus or état marbre involving the basal ganglia in
1893 (Levene, 1995) and Bressler first described ulegyria or gyral scarring in 1899.
Sir William Osler (1849-1919) described by many as the most influential physician in history,
was the first to use the name cerebral palsy, in a series of five lectures entitled, "The cerebral
palsies of children". Osler also described associations between cerebral palsy and difficult
delivery, asphyxia requiring prolonged resuscitation, neonatal seizures and arachnoid or
subarachnoid bleeding.
Sigmund Freud (1856-1939) was the next physician to write on cerebral palsy and he devoted
several volumes to the study of this condition (Freud & Rie, 1891; Freud, 1893, 1897). He
agreed with William Little that difficult birth was a cause of cerebral palsy, but went on to
suggest that difficult birth might more often be a symptom rather than a cause of the
condition. Freud, who had originally trained as a neurologist with a strong background in
9
neuroanatomy, stressed the lack of correlation of clinical findings with either purported
aetiology or neuropathological findings. It was Freud who first proposed that antenatal fetal
exposure to asphyxia could also result in cerebral palsy (Freud, 1897). He also described the
relationship between lesions in the brain and the degree of spasticity and paralysis in the
body, noting that more superficial brain lesions were more likely to cause problems in the
lower extremities.
Even though there were no reliable clinical data to support a diagnosis of “fetal distress”,
from 1862 until the late 1980’s it was commonly believed that the major cause of cerebral
palsy was asphyxia occurring during the intrapartum period (Little, 1862; Lilienfeld &
Pasamanick, 1955; Eastman et al., 1962; Steer & Bonney, 1962). Even in the mid-1970’s it
was believed that more than half of the cases of mental retardation were due to intrapartum
asphyxia and that electronic FHR monitoring could potentially prevent it (Quilligan & Paul,
1975).
1.2.3 Current view of fetal brain injury during labour
The theories of the last 150 years were challenged during the last two decades by
epidemiological studies suggesting that intrapartum hypoxia was not the cause of the majority
of cases of cerebral palsy. In 1988 both a cohort study from North America (Nelson, 1988)
and case-control study from Western Australia (Blair & Stanley, 1988) reported that in 90%
of cases of cerebral palsy, intrapartum hypoxia could not be the cause of cerebral palsy and in
the remaining 10% intrapartum signs compatible with damaging hypoxia may have had
antenatal or intrapartum origins. Similar results were also reported in another
geographically-based cohort study from Oxford, in the United Kingdom (Yudkin et al., 1995).
These three studies and a number of other epidemiological studies suggested that a large
proportion of cases of cerebral palsy were associated with maternal and antenatal factors
such as prematurity, intrauterine growth restriction, intrauterine infection, fetal coagulation
disorders, multiple pregnancy, antepartum haemorrhage, breech presentation, and
chromosomal or congenital anomalies (Nelson & Ellenberg, 1984; Stanley & Alberman,
1984; Nelson & Ellenberg, 1986; Blair & Stanley, 1988; Naeye et al., 1989; Nelson, 1989; Blair
& Stanley, 1990, 1993; PSANZ, 1995; Volpe, 1995b; Evrard et al., 1996; Grether & Nelson,
1997; Nelson et al., 1998; Nelson & Grether, 1998).
This body of epidemiological evidence led the American College of Obstetricians and
Gynecologists (ACOG), the Society of Obstetricians and Gynaecologists of Canada (SCOG)
and the Perinatal Society of Australia and New Zealand (PSANZ) to release policy
10
statements stating that a number of criteria were required to be met to link perinatal asphyxia
to neurological deficit. As early as 1992, ACOG issued a technical bulletin concluding that
for perinatal asphyxia to be linked to a neurological deficit in the child, all of the following
criteria must be present (ACOG, 1992):
1. Profound umbilical artery metabolic or mixed acidaemia (pH<7.00),
2. Persistence of an Apgar score of 0-3 for longer than 5 minutes,
3. Neonatal neurological sequelae including seizures, coma or hypotonia, and
4. Multiple organ system dysfunction (eg. cardiovascular, gastrointestinal, haematologic,
pulmonary or renal).
In 1995, the Task Force on Cerebral Palsy and Neonatal Asphyxia of the Society of
Obstetricians and Gynaecologists of Canada issued a policy statement (SOGC, 1996a) which
stated that the same criteria as those described by the ACOG should be used for defining a
potential link between intrapartum events and neurologic outcome. In addition to the four
ACOG criteria, they added a fifth criterion that the umbilical artery base deficit had to be
greater than 16mmol/L. The Canadian task force stated that if all these criteria were not
present, one could not conclude that hypoxic acidaemia existed nor that it had the potential
to cause neurological deficits during the intrapartum period.
In 1999, the Consensus Statement of the Perinatal Society of Australia and New Zealand
regarding the intrapartum origin of cerebral palsy (MacLennan, 1999) was published and has
now been endorsed by 21 obstetric and paediatric societies from around the world. This
statement defined criteria required to suggest the occurrence of damaging intrapartum
hypoxia sufficient to cause permanent neurological impairment. These criteria are presented
in Table 1.2 and included three essential criteria and five suggestive criteria which could be
used to help time the hypoxic event to the intrapartum period.
Cerebral palsy is not the only form of brain injury which may result from intrapartum
hypoxia. Early onset neonatal encephalopathy can also result from intrapartum hypoxia and
ischaemia and has a prevalence that varies widely in the literature according to the definition
used and the population to which that definition is applied. As a result the prevalence quoted
in the literature ranges from 1.8 to 7.7 per 1000 term live births (Finer et al., 1981; Ergander et
al., 1983; Levene et al., 1985; Nelson & Leviton, 1991; Adamson et al., 1995; Thornberg et al.,
1995). The United States Collaborative Perinatal Project studied 39,000 infants born at birth
weights greater than 2500g and found that 70% of those who showed early onset neonatal
encephalopathy died or were disabled (Ellenberg & Nelson, 1988). Mortality from neonatal
11
encephalopathy varies in different populations from 7% to 30.5% (Brown et al., 1974; Ellis et
al., 1999) and the spectra of impairment which can result from neonatal encephalopathy
include cerebral palsy, cerebral blindness, cognitive impairment, developmental delay,
convulsive disorders and severe hearing loss (Robertson et al., 1989; Dixon et al., 2002).
Table 1.2 Criteria to define an acute intrapartum hypoxic event
Essential Criteria
1
Metabolic acidaemia in intrapartum fetal scalp, umbilical cord arterial or very early neonatal blood samples (pH<7.00 and base deficit >12mmol/l)
2 Early onset moderate or severe neonatal encephalopathy in neonates ≥ 34 weeks
3 Cerebral palsy of the spastic quadriplegic or dyskinetic type
Non-specific criteria to time the hypoxic event to the intrapartum period
4 A sentinel hypoxic event occurring immediately before or during labour
5
A sudden, rapid, and sustained deterioration of the fetal heart rate pattern usually after the sentinel hypoxic event where the pattern was previously normal
6 Apgar scores of 0-6 for longer than 5 minutes
7 Early evidence of multisystemic involvement
8 Early imaging evidence of acute cerebral abnormality (Adapted from MacLennan, 1999) Similar to cerebral palsy, for many years it was accepted that fetal asphyxia during labour was
the major cause of neonatal encephalopathy despite the fact that the evidence for this was
sparse (Edwards & Nelson, 1998). Again, during the last decade, epidemiological studies
suggested that the primary aetiology of neonatal encephalopathy was antenatal rather than
intrapartum events (Nelson & Leviton, 1991; Yudkin et al., 1994; Badawi et al., 1998a). The
independent risk factors for neonatal encephalopathy occurring before conception or during
the antepartum period included socioeconomic status, family history of seizure or other
neurologic disease, conception after infertility treatment, maternal thyroid disease, severe pre-
eclampsia, bleeding in pregnancy, viral illness, having an abnormal placenta, intrauterine
growth restriction, and postmaturity (Edwards & Nelson, 1998).
Some caution is required in the interpretation of the epidemiological studies of neonatal
encephalopathy. First, the definition of encephalopathy varies between publications. In the
two studies by Badawi et al. (1998a,b) the diagnosis of encephalopathy is wider and less
restrictive than other studies and the long term prognosis of this wider definition is uncertain.
12
For comparison, in a consecutive series of 56 infants at the Hammersmith Hospital in
London diagnosed as encephalopathic on criteria similar to those used by Badawi et al.,
almost half would have been classified as clinically normal at 18-24 months (Edwards &
Nelson, 1998). Second, Badawi et al.(1998a,b) included infants with obvious neuro-
developmental and chromosome abnormalities, and those who presented with the disorder
late in the first week of life. The inclusion of 37 infants in the cohort with birth defects (23%
of the cases) clearly has implications for the subsequent analysis of the likely time of the
insult (Ellis & de Costello, 1999). Furthermore, infants with problems such as low tone and
feeding difficulties, which are common to many disorders, were included as having
encephalopathy. Therefore, the population selected in Badawi’s studies would have reduced
the proportion of infants in whom difficulties during birth were the most important factor,
and increased the chance that earlier antenatal factors would be significant. It is worthy to
note that while Badawi et al. (1998b) attribute the cause of neonatal encephalopathy to
antenatal factors rather than any intrapartum abnormality, they reported significant
differences between cases and controls in the frequency of post maturity beyond 42 weeks,
acute intrapartum events, Apgar score, onset of respiration, need for resuscitation, and birth
trauma. Furthermore, they identified an inverse association between elective caesarean
section and neonatal encephalopathy (Badawi et al., 1998b), a relation also suggested by
Gaffney and colleagues (Gaffney et al., 1994a). Together, these observations suggest that
intrapartum events may have a greater contribution to neonatal encephalopathy than
reported by the authors.
Other authors have presented epidemiological evidence to suggest that events during labour
are associated with neonatal outcome (Gaffney et al., 1994a; Nelson & Grether, 1998; Heller
et al., 2000; Draper et al., 2002). Gaffney et al. reported a cohort of children with cerebral
palsy with and without neonatal encephalopathy at birth. The maternal and antenatal
characteristics were similar in the two groups of children; however, they did find that the
seriousness of intrapartum complications and the pattern and severity of motor and
intellectual deficit were significantly worse in the neonatal encephalopathy group, strongly
suggesting that events during birth do affect infant outcome (Gaffney et al., 1994a). Nelson et
al. (1998) have noted that a tight nuchal cord at delivery was seen significantly more often in
infants with neonatal encephalopathy than in controls, which lends further support to the
explanation that events late in labour are important in neonatal encephalopathy (Nelson &
Grether, 1998). There are also data which suggest that infants born at night are more than
twice as likely to die from asphyxia than those born during the day, a finding that might be
attributable to tiredness and inexperience of night time staff, or a delay in appropriate
13
treatment (Heller et al., 2000). Evidence from confidential inquiries into neonatal
encephalopathy and neonatal deaths in term infants in the United Kingdom documents a
high proportion of suboptimal care during labour (Draper et al., 2002).
Over the last three years a number of studies have been published which lend support to the
original hypothesis that hypoxic-ischaemic events in the perinatal period are the main cause
of early neonatal encephalopathy, seizures or both (Biagioni et al., 2001; Maalouf et al., 2001;
Cowan et al., 2003) and perhaps play a more important role in the origin of cerebral palsy
than postulated over the last two decades (Hagberg et al., 2001). The most recent publication
by Cowan et al. (2003) was a review of 351 neonates referred to two tertiary centres for the
assessment of neonatal encephalopathy, seizures or both. In this study magnetic resonance
imaging (MRI) found evidence of an acute insult without established injury or atrophy in
80% of infants with neonatal encephalopathy and 69% of infants with seizures. Similar
results have been reported in other studies using MRI to assess neonates with neonatal
encephalopathy (Biagioni et al., 2001; Maalouf et al., 2001). The series reported by Cowan et
al. (2003) was not population-based, however this disadvantage is somewhat offset by the
detailed investigations and specific diagnosis for all of the 351 infants, ensuring that there was
no ambiguity about their inclusion in a study of term infants with encephalopathy. The
authors commented that while it is accepted that a few normal term infants will have
established lesions on MRI at birth, the low rate of established lesions in their study suggests
that there was no relation between these lesions and presentation with encephalopathy. This
observation is in accordance with data from Hagberg and colleagues' who noted that term
infants later presenting with cerebral palsy of apparent antenatal origin rarely had symptoms
as a neonate (Hagberg et al., 2001).
There have been concerns expressed about the prognostic significance of abnormalities
identified on neonatal MRI, particularly in neonates with encephalopathy, where there is a
high incidence of abnormalities on MRI. In the study by Cowan et al. (2003) all of the babies
with hypoxic-ischaemic patterns of lesions on MRI have been followed up for a minimum of
18 months; 34% died and 43% developed cerebral palsy. The location of injury in those who
died was largely central grey matter and the brain stem. No infant with only cortical or white
matter injury on MRI scan died, and only 2 with focal infarction died. The severity of
cerebral palsy in the surviving infants was consistent with published data that relates to
pattern of injury with imaging findings (Rutherford et al., 1996; Mercuri et al., 1999; Cowan,
2000). One long-term follow-up study has been performed on children with neonatal
encephalopathy who underwent MRI within their first week of life (Barnett et al., 2002). In
14
that study 68 term infants with NE were assessed between 5 and 6 years of age. Fifteen of
the 68 infants (22%) died in the neonatal period. Of the 53 surviving infants, 19 (36%)
developed cerebral palsy. The remaining 34 infants were considered normal at 2 years of age
but when assessed at school age, 8 (15%) had minor neurological dysfunction and/or
perceptual-motor difficulties, 1 (2%) had only cognitive impairment and 25 (47%) were
normal. Again in this study, the outcome largely reflected the pattern of lesions on MRI
imaging; 83% of those with a normal outcome had normal scans or minimal white matter
lesions and 80% of those with minor neurological dysfunction and/or perceptual motor
difficulties had mild or moderate basal ganglia lesions or more marked white matter lesions.
Taken together, recent studies utilizing MRI imaging suggest that MRI findings assist in the
prediction of outcome better than reliance on clinical assessment and Apgar score, which are
less specific (Barnett et al., 2002; Mercuri et al., 2002).
There are two recent population-based cohort studies that add useful information to the
debate regarding the potential intrapartum origin of cerebral palsy. The first study is a
population-based cohort of children with cerebral palsy from Sweden (Hagberg et al., 2001)
which encompassed the western health care region of Sweden, a total population of 2.1
million. Unlike many of the previous population-based cohort studies of cerebral palsy,
postnatal computer tomography and/or MRI was available for 90% of the 241 cases of
cerebral palsy identified in the population over a 4 year period (1991-1994). Of the cases of
cerebral palsy, 34% were assessed to have an antenatal aetiology, 46% had a perinatal
aetiology (intrapartum + neonatal) and 19% were unclassifiable. In term children who
developed cerebral palsy, hypoxic-ischaemic encephalopathy (HIE) was the single most
common aetiology present in 79% of cases and the birth asphyxia responsible for the HIE
began in labour in 86% of these cases. The outcomes in this study were graded; normal with
no seizures, seizures and not disabled, seizures and disabled, four-limb cerebral palsy and
death. Across this range of outcomes a gradient of severity was apparent in Apgar scores,
acidosis, renal dysfunction and the need for ventilation. Overall in this cohort, birth asphyxia
severe enough to cause cerebral palsy was recorded and documented in 28% of cases.
The second recent study suggesting an intrapartum origin of cerebral palsy is a population
cohort of children with neonatal encephalopathy from the South West Thames Region in the
United Kingdom (Evans et al., 2001). In their cohort of 143 cases of neonatal
encephalopathy (NE), which deliberately used an over-inclusive case definition similar to that
of Badawi et al. (1998), there were only seven cases (5%) with well-defined conditions that
were clearly unrelated to intrapartum asphyxia. Of the 15 NE deaths, 13 (87%) were
15
attributed to HIE; there was one dysmorphic baby and one with a probable metabolic
disorder. There were 16 cases of cerebral palsy in this cohort, giving a prevalence of cerebral
palsy of 12%, and antenatal complications or risk factors were identified in only 3 cases
(19%). Of the 16 cases of cerebral palsy, 13 had four-limb cerebral palsy of which 12 had
evidence of HIE based on their neonatal illness, the occurrence of acidosis and renal
dysfunction. The authors concluded that in term infants, neonatal encephalopathy with
seizures is commonly the result of an acute insult, rather than a direct reflection of
antepartum events, and it carries a high risk of death or cerebral palsy (Evans et al., 2001).
1.2.4 Conclusion
In summary, there is a vigorous debate about how important birth asphyxia really is as a
cause of either neonatal encephalopathy or cerebral palsy. The question is not whether birth
asphyxia happens or whether it can cause brain injury; both of these occurrences are real.
The issues are; first, what proportion of neurological impairment in children is due to
perinatal hypoxia-ischaemia and how much is due to entirely different causes? Second, how
many infants with asphyxia also have significant predisposing factors? Third, what are the
non-asphyxial factors which contribute to or determine unfavourable outcome? Considering
all of the published literature over the last three decades, between 5% and 95% of neonatal
encephalopathy is solely due to perinatal hypoxia-ischaemia and between 5% and 28% of
cerebral palsy has an intrapartum origin.
Although as an obstetrician I would like to believe that the great majority of adverse events
occur antenatally and few occur during the intrapartum period, there is growing evidence that
intrapartum events may be more important with regard to brain injury. The answers to the
questions posed above are yet to be established but they probably lie somewhere between
two divergent opinions. It is only with further large prospective population-based studies
utilising both advanced neonatal imaging with MRI and detailed follow-up with blinded
assessors, that the truth will be established.
16
1.3 Mechanisms of brain injury
During the 1950’s, it was recognised that animal models were required to permit systematic
examination of the effects of asphyxia and ischaemia on the fetus. At a symposium marking
the beginning of studies of the fetus in the monkey colony in Puerto Rico, Dr Baillie, the
Director of the National Institute of Neurological Disease, stated “Medical research in regard
to cerebral palsy and other neurological disorders has been relatively neglected. The focus
should be on those adverse biological factors, which operate in the perinatal period. The
proper point of departure is through controlled animal observations” (Windle, 1956).
Since 1956, there have been a number of research models utilised to examine the effect of
asphyxial exposure in the fetal monkey and fetal lamb. More recently, other animals such as
the rat, guinea pig and gerbil have also been utilised. Asphyxia has been induced by maternal
hypoxaemia, reduced uteroplacental blood flow, and umbilical cord occlusion whilst cerebral
ischaemia has been induced by carotid artery occlusion.
Animal models of asphyxia have been used to address two issues; first, to determine the fetal
response to asphyxial exposure, and second, to investigate the pattern and frequency of brain
injury after an asphyxial exposure.
1.3.1 The fetal response to asphyxia
Fetal survival after perinatal hypoxia-ischaemia depends in part on maintaining a balance
between oxygen supply and demand. It is well established that at extreme levels of
hypoxaemia the fall in oxygen delivery inhibits aerobic metabolism and eventually leads to a
fall in the concentration of high-energy phosphates in all tissues. This however represents
the final stage. Before this, during more moderate reductions in oxygen delivery, there is
evidence that the fetus is capable of mounting a complex response involving compensatory
vascular and metabolic mechanisms, which, at least in the short term, might preserve
oxygenation and function in essential organs such as the heart and brain (Jensen & Lang,
1992). The fetal cardiovascular responses to asphyxia have been thoroughly investigated in
the fetal lamb using a variety of different models of asphyxia including maternal hypoxaemia
(Cohn et al., 1974; Peeters et al., 1979; Richardson et al., 1989; Richardson et al., 1993; Asano et
al., 1994), reduced utero-placental blood flow (Field et al., 1990; Jensen et al., 1991; Reid et al.,
1991; Ball et al., 1994), and cord occlusion (Itskovitz et al., 1987; Ball et al., 1994; Kaneko et al.,
17
2003). There has been a remarkable consistency in the fetal cardiovascular response to
asphyxia seen in these different models as demonstrated in Table 1.3.
Table 1.3 Cardiovascular responses to an asphyxial exposure
Blood Flow
Reference:
Mod
el*
Dur
atio
n (m
in)
Mea
n A
rteria
l Pr
essu
re
Brai
n
Hea
rt
Adr
enal
Kid
ney
Car
cass
Cohn et al, 1974 1 4-44 ↑ ↑ ↑ ↑ ↓ ↓
Peeters et al, 1979 1 60 ↑ ↑ ↑ ↑ ↓ ↓
Richardson et al, 1989 1 420 ↑ ↑
Field et al, 1990 2 90 ↑ ↑ ↑ ↑ ↓ ↓
Reid et al, 1991 2 20 ↑ ↑ ↑ ↑ ↓ ↓
Jensen et al, 1991 2 35 ↑ ↑ ↑ ↑ φ ↓
Ball et al, 1994 2 90 φ φ ↑ ↑ ↓ ↓
Newman et al, 2000 2 60 φ φ ↓
Itskovitz et al,1987 3 5 ↑ ↑ ↑ ↑ φ φ
Ball et al, 1994 3 90 ↑ ↑ φ ↑ ↓ ↓ *Model 1: maternal hypoxaemia; Model 2: reduced utero-placental blood flow; Model 3: umbilical cord occlusion. The up arrow (↑) indicates a significant increase, the down arrow (↓) a significant decrease, and the φ represents a change that was not statistically significant. (Adapted from Low, 1998)
The late gestation fetal sheep mounts a rapid response to an episode of acute hypoxia with
bradycardia and redistribution of the combined ventricular output to the heart, brain and
adrenal glands at the expense of the fetal carcass (Campbell et al., 1967; Cohn et al., 1974;
Peeters et al., 1979; Giussani et al., 1994). The initial response is an increase in arterial blood
pressure due to increased systemic vascular resistance mediated by the autonomic nervous
system (Jensen & Lang, 1992). This response is initiated by arterial chemoreceptors (Bartelds
et al., 1993) and then augmented by an increase in plasma catecholamines, ACTH, cortisol
and possibly by arginine vasopressin, angiotensin and circulating endogenous opiates (Rurak,
1978; LaGamma et al., 1982; Jones et al., 1988; Challis et al., 1989; Giussani et al., 1994). The
18
fetus compensates by increasing blood flow through the ductus venosus, thus reducing blood
flow through the liver. Therefore, a fall in arterial oxygen content results in a response that
reduces the delivery of oxygen to organs such as the gut and carcass, while protecting the
heart, adrenals and brain.
During hypoxic insults in late gestation fetal sheep there is an increase in cerebral blood flow
to all areas of the fetal brain. After both two and four minutes of asphyxia there is a 2.5 fold
increase in cortical blood flow. The increases in blood flow to the brainstem and subcortex
are relatively more pronounced (Kaneko et al., 2003). With more prolonged insults, the
increases in cerebral blood flow are of a smaller magnitude (Richardson et al., 1989). Of note,
the lowest increase in blood flow in the subcortical regions occurs in the corpus striatum
(Kaneko et al., 2003) and it is this structure that shows neuronal loss in the near-term ovine
fetus after repetitive cord occlusion of a severe degree (Mallard et al., 1995b). The increase in
cerebral blood flow is thought to result from both a significant increase in perfusion pressure
(Kaneko et al., 2003) and cerebral vasodilation from local, circulating and neurogenic factors
that affect cerebral vasculature (Licata et al., 1975; Kontos, 1981).
During the first 60 seconds of an acute hypoxic episode in the late gestation fetal sheep the
oxygen uptake in the fetal brain is well maintained (Richardson et al., 1996). After this time
there is a progressive reduction in oxygen delivery and fractional oxygen extraction despite
the marked increase in cerebral blood flow (Kaneko et al., 2003). It has been shown that the
cortex and cerebellum reduce their oxygen delivery by one third after two minutes of acute
hypoxia whereas the subcortical regions and the brainstem are able to maintain oxygen
delivery for longer periods of time (Kaneko et al., 2003).
Despite maintaining or partially maintaining oxygen delivery to the near-term fetal brain
during hypoxia, the fractional oxygen extraction has been shown to be reduced toward zero
after two minutes of an acute hypoxic episode whereas the glucose fractional extraction is
doubled over the same time period (Chao et al., 1989; Kaneko et al., 2003). This suggests that
oxygen availability is the rate limiting step for oxygen consumption and in the absence of
adequate oxygen there is a rapid switch to anaerobic metabolism in the fetal brain. Lactate is
the end product of anaerobic metabolism, and under normoxic conditions the fractional
extraction of lactate is close to zero. During hypoxia a small but significant efflux of lactate
has been demonstrated from the ovine fetal brain (Kaneko et al., 2003). The limited size of
the lactate efflux may relate to constraints of the blood brain barrier transport systems (Chao
et al., 1989).
19
Overall, during an acute hypoxic event it has been calculated that there is an 80% decrease in
total energy production in the fetal brain after 2 minutes of cord occlusion (Kaneko et al.,
2003). This is countered by a decrease in synaptic transmission in the fetal brain, reflected by
a flat isoelectric electroencephalogram which has been suggested to reduce the total energy
requirements of the fetal brain by 50% (Astrup, 1982; Jones & Traystman, 1984). The twin
strategies of reducing energy turnover and maximising the efficiency of ATP production
mean that supply and demand are balanced, and ATP levels remain stable (Connett et al.,
1990; Newman et al., 2000).
These cardiovascular and metabolic responses to asphyxia are important to the integrity of
the central nervous system during the compensatory phase of an asphyxial exposure. If the
asphyxial exposure persists, the fetus will cross a threshold after which decompensation
begins to occur. When fetal oxygen saturation falls below 30%, a progressive metabolic
acidosis will develop. The threshold of cerebral dysfunction, characterised by suppression of
electrocortical activity and seizures in the fetal lamb, occurs when metabolic acidosis is severe
(base deficit greater than 16mmol/L) (Low, 1998). Decompensation with severe metabolic
acidosis is associated with a fall of arterial blood pressure due either to a decrease in cardiac
output or a failure to maintain peripheral vascular resistance. The combination of asphyxia
and ischaemia due to hypotension results in a decrease of cerebral oxygen consumption and,
if sustained, brain damage (Maulik, 1998).
1.3.2 Pattern and frequency of brain injury after asphyxia
Since the first animal studies of asphyxia were performed in the 1950’s, it has been
recognised that specific patterns of perinatal brain damage are associated with different types
of asphyxial insult. This is illustrated by the work of Myers et al. who described four patterns
of perinatal brain damage in primates associated with different categories of insults (Myers,
1975). These are summarised in Table 1.4. Anoxia or total asphyxia, whether in the
newborn or the adult, resulted in injury in the brainstem. In those animals that survived,
cerebral structures outside the thalamus and brainstem were spared. In contrast, situations
leading to hypoxia associated with severe acidosis (usually of a mixed respiratory and
metabolic type), cause brain oedema. When this oedema is limited in distribution, the
damage is restricted to specific cortical loci. However, when oedema becomes more
generalised, more extensive regions of the hemispheres are damaged until the entire
cerebrum may become necrotic. When the experimental model led to hypoxia without
acidosis, damage was restricted to the white matter, characterised by periventricular white
20
matter haemorrhage and/or focal areas of periventricular leukomalacia. The fourth pattern
described by Myers was that which occurred with combined episodes of hypoxia plus anoxia
with acidosis; these circumstances favoured a predominance of lesions that affected the basal
ganglia.
Table 1.4 Four patterns of perinatal brain damage
Neuropathology
Pattern
Corte
x
Whi
te
Mat
ter
Basa
l G
angl
ia,
Thala
mus
Brain
stem
Anoxia or total asphyxia + ++
Hypoxia with severe acidosis ++
Hypoxia without acidosis ++
Combined episodes of hypoxia, anoxia and acidosis ++ (Adapted from Myers et al., 1975)
Many different animal models have been utilised to investigate the occurrence of cerebral
dysfunction and brain damage after an asphyxial exposure. These studies can be broadly
categorised as cerebral ischaemia models, global asphyxia models and partial asphyxia
models.
1.3.2.1 Cerebral ischaemia models
Cerebral ischaemia has been examined using a carotid artery occlusion model in chronically
instrumented fetal lambs and rats (Williams et al., 1992; Mallard et al., 1993; Uehara et al.,
1999). The results of these studies are summarised in Table 1.5. Ten to twenty minutes of
ischaemia resulted in brief depression of the electroencephalogram (EEG) which then
progressively recovered. Mild selective neuronal necrosis was seen on post mortem
examination. After 30-40 minutes of ischaemia, EEG activity remained depressed for 8
hours, followed by a transition to low-frequency epileptiform activity that reached maximum
intensity at 10 hours. This sequence was associated with laminar necrosis of the parasagittal
cortex, and with necrosis in the hippocampus, basal ganglia and thalamus (Williams et al.,
1992).
The electrophysiologic and histologic consequence of isolated brief episodes of reversible
cerebral ischaemia of 10 minutes at 1- and 5-hour intervals has also been examined. The
21
effect of frequent episodes of brief cerebral ischaemia alters the pattern of brain injury with
basal ganglia damage a major feature (Mallard et al., 1993).
Table 1.5 Brain damage after cerebral ischaemia
Neuropathology
Reference Model* Duration Cerebrum Basal Ganglia Thalamus
Williams et al, 1992 1 10 minutes +
Williams et al, 1992 1 40 minutes +++ +
Mallard et al , 1993 1 10 minutes x 3 ++ +++
Uehara et al, 1999 2 2 days ++ * Model 1: carotid artery occlusion (lamb); Model 2: carotid artery occlusion (rat). (Adapted from Low, 1998)
Prolonged carotid artery occlusion has also been examined in a rat model where both carotid
arteries were ligated for 2 days. When the brains were assessed, 91% had evidence of white
matter injury including coagulation necrosis and cystic lesions in and around the internal
capsule while only 9% had evidence of cerebral infarction and 23% had evidence of
ischaemic neurons in the cortex (Uehara et al., 1999).
Therefore, considering these studies it appears that both the duration and pattern of
ischaemia are important factors regarding the resultant neuropathology. Repeated brief
episodes of ischaemia appear to sensitise the fetal brain to neuronal injury, particularly if the
episodes were frequent.
1.3.2.2 Global asphyxia models
A number of series of experiments in the monkey and lamb have been performed, using a
variety of models, to examine the effect of global asphyxia on the fetal brain (Ranck &
Windle, 1959; Mallard et al., 1992; Mallard et al., 1995a; de Haan et al., 1996; de Haan et al.,
1997b; Ohyu et al., 1999; Loeliger et al., 2003). These studies are summarised in Table 1.6.
Ranck and Windle performed the initial studies with the Mucacca mulatta monkey where they
induced global asphyxia by placental separation. They found that global asphyxia for more
than 10 minutes resulted in profound hypoxaemia with severe metabolic acidosis and
hypotension. The resultant neuropathology included neuronal necrosis principally in the
brain stem, basal ganglia, and thalamus (Ranck & Windle, 1959). On the basis of their
22
Table 1.6 Brain damage after global asphyxia
Fetal Response Neuropathology
Reference Model* Duration (minutes) Acidosis Hypotension Cerebrum Basal Ganglia Thalamus Brain Stem
Ranck & Windle, 1959 1 8-30 + + + ++ +++
Mallard et al., 1992 2 10 + + ++ +
Mallard et al. 1995a 2 Recurrent 5 min every 30 min + ++
de Haan et al., 1996 2 Recurrent 5 min every 30 min + + + ++
de Haan et al., 1997b 2 Recurrent 1 min out of 2.5 min or 2 min out of 5 min
+ + + +
Ohyu et al., 1999 2 Recurrent 3 min every 8 min + +
Marumo et al., 2001 2 Recurrent 3 min every 8 min + +/- + +
Loeliger et al., 2003 2 Recurrent 2 min every 30 min day 1, 4 min every 30 min day 2
+ - ++ +
* Model 1: placental separation (monkey); Model 2:– cord occlusion (lamb); +/- present in some but not all fetuses (Adapted from Low, 1998)
23
studies, it was concluded that global asphyxia for less than 8 minutes may not cause brain
damage, while global asphyxia for more than 10 minutes invariably resulted in
neuropathology. This pattern of brain damage after 10 minutes of global asphyxia was
confirmed by 10 minutes of cord occlusion in chronically instrumented fetal sheep (Mallard et
al., 1992). Post mortem examination of these fetuses demonstrated neuronal necrosis in the
hippocampus and to a lesser degree, in the basal ganglia and thalamus. There was a
correlation between hypotension and the magnitude of neuronal necrosis.
Repeated short episodes of global asphyxia have been investigated by a number of groups,
and are associated with different patterns of neuronal loss when compared with prolonged
global asphyxia. Mallard et al., using five minute complete cord occlusion repeated four times
at 30-minute intervals, found the areas principally affected were the basal ganglia and
specifically the striatal projection neurons (Mallard et al., 1995b). De Haan et al. utilised the
same occlusion protocol and found selective neuronal loss throughout the brain, with the
most extensive damage in the putamen. The second most damaged region was the caudate
nucleus of the striatum, followed by the hippocampus, cortex, thalamus and cerebellum (de
Haan et al., 1997c). Fetal EEG activity decreased and cerebral impedance increased during
the occlusions and the maximum spike and seizure activity occurred 5 to 10 hours after
asphyxia (de Haan et al., 1997c).
In further studies, De Haan and Gunn studied 2 groups of lambs; group one experienced
cord occlusion for 1 minute out of every 2.5 minutes and group two underwent occlusion for
2 out of every 5 minutes over several hours (de Haan et al., 1996). Both protocols resulted in
severe acidosis and hypotension and intermittent cord occlusion was continued until mean
arterial pressure was less than 20mmHg or it failed to recover to baseline before the next
occlusion. Ongoing asphyxia was associated with progressive suppression of the EEG,
which occurred faster and with more epileptiform activity in group 2. The predominant
neuropathology was selective mild neuronal necrosis throughout the fetal brain (de Haan et
al., 1996); however, in six of the 14 surviving asphyxiated fetuses, there were focal infarcts in
the parasagittal cortex, thalamus, and cerebellum. Infarction was associated with a longer
period of arterial pressure below baseline levels, with more epileptiform activity, and a slower
normalisation of the EEG (de Haan et al., 1997b). The authors felt that the histologic
damage that was found mimicked that of birth asphyxia in humans, and that their data
suggested brief repeated insults interact, leading to cardiac compromise and cumulative cell
membrane damage in the fetal cerebrum.
24
Ohyu et al. (1999) utilised yet another intermittent umbilical cord occlusion protocol during
which the cord was occluded for 3 minutes every 8 minutes. Interestingly, unlike the
majority of other intermittent cord occlusion models which show injury in the vast majority
of cord occluded fetuses, brain injury was not found in all of the fetuses exposed to this
pattern of cord occlusion. Seven of the 11 (63%) occluded fetuses had white matter and
cortical injury, six (55%) had evidence of thalamic injury and 4 (36%) had no histologic
evidence of damage (Ohyu et al., 1999).
Similar results were reported by Marumo et al. (2000) who used the same cord occlusion
protocol as Ohyu’s group. They found that one third of the occluded fetuses had
periventricular white matter injury, one third had cortical and thalamic injury, and one third
had no or minimal brain lesions. The pattern of neuronal injury was associated with fetal
arterial pressure responses and lipid peroxide concentrations. The fetuses with white matter
injury had higher arterial pressures and lipid peroxide levels prior to the onset of cord
occlusion and the fetuses with thalamic injury had the most severe hypotension during
episodes of asphyxia. Of note, both Marumo et al. and Ohyu et al. delivered most of their
fetuses 1 day after the asphyxial insult rather than waiting three days, the time used by the
majority of other investigators. This may partially explain why they found no neuronal injury
in some of their occluded fetuses.
Loeliger et al. specifically designed their recurrent brief global asphyxia study to induce white
matter injury (Loeliger et al., 2003). Their model was successful in inducing injury to white
matter, layer six of the cerebral cortex, and the caudate-putamen complex. Brain damage was
observed in all fetuses with the most extensive damage occurring in fetuses with the greatest
increase in glutamate levels. No association was found with fetal hypotension.
The preterm fetus appears to have a different response to global asphyxia than near-term
fetuses. At mid-gestation, before myelination has begun, 10 minutes (Mallard et al., 1994) and
20 minutes (Keunen et al., 1997) of cord occlusions produced no brain damage in fetal lambs.
It appears that the mid-gestation fetus may be more resistant to global asphyxia. On the
basis of these studies, the effect of global asphyxia on the fetal brain appears to be dependent
on the nature (prolonged or intermittent) and duration of the asphyxia, and the gestational
age of the fetus.
25
1.3.2.3 Partial asphyxia models
The effects of sustained partial asphyxia have been examined in fetal monkey and lamb
models and the results of these studies are summarised in Table 1.7 (Myers, 1972; Ting et al.,
1983; Clapp et al., 1988; Gunn et al., 1992; de Haan et al., 1993; Ikeda et al., 1998a).
Table 1.7 Brain damage after partial fetal asphyxia
Fetal Response NeuropathologyRe
fere
nce
Mod
el*
Dur
atio
n (m
inut
es)
Acid
osis
Hyp
oten
sion
Cere
brum
Basa
l Gan
glia
Thala
mus
Myers, 1972 1 60-180 + + +++ ++
Ting et al., 1983 2 120 + + ++ ++
Gunn et al., 1992 3 60 + + ++ ++
de Haan et al., 1993 3 60 + φ ++
Clapp et al., 1988 4 1 min out of 3 min for 120 min φ ++
Ikeda et al., 1998a 4 60 + + ++ ++
* Model 1: reduced uteroplacental blood flow (monkey); Model 2: maternal hypoxaemia-fetal hypovolemia (lamb); Model 3: reduced uteroplacental blood flow (lamb); Model 4: partial cord occlusion (lamb); φ, change not statistically significant. (Adapted from Low, 1998) Myers in 1972 exposed fetal monkeys to partial asphyxia by impairing utero-placental blood
flow for one to three hours. All of the monkeys experienced severe acidosis, but only some
had evidence of neuropathology. Those monkeys with neuropathology had experienced
severe acidosis with concurrent hypotension. The neuropathology ranged from generalised
neuronal necrosis to focal neuronal necrosis principally involving the parasagittal regions and
the junctional zones between the occipital and parietal lobes. Neuronal necrosis also
occurred in the basal ganglia and thalamus (Myers, 1972).
26
Ting et al. utilised a different model to investigate partial asphyxia in mid-gestational fetal
lambs; they exposed the fetuses to two hours of maternal hypoxaemia and fetal hypovolemia
(Ting et al., 1983). Subsequent post-mortem examination demonstrated that some lambs had
brain damage; the fetuses with brain damage had experienced moderate acidosis with
concurrent hypotension. The extent of neuropathology in the white matter, cerebral cortex
and basal ganglia correlated with the severity of hypotension.
Gunn et al. investigated partial asphyxia in the late gestation fetal lamb and found similar
results to both the monkey and mid-gestational fetal lamb models (Gunn et al., 1992). They
exposed 14 fetuses to partial asphyxia by reducing uterine blood flow such that the fetal
arterial oxygen content was <1.5mmol/L for 60 minutes (Gunn et al., 1992). Only eight
fetuses had evidence of brain damage, and these had severe acidosis (pH<7.0) with
concurrent hypotension. The predominant neuronal necrosis was in the parasagittal cortex,
and to a lesser extent in the basal ganglia; there was a strong correlation between the severity
of the hypotension and the extent of the neuropathology. They also found an association
between the reduction in EEG intensity during the insult and the severity of neuronal injury
(Gunn et al., 1992). Gunn et al. also compared neuropathology induced by 60 minutes of
reduced uterine artery blood flow with that induced by 30 minutes of cerebral ischaemia
from carotid artery occlusion (Williams et al., 1990; Gunn et al., 1992). They found greater,
but patchy damage to the striatum, and although the overall level of damage in the thalamus
was not significantly different, severe focal damage was seen in one fetus that was never
found in the ischaemic model. These differences may reflect greater susceptibility of the
basal ganglia to acidotic insults. Alternatively, asphyxia may be accompanied by differential
effects on oxygen delivery to cerebral regions. During transient cerebral ischaemia, there is
also likely to be residual anastomotic flow to the mid and hindbrains (Williams et al., 1990).
De Haan et al. performed an interesting project where severe acidosis was induced, without
hypotension, using a partial asphyxia model. Partial asphyxia was induced by reducing
uterine blood flow for 60 minutes to expose the fetuses to severe acidosis (pH<7.0), but in
their model, there was no change in arterial pressure compared with the control group (de
Haan et al., 1993). Without concurrent hypotension, only a few fetuses showed neuronal
necrosis that was principally in the cerebellum and to a lesser extent in the cerebral cortex.
Several different partial cord occlusion models have been employed to induce neuronal
damage in the late gestation fetal lamb. Clapp et al. repeated 1-minute partial cord occlusions
every three minutes for two hours. They found that this model did not induce acidosis, but it
did alter fetal heart rate patterns and fetal EEG. Eight of the nine fetuses subjected to
27
repeated cord occlusion were found to have histological damage in their cerebral white
matter (Clapp et al., 1988).
Ikeda et al. utilised a very different model in which partial umbilical cord occlusion lasted for
60 minutes. This induced severe acidosis (pH<6.9, base excess >20mmol/L) and there was a
wide variety of neuropathology ranging from almost total infarction of cortical and
subcortical structures to extremely subtle and patchy white matter alterations characterised by
slight vacuolisation of the white matter or slight to moderate increases in cellularity confined
to the junction of cerebral cortex and white matter. Even fetuses that showed full recovery
of all physiologic parameters, including EEG activity, demonstrated subtle but distinct white
matter lesions. The gray matter, including the hippocampal neurons, was generally spared in
these cases. EEG parameters, duration of hypotension during asphyxia, and delayed
recovery of blood lactate concentrations correlated well with the histologic grading of brain
damage (Ikeda et al., 1998a).
Most of the animal research with partial asphyxia has been performed on near-term fetuses.
Rees at al. studied the mid-gestation fetal sheep using a model which reduced uteroplacental
blood flow by 50% for 6 or 12 hours at 84 days of gestation (term 147 days; Rees et al, 1997).
In contrast to global asphyxia, which appeared to induce no damage in the preterm fetus
after 10 or 20 minutes of asphyxia, partial asphyxia resulted in white matter damage and
neuronal death in the hippocampus and to a lesser extent in the cerebral cortex and
cerebellum. Partial asphyxia also retarded neuronal migration and the growth of neural
processes in the hippocampus where development is well established at this age.
1.3.2.4 Summary of pattern and frequency of brain injury after asphyxia
Animal models of hypoxia and ischaemia confirm that cerebral ischaemia or global asphyxia
of ten minutes duration is consistently associated with brain injury. The studies using
repeated short episodes of ischaemia or global asphyxia have found that the pattern of brain
injury is dependent on the nature and duration of asphyxia, and the gestational age of the
fetus. Sustained partial asphyxia of 1 to 3 hours duration may or may not be associated with
brain damage, with the severity of the brain injury correlating with the severity of fetal
hypotension.
The sequence of events during global asphyxia or cerebrovascular occlusion provides the
fetus with limited compensation prior to the threshold of brain damage. However, although
global asphyxia or cerebrovascular occlusion may occur, they are an infrequent cause of brain
damage in the surviving human newborn (Low, 1998). With partial asphyxia, the fetal brain
28
is protected for variable periods (hours) before the threshold of metabolic acidosis and
hypotension leading to brain damage is reached. An explanation for the variability of
outcome seen after fetal asphyxial exposure is summarised in Figure 1.1. The fetus may be
exposed to asphyxia with a developing metabolic acidosis; however, if the fetal compensatory
cardiovascular response is maintained with increased blood flow to the brain, heart and
adrenals, then oxygen supply to these organ systems will be sustained and cerebral and other
organ system dysfunction or damage will not occur. If the asphyxia persists with progression
of the metabolic acidosis, then fetal decompensation with hypotension, decreased blood flow
and decreased oxygen supply to the brain will develop. The resulting tissue oxygen debt and
anaerobic metabolism will initiate a cascade of biochemical events leading to brain cell
dysfunction and death (Low, 1998). It is likely that this pathophysiological sequence of
events in response to asphyxia operates in the human fetus; however unlike in the animal
studies, in the human fetus the duration of the exposure or whether the exposure is
continuous or intermittent is rarely known.
1.3.3 Molecular mechanisms of perinatal brain injury
Perinatal brain injury has multiple causes including hypoxia-ischaemia, haemorrhage,
infection, and metabolic derangement. Of these, hypoxia-ischaemia predominates and is
generally accepted as the single most important cause of brain injury in the newborn with
consequences that are potentially devastating and lifelong (du Plessis & Volpe, 2002).
Our understanding of the fundamental mechanisms underlying hypoxic-ischaemic brain
injury has advanced rapidly in recent years. A number of the features of the pathophysiology
and clinical consequences of cerebral hypoxia-ischaemia are unique to the immature brain.
Particularly important in this regard is the critical interface between mechanisms of normal
development and injury to the immature brain, as well as the poorly understood but
important role of developmental plasticity in long-term outcome (du Plessis & Volpe, 2002).
Hypoxia-ischaemia leads to different neuropathological manifestations in term and preterm
infants. In term infants, neuronal injury is the primary manifestation of hypoxia-ischaemia
whereas in the preterm infant, oligodendroglial white matter injury predominates (Volpe,
2001b). The biochemical mechanisms leading from the hypoxic-ischaemic insult to neuronal
injury in the term infant are better understood than those resulting in oligodendroglial injury
in the premature infant.
FETAL
EXPOSUREASPHYXIA
Anoxia
+
Minutes
Hypoxia
+
Hours
HypercapniaRespiratory
Acidosis
HypoxemiaMetabolic Acidosis
FETAL
RESPONSECompensation Decompensation
Blood Pressure
Cerebral Blood Flow
Cerebral O2Consumption
OUTCOME
Cerebral Dysfunction None / MinorModerate /
Severe
Brain Damage Nil +
Mortality Nil +
Figure 1.1 Fetal pathophysiology after an asphyxial exposure. (Adapted from Low, 1998)
29
30
The most recent advances in the understanding of hypoxic-ischaemic brain injury involve the
delayed mechanisms of brain injury, the role of inflammatory processes in this injury and the
pathogenesis of injury to the immature oligodendrocyte.
1.3.3.1 Biphasic energy failure
It is now well established that the cerebral energy failure that results from hypoxic- ischaemic
insults occurs in two phases (Lorek et al., 1994; Roth et al., 1997; Inder & Volpe, 2000).
During the hypoxic-ischaemic insult there is a primary phase of energy failure, which is
followed after reperfusion by at least partial energy recovery. After a latent period, typically
between 8 and 48 hours, a secondary phase of energy failure may develop despite apparently
adequate cerebral perfusion and oxygenation. During the latent phase, processes of delayed
cellular injury become apparent and present attractive targets for clinical interventions to
interrupt this deleterious cascade of events even after termination of the insult.
During the primary energy failure phase, cell loss is related directly to hypoxia with
exhaustion of energy metabolism. Three mechanisms are thought to be involved in cell
death during this phase: (1) depolarization due to hypoxia, causing an influx of sodium,
chloride and water into the cell which if severe enough will result in lysis and cell death; (2)
intracellular calcium accumulation; (3) damage of cell membranes by oxygen free radicals in
the immediate reoxygenation-reperfusion phase (Saugstad, 2001).
Secondary or delayed neuronal death is triggered by events occurring in the primary phase
and is associated with hyperexcitability and cytotoxic oedema for between six and 100 hours
after the insult. The mechanisms involved in delayed neuronal injury are thought to include:
(1) excito-toxic amino acids; (2) apoptosis; and (3) cytotoxic activation of activated microglia
(Lorek et al., 1994; Inder & Volpe, 2000; Saugstad, 2001).
1.3.3.2 Pathogenesis of neuronal injury
Neurons have a high energy demand driven by their continuous activities essential for cellular
function and survival, but they possess inadequate reservoirs of oxygen and substrates for
energy production (Siesjo, 1992). Because neurons do not contain any glycogen or fat, they
depend exclusively on a continuous supply of glucose to fulfil their energy needs. By limiting
the availability of oxygen and glucose, the two obligatory substrates for brain energy
metabolism, hypoxia-ischaemia has a greater potential for neuronal injury than hypoxia alone.
There appears to be two perfusion thresholds for neuronal compromise, one for the
cessation of electrical activity and the other at a lower level for the loss of ionic homeostasis
31
which also serves as a threshold at which neuronal energy failure occurs (Branston et al.,
1974; Heiss et al., 1976; Siesjo, 1992).
Adenosine triphosphate (ATP) is the principal source of energy in a cell. The other high-
energy phosphate compound important as a source of energy in the mammalian brain is
phosphocreatinine. Oxidative phosphorylation is the primary ATP-generating mechanism of
the brain and is responsible for 95% of ATP production. This process occurs in the
neuronal cytosol where glycolysis of glucose generates pyruvate, which is further metabolised
in the Krebs cycle in the mitochondrial matrix to yield carbon dioxide and high-energy
electrons. These electrons are then transported by a complex system of electron carriers in
the respiratory chain in the mitochondrial inner membrane where they are eventually
combined with molecular oxygen. At the end of the respiratory chain an enormous amount
of energy is unleashed which is chemically stored by the formation of ATP. The Krebs cycle
and the mitochondrial respiratory chain require oxygen; from the combustion of one
molecule of glucose, oxidative phosphorylation generates 38 molecules of ATP whereas
anaerobic glycolysis, the alternative pathway utilised during hypoxia, yields only two
molecules of ATP. Thus oxidative phosphorylation is 16-17 times more efficient in
replenishing cellular energy supply than anaerobic metabolism.
Depletion of high-energy phosphates almost certainly initiates a cascade of events leading to
neuronal death after ischaemia and deprivation of oxygen and glucose (Volpe, 2001b). At a
cellular level, cerebral hypoxia-ischaemia sets in motion a cascade of biochemical events
commencing with a shift from oxidative to anaerobic metabolism, which leads to an
accumulation of nicotine adenine dinucleotide (NADH), flavin adenine dinucleotide
(FADH), lactic acid and hydrogen ions (Palmer et al., 1990). If the asphyxial insult persists,
the fetus is unable to maintain circulatory centralisation, and cardiac output and cerebral
perfusion fall. Owing to the acute reduction in oxygen supply, oxidative phosphorylation
and ATP production in the brain are diminished (Berger et al., 1992; Yager et al., 1992; Berger
et al., 1994). As a result, the sodium-potassium pumps in the cell membranes are deprived of
energy needed to maintain ionic gradients. With a reduced membrane potential, large
numbers of calcium ions flow through voltage-dependent ion channels into the cell down an
extreme extra- to intracellular concentration gradient. Intracellular accumulation of sodium
and chloride ions leads to swelling of the cells as water enters by osmosis leading to cytotoxic
cell oedema (Vannucci et al., 1993).
32
There are many consequences of the excessive increase in levels of intracellular calcium
which are summarised in Figures 1.2 and 1.3. (Rehncrona et al., 1982; de Courten-Myers et al.,
1989; Huang & Gibson, 1989; Mattson & Cheng, 1993; Berger & Garnier, 2000; Volpe,
2001b). One damaging effect is the activation of phospholipases A2 and C. These reactions
lead to membrane phospholipid hydrolysis, induction of free radicals, disruption of cell and
organelle membranes, increased membrane permeability and alterations in ionic distribution
(de Courten-Myers et al., 1989). Phospholipase C also catalyses reactions leading to the
production of inositol triphosphate, a second messenger that releases calcium from the
endoplasmic reticulum, and diacyl glycerol, which decreases calcium-sodium exchange
(Rehncrona et al., 1982; Huang & Gibson, 1989). Both these reactions further augment
calcium concentrations in the cell and amplify its deleterious effects, creating a vicious cycle
that eventually destroys the cell.
During ischaemia, besides an influx of calcium ions into the cells via voltage-dependent
calcium channels, additional calcium enters the cell through glutamate-regulated ion channels.
Glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles during
ischaemia following anoxic cell depolarization. Extracellular glutamate accumulates during
hypoxia-ischaemia not only because of enhanced release but also impaired uptake both by
astrocytes and by presynaptic nerve endings (Rosenberg, 1997). The presence of increased
levels of glutamate in the synaptic cleft causes a further uptake of calcium in the postsynaptic
cells which leads to additional damage (Figure 1.2 A and Figure 1.3; Berger & Garnier, 2000).
Glutamate also leads to toxicity of immature oligodendroglia by both receptor and non-
receptor mediated mechanisms (Volpe, 2001b).
The acute lack of cellular energy during ischaemia induces almost complete inhibition of
cerebral protein biosynthesis. Once the ischaemic period is over, protein biosynthesis returns
to pre-ischaemic levels in non-vulnerable regions of the brain, while in more susceptible areas
it remains inhibited (Gitto et al., 2002). The inhibition of protein synthesis appears to be an
early indicator of subsequent neuronal cell death.
A second wave of neuronal cell damage occurs during the reperfusion phase after
termination of the hypoxic-ischaemic insult (Buonocore et al., 2001). It is during this phase
that the principal mechanisms leading to cell death operate. The cascade of events during the
reperfusion phase involves accumulation of cytosolic calcium and the production of oxygen
radicals, synthesis of nitric oxide, inflammatory reactions and an imbalance between the
excitatory and inhibitory neurotransmitter systems (Figure 1.3). Part of the secondary
neuronal cell damage may be caused by induction of apoptosis (Berger & Garnier, 2000).
33
GlutamateRelease
MembraneDepolarization
GlutamateUptake
ExtracellularGlutamate
ATP
IntracellularCa2+
A
ATP
Ca2+
Nitric OxideSynthase
Xanthineoxidase
Lipases Proteases MicrotubuleDisassembly
Nucleases UncoupleOxidative
Phosphorylation
Adenosine
Hypoxanthine
Xanthine
Oxygen FreeRadicals
Arachidonate
Eicosanoids
Degradation ofNeurofilaments
MembraneInjury
CytoskeletonDisruption
Cell Death
B
Figure 1.2 Hypoxia-ischaemia induced energy depletion and the cascade to neuronal death.
(A) Relationship between energy depletion, activation of glutamate receptors and
accumulation of intracellular calcium. (B) Deleterious effects of increased intracellular
calcium; note the critical role of oxygen free radicals. (Adapted from Volpe, 2001b)
33
Figure 1.3 Pathways of hypoxic mediated neuronal cell death.
During hypoxia ATP stores become depleted resulting in an increase in pre-synaptic release
of excitatory amino acids, primarily glutamate which acts on N-methyl-D-aspartate (NMDA),
α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and kainite (KA) receptors
causing an influx of calcium ions (Ca2+). High intracellular Ca2+ levels trigger a cascade of
intracellular reactions resulting in excess activation of kinases, proteases, mitochondrial
dysfunction and consequently additional energy failure, ultimately resulting in cell death of a
necrotic nature. During this process lactic acid, oxygen free radicals (OFR) and nitric oxide
(NO) may be produced and accumulate within the cell. Factors such as these can contribute
to damage of not only the compromised neuron but also neighbouring cells and the blood-
brain barrier, and are thought to play an important role in reperfusion injury..
Hypoxic insults may also result in apoptotic cell death by either the death receptor or
mitochondrial pathways. Binding of ligands to membrane bound death receptors (Fas)
results in the activation of pro-caspases 8 and 3 to initiate cell death and may interact with the
mitochondrial pathway by the pro-apoptotic protein BID. The mitochondrial pathway
responds to stress during which changes in the expression of the pro-apoptotic protein BAX
and the anti-apoptotic protein Bcl-2 permits cytochrome c to leave the mitochondria,
interacting and activating Apaf-1 and pro-caspase 9. Caspase 9 then activates caspase 3
which initiates apoptosis. It is thought that the mitochondrial pathway plays the largest role
in hypoxic apoptotic cell death.
(Adapted from Mattson and Cheng, 1993; Herr and Debatin, 2001; and Duncan 2002)
Bloodbrain
barrier
Glutamate
AMPA
Ca2+
Ca2+
NMDA
Ca2+ Ca2+
KACa2+
Ca2+
Hypoxia+
↓Glucose
ATP
Ca2+
Energy
Necrotic Cell Death
SynapticCleft
Neuron
NeighbouringCell
CellDeath
OFR
OFRNO
NO
Ca2+
MitochondrialDysfunction
KinasesProteases
LactateAxon
Fas
Apoptotic Cell Death
Fas
Cytochrome c
Bax
Bcl-2
Caspase 8
Caspase 3
BID
Apaf-1c
Caspase 9
pro
pro
pro
pro
34
35
There is a large amount of data to suggest that oxidative stress plays a role in the initiation of
apoptosis (MacManus et al., 1993; Fernandez et al., 1995; Polyak et al., 1997). Studies have
shown also that free radicals are important in initiating delayed cell death via apoptosis after
cerebral ischaemia (Buttke & Sandstrom, 1994; Saikumar et al., 1998). Free radical mediated
cell death also appears to be the final common pathway for oligodendroglial cell death
(Volpe, 2001b).
The notion of early necrosis and more delayed apoptosis has recently been challenged by
evidence that suggests that the distinction and interaction between these two modes of cell
death is more complex than previously thought. Instead of the two temporally distinct
modes of cell death, an apoptotic-necrotic continuum has been described with an
intermediate group of hybrid cells having features of both necrosis and apoptosis (Nakajima
et al., 2000).
Recent studies have provided additional information regarding the timing of apoptosis in
brain injury. Caspase-3 activation, the pivotal apoptotic event, occurs between 24 hours and
7 days post-insult suggesting a prolonged role for apoptosis in neonatal brain injury. The rate
of apoptosis also differs across various brain regions being maximal in the thalamus and
hippocampus between 24 and 72 hours but delayed for up to seven days in the basal ganglia
and neocortex (Nakajima et al., 2000). Although it has been suggested that apoptosis is
delayed by up to 24 hours after an acute insult, critical apoptotic processes are in progress as
early as six hours post-insult. These include elevation of the Fas death receptor protein,
accumulation of cytosolic cytochrome c, caspase-8 activation and peri-nuclear clustering of
mitochondria (Figure 1.3; Northington et al, 2001).
1.3.3.3 The role of inflammatory mechanisms
The fetal inflammatory response appears to be important in the genesis of hypoxic-ischaemic
brain injury (Volpe, 2001b). Recent epidemiological and experimental studies have
demonstrated a strong association between inflammation and the development of
periventricular leukomalacia (PVL) and cerebral palsy, especially in the preterm infant
(Nelson et al., 1998). It is known that maternal, neonatal and fetal infections are associated
with increases in circulating cytokines, but this can also be triggered by ischaemia.
Pro-inflammatory cytokines may cause brain injury through several different mechanisms.
Inflammatory cytokines have direct cerebral cytotoxicity, inhibiting the differentiation of
oligodendrocyte precursors (Cammer & Zhang, 1999), stimulating oligodendrocyte apoptosis
(Selmaj et al., 1991; Louis et al., 1993; Selmaj et al., 1998), and causing vacuolar myelin
36
degeneration (Probert et al., 1995; Taupin et al., 1997). Cytokines may also have potent
vasomotor and vaso-occlusive effects (Brian et al., 1995; Brian & Faraci, 1998; Eklind et al.,
2001). Recent data suggest that cytokine toxicity may also be mediated through disturbances
in glutamate transport (Liao & Chen, 2001).
Studies suggest that the relationship between circulating inflammatory substances and
hypoxia-ischaemia in white matter injury may operate through a ‘two-hit’ mechanism. In
rats, the presence of an inflammatory stimulus prior to an hypoxic-ischaemic insult markedly
increased the vulnerability of the brain to hypoxic-ischaemic injury (Eklind et al., 2001).
Similarly, in an autopsy study of human neonates with PVL, infants with a combination of
infection and PVL had higher levels of cytokines than those with PVL without infection.
Together these data suggest that preceding infection with cytokine production amplifies the
effect of insults such as hypoxia-ischaemia (Kadhim et al., 2001).
Further evidence that inflammatory mechanisms are involved in the genesis of hypoxic-
ischaemic brain injury is provided by the growing body of evidence that manipulation of
inflammatory processes may offer protective effects in hypoxic-ischaemic brain injury. A
beneficial effect of induced neutropenia prior to hypoxia-ischaemia has been shown in an
animal model of perinatal hypoxic-ischaemic brain injury (Hudome et al., 1997). Platelet-
activating factor (PAF) levels increase with ischaemia reperfusion and PAF is important in
induction of leucocyte adhesion molecules and thereby subsequent events in the
inflammatory cascade. In the immature rat, administration of a PAF antagonist has been
shown to decrease infarct size both before the insult and during the reperfusion phase (Liu et
al., 1996). Studies using adult animal models of cerebral ischaemia suggest that the use of
specific antibodies or drugs directed at leukocytic adhesion molecules or various cytokines
may make an important contribution to neuro-protection (Zhang et al., 1994; Jiang et al.,
1995; Eun et al., 2000). A critical product of microglia/macrophages is interleukin-1β (IL-1β)
which in turn induces formation of other pro-inflammatory cytokines including tumour
necrosis factor-α (TNF-α). The use of an antagonist of the IL-1 receptor both pre-insult and
upon reperfusion has had protective effects in hypoxic-ischaemic brain injury in the neonatal
rat (Hagberg et al., 1996). Similarly, pentoxifylline, a phosphodiesterase inhibitor that inhibits
production of TNF-α, has been shown to ameliorate hypoxic-ischaemic brain injury when
administered immediately before hypoxic-ischaemic brain injury (Eun et al., 2000).
37
1.3.4 Summary of mechanisms of brain injury
Fifty years of research have consistently produced evidence that hypoxia and ischaemia can
produce prenatal brain injury. The pattern of damage is dependent on many factors
including the duration of the insult, the gestational age at exposure and the nature of the
insult (single or repeated exposure, partial or global asphyxia). Molecular mechanisms
involved in brain injury are complex and include cascades of events inducing the release of
glutamate, an increase in intracellular calcium, the production of oxygen free radicals, and
both necrotic and apoptotic cell death. Recent evidence also suggests that the fetal
inflammatory response is important in the genesis of hypoxic-ischaemic brain injury.
It is now well established that the cerebral energy failure that results from hypoxic-ischaemic
insults occurs in two phases in both animal and human newborns. The secondary phase can
result in neuronal injury and cell death that continues for up to seven days after the insult.
Both the delayed mechanisms of brain injury and the fetal inflammatory response are
attractive targets for clinical intervention to minimise the implications of hypoxic-ischaemic
brain injury.
38
1.4 Current techniques for intrapartum fetal surveillance
1.4.1 What are we aiming to prevent with intrapartum monitoring?
When reviewing our current techniques for intrapartum surveillance it is important to be
clear about what clinicians are attempting to prevent with intrapartum fetal monitoring.
1.4.1.1 Perinatal death, cerebral palsy and neurodevelopmental disability
The aim of intrapartum fetal monitoring is to reduce the incidence of three important
adverse clinical outcomes of fetal hypoxia; perinatal death, cerebral palsy and
neurodevelopmental disability. All of these outcomes are rare and both cerebral palsy and
neurodevelopmental disability only become apparent with the passage of time. Approximate
incidence figures for these conditions in the developed world are summarised in Table 1.8.
Table 1.8 Important adverse clinical outcomes
Condition Prevalence Source
Perinatal mortality 8 per 1000a MCHRC, 2000
Cerebral palsy 1.1 per 1000c Nelson et al., 1996
Perinatal mortality with intrapartum origin 0.8 per 1000a MCHRC, 2000
Cerebral palsy with potential intrapartum origin
0.1 per 1000c
0.2 per 1000a
Nelson et al., 1996 Watson et al., 1999
Neonatal encephalopathy 7 per 1000b Spencer et al., 1997
Neonatal seizures 11 per 1000a Thacker et al., 2001
Severe metabolic acidosis 20 per 1000d Low, 1998 a per 1000 live births b includes all grades of encephalopathy c per 1000 children who survived to three years of age (includes all birth weights) d pH<7.0, base deficit >12 mmol/L (Adapted from RCOG, 2001) It is important to note that only 10% of all perinatal mortality occurs during labour
(prevalence 0.8 per 1000 live births) and similarly it is thought only 10% of cases of cerebral
palsy have an intrapartum origin (prevalence 0.1-0.2 per 1000 children surviving to 3 years of
age) (Nelson et al., 1996; MacLennan, 1999; Watson et al., 1999; MCHRC, 2000; RCOG,
2001).
39
Other important neonatal outcomes which warrant prevention include neonatal
encephalopathy, neonatal convulsions and severe metabolic acidosis. The prevalence of
these outcomes is much more common than death and cerebral palsy, occurring in 2-3% of
all live births (Table 1.8)
1.4.1.2 Neonatal encephalopathy
In addition to asphyxia and hypoxic-ischaemic injury, a diverse group of disorders can result
in neonatal encephalopathy including maternal and fetal infections, maternal thyroid and
nutritional deficiencies, placental disorders and genetic diseases (Smith et al., 1996; Grether &
Nelson, 1997; Badawi et al., 1998b, a; Ellis et al., 2000; Impey et al., 2001; Richer et al., 2001;
Geerdink et al., 2002; Johnston, 2003). Although as discussed in section 1.1.4 there is debate
about the exact contribution of hypoxic-ischaemic events in labour to the incidence of
neonatal encephalopathy, there is general agreement that this condition is worthy of
prevention if possible.
Early onset neonatal encephalopathy is a good predictor of early developmental outcomes
(Dixon et al., 2002) and one of the better predictors of long-term outcome (Robertson et al.,
1989; Low, 1998). In a meta-analysis of five studies of term newborns with encephalopathy,
good outcomes were reported for newborns with minor encephalopathy, whereas the risk of
death or severe handicap in moderate encephalopathy was 5.6% and 20% and in severe
neonatal encephalopathy it was 60% and 72% respectively (Pelowski & Finer, 1992).
When neonates with neonatal encephalopathy were reviewed between one and two years of
age, they were found to have both statistically and clinically significant deficits in all areas of
development with the largest deficits seen in speech and hearing, which are crucial areas for
all aspects of development and learning (Dixon et al., 2002). These deficits persisted even
when children with cerebral palsy were excluded from the analysis.
The prevalence of cerebral palsy amongst children who experience neonatal encephalopathy
varies both between and within cohorts, based on the severity of the encephalopathy. The
overall prevalence of cerebral palsy in children with encephalopathy at birth ranges from 10%
in a Western Australian cohort to 22% in a cohort in Kathmandu (Ellis et al., 1999; Badawi et
al., 2001). Children with severe neonatal encephalopathy have the worst prognosis with
reported rates of cerebral palsy of 23% and abnormal development in 22% of those who
survive the neonatal period. This is to be compared with those who survive moderate
encephalopathy at birth, who have reported rates of cerebral palsy of 10%; abnormal
40
development occurs in 12% of these cases and 77% have normal developmental assessment
at two years of age (Dixon et al., 2002).
The deficits identified in short term follow-up of children with neonatal encephalopathy
continue to be evident when these children are reviewed later in childhood. Long term
follow-up studies at eight years of age suggest that intellectual, visual-motor integration,
receptive vocabulary scores, reading, spelling, and arithmetic grade levels for those with
moderate or severe encephalopathy, were significantly below (p<0.01) those in the mild
encephalopathy or peer comparison groups (Robertson et al., 1989).
1.4.1.3 Neonatal seizures
Intrapartum asphyxia is considered a major contributor to the occurrence of early neonatal
seizures (Derham et al., 1985; Low et al., 1985; Minchom et al., 1987; Patterson et al., 1989;
Lien et al., 1995) which occur in approximately 11 per 1000 live births (Thacker et al., 2001).
A 1985 consensus statement of the National Institutes of Child Health and Human
Development concluded that seizures were the best predictor of later neurologic damage in
the term infant (Freeman, 1985). More recent studies of children with neonatal
encephalopathy have shown that children with a history of encephalopathy and seizures were
more than three times more likely to develop cerebral palsy (16%) compared with those
without a history of seizures (OR 3.4; 95%CI 1.14-10.2; Dixon et al, 2002). Even seemingly
normal survivors of neonatal seizures may have a higher incidence of specific learning
disabilities and poor social adjustment when assessed during the late teenage years (Temple et
al., 1995).
1.4.1.4 Severe metabolic acidosis
Severe metabolic acidosis (pH<7.0, base excess<-12mmol/L) in umbilical arterial blood is a
sensitive reflection of birth asphyxia (MacLennan, 1999) and, ideally, intrapartum monitoring
should reduce the incidence of this condition. A number of studies have examined the
relationship between acidaemia at birth and both short- (Gilstrap et al., 1989; Low et al., 1995;
van den Berg et al., 1996) and long-term complications (Low et al., 1983, 1988; Ruth & Raivio,
1988; Low et al., 1994; Socol et al., 1994; Nagel et al., 1995; Ingemarsson et al., 1997). In short
term studies, babies with severe acidosis (pH<7.0) were significantly more likely to suffer
neonatal complications (Gilstrap et al., 1989; Low et al., 1995; van den Berg et al., 1996), and
in one study this relationship was found for those babies with metabolic acidosis rather than
severe respiratory acidosis (Low et al., 1994). The incidence of severe metabolic acidosis at
birth is consistently reported at approximately 2% of live births (Nagel et al., 1995; Low,
41
1998; MacLennan, 1999). In the series reported by Nagel et al. 77% of babies born with
severe metabolic acidosis were admitted to the neonatal intensive care unit, 27% required
ventilation and 7% died. Low et al. (1994) examined in detail the neonatal complications of
babies with severe metabolic acidosis and found that 78% of those with severe acidosis had
neonatal complications, compared with 27% in the babies with normal cord blood gas
analysis (p<0.001). More importantly they found that babies with severe metabolic acidosis
at birth were more likely to have complications in three or more organ systems (severe
acidosis 38% vs. normal blood gas analysis 6%, p<0.001). The specific neonatal
complications assessed in this study are summarised in Table 1.9. Of note, 61% of babies
with severe acidosis at birth had neonatal encephalopathy, with 42% having moderate or
severe encephalopathy.
Table 1.9 Short term neonatal complications of severe metabolic acidosis at birth
Umbilical Cord Blood Gas Analysis Neonatal Complication
Severe acidosisa Normalb p-value
Encephalopathy 61% 15% <0.001
Moderate or severe encephalopathy 42% 6% <0.001
Cardiovascular complications 59% 18% <0.001
Renal complications 24% 3% <0.010
Moderate or severe renal complications 16% 0% <0.010 aSevere acidosis, metabolic acidosis with base deficit >16mmol/L, mean pH 6.97, SD 0.13 bNormal, base deficit <16mmol/L, mean pH 7.25, SD 0.05 (Adapted from Low et al, 1994) In addition to an increased incidence of neonatal complications at birth, babies with severe
metabolic acidosis at birth have an increased incidence of neurodevelopmental deficits during
early infancy. When babies with severe metabolic acidosis at delivery were reviewed at one
year of age, the incidence of both major and minor motor and cognitive deficits were
significantly greater in the group with severe acidosis at birth than the control group, which
had normal acid-base status at birth (Low et al., 1988). The incidence of major deficits in the
group with severe acidosis at birth was 14% which was significantly greater than the 1%
incidence in the control group (p<0.01). Similarly the incidence of minor deficits in the
acidosis group was 27% which was significantly greater than the 6% in the control group
(p<0.01).
42
In long term follow-up studies, the association between acidaemia and neurodevelopmental
outcome is poor, with all but one study reporting no significant association between
acidaemia and neurodevelopmental disability (Low et al., 1983, 1988; Ruth & Raivio, 1988;
Dennis et al., 1989; Socol et al., 1994). The only study which did identify a significant
association between acidaemia at birth and neurodevelopmental disability found that babies
with umbilical arterial pH less than the first centile (umbilical arterial pH<7.05), when
compared to a control group with umbilical arterial pH>7.1, were more likely to have
abnormal developmental assessment at four years of age (24% vs. 13%, p=0.05; Ingemarsson
et al, 1997). Considering the acidotic babies, there was a significant increase in the number of
children with speech problems on developmental screening at 4 years (19% vs. 8%, p=0.03).
The risks for babies with severe acidosis at birth were worse in the subgroup that had a
pathological intrapartum FHR pattern which lasted longer than 60 minutes. In this
subgroup, both the incidence of speech problems and attention deficits were increased
compared to the acidotic babies with pathologic FHR patterns with durations less than 60
minutes (Attention deficit 18% vs. 6%, p=0.06; speech problems 32% vs. 14%, p=0.03;
Ingemarsson et al, 1997).
1.4.1.5 Summary
Intrapartum fetal surveillance is designed to reduce the incidence of perinatal death, cerebral
palsy, neurodevelopmental disability, neonatal encephalopathy, neonatal seizures and severe
metabolic acidosis at birth. Although it is universally accepted that perinatal death and
cerebral palsy are outcomes worthy of prevention, their combined prevalence relating to
intrapartum events is uncommon, occurring in approximately 0.1% of live births. Neonatal
encephalopathy and severe metabolic acidosis at birth are significantly more common than
cerebral palsy and perinatal death, occurring in 2-3% of live births, and have both short-,
medium- and long-term implications worthy of prevention.
43
1.4.2 Current monitoring techniques and their limitations
The three techniques which are currently widely used for the assessment of fetal wellbeing in
labour are FHR monitoring, fetal scalp blood gas analysis and umbilical cord blood gas
analysis.
The majority of women in labour in the developed world have FHR monitoring as the
principal method of intrapartum fetal surveillance. Intrapartum FHR monitoring is
performed as either intermittent auscultation or continuous electronic fetal heart rate
monitoring (EFM) with the selection of monitoring technique dependent on many issues. In
most obstetric centres, women with pregnancies deemed to be “high risk” have continuous
electronic FHR monitoring and those deemed to be “low risk” undergo intermittent FHR
auscultation. The specific criteria required for a pregnancy to be classified as “high risk” or
“low risk” varies between obstetric units, however there is remarkable consistency between
the recommendations of professional obstetric societies of developed countries (ACOG,
1995; RCOG, 2001; Liston et al., 2002a; Liston et al., 2002b; RANZCOG, 2002). The choice
of FHR monitoring technique is also influenced by issues such as hospital staffing, midwifery
experience, patient request, availability of equipment, and the unit’s philosophical opinion
regarding fetal heart rate monitoring.
When abnormalities are identified with intrapartum FHR monitoring a cascade of events is
initiated. The options for further fetal assessment vary significantly between obstetric
centres. In some centres delivery is expedited with either an instrumental vaginal delivery or
caesarean section. In other centres fetal scalp blood sampling is performed to assess the fetal
acid-base status. Once the baby is delivered, the fetal response to labour and birth is assessed
with clinical assessment, including Apgar scores. Umbilical cord acid-base assessment is
performed in some centres to determine if birth asphyxia has occurred.
In the next three subsections I will expand on FHR monitoring, fetal scalp blood sampling
for pH assessment, and umbilical cord acid-base assessment at birth. In particular I will
discuss their ability to predict or prevent fetal compromise with specific attention to the
prediction and prevention of fetal neurologic injury. Each subsection will conclude with a
discussion of the limitations of each assessment modality.
44
1.4.2.1 Fetal heart rate monitoring
Fetal heart rate monitoring was introduced into clinical practice and patient management
over three decades ago. It continues to be the predominant method of intrapartum fetal
surveillance despite questions about its efficacy and outcomes associated with its use
(Freeman, 2002).
1.4.2.1.1 Historical perspective
The era of modern obstetrics began in 1821 with the discovery of the ability to auscultate the
fetal heart by Jean Alexandre Lejumeau Vicomte de Kergaradec (Kergaradec, 1822). He was
genius enough not only to recognize the origin of the fetal heart tones, but also more
significantly, to anticipate the practical possibilities that could derive from its auscultation.
He stated, “From the changes occurring in strength and rate of fetal heart beats, wouldn’t it
be possible to know about the status of health or sickness of the fetus?” (Kergaradec, 1822)
Over the next thirty years, the fetal stethoscope allowed many discoveries including the
relationship between FHR accelerations and fetal movement, the association between FHR
and maternal temperature and the association between FHR and maternal blood pressure. It
also made possible the diagnosis of fetal life or death, twins, and the determination of fetal
presentation and position. It was during the 1840’s that the adverse fetal and neonatal
consequences of both late decelerations and progressive and long-lasting bradycardia were
described during labour (Sureau, 1996). In 1847, Depaul claimed that auscultation should be
used for every delivery and not be restricted to “high risk cases” (Depaul, 1847). In 1893,
Von Winckel described the criteria for fetal distress (Von Winckel, 1893). These criteria
included tachycardia (FHR more than 160bpm), bradycardia (FHR less than 100bpm),
irregular fetal heart rate, passage of meconium and gross alteration of fetal movement. By
the beginning of the 20th century, auscultation of the fetal heart was an established practice in
Europe (Sureau, 1996). The criteria set for the normal FHR in the latter part of the 19th
century remained virtually unchanged until the 1950’s.
The technology for electronic fetal heart rate monitoring was first developed in the 1950’s
and became commercially available in the 1960’s. During the 1960’s theories regarding the
cause of FHR patterns were formulated and a limited amount of experimental and empirical
evidence was produced to support these theories (Hon, 1968). While the original aim of
intrapartum EFM was to prevent harm, it was introduced into delivery suites in the 1950’s
and 60’s with the emphasis on improving fetal birth outcomes by detecting fetal hypoxia
before it led to death or disability. Like intermittent auscultation in the 19th century,
45
continuous EFM was introduced into clinical practice before its effectiveness had been fully
evaluated scientifically (RCOG, 2001).
The initial studies supporting the use of intrapartum fetal heart rate monitoring were
retrospective observational studies published between 1972 and 1976 which reported a
decrease in perinatal mortality in those women who had continuous EFM as opposed to
those who had selective EFM or no EFM at all (Koh et al., 1975; Shenker et al., 1975; Tipton
& Lewis, 1975; Hochuli, 1976; Lee & Baggish, 1976; Beard et al., 1977; Johnstone et al., 1978;
Weinraub et al., 1978). Similarly, studies comparing fetal outcomes at two serial time periods
covering the introduction of EFM into the United States reported a virtual disappearance of
intrapartum stillbirths once EFM was instituted (Parer, 1979; Yeh et al., 1982). Before the
introduction of FHR monitoring, approximately one third of all stillbirths, or three per 1000
births, occurred in the intrapartum period (Lilien, 1970). While these studies were
encouraging, the methodological biases of observational studies prompted the need for
randomised controlled trial evidence to more rigorously evaluate the use of intrapartum EFM
for preventing perinatal mortality and morbidity.
Thirteen randomized clinical trials have assessed the efficacy and safety of EFM, comparing
EFM to intermittent auscultation (Haverkamp et al., 1976; Renou et al., 1976; Kelso et al.,
1978; Haverkamp et al., 1979; Langendoerfer et al., 1980; Wood et al., 1981; MacDonald et al.,
1985; Neldam et al., 1986; Luthy et al., 1987; Shy et al., 1990; Vintzileos et al., 1993; Herbst &
Ingemarsson, 1994; Mahomed et al., 1994). Nine of these trials have been utilised in the three
systematic reviews comparing EFM to intermittent auscultation and include over 18,000
high- or low-risk pregnancies from seven clinical centres in the United States, Europe, and
Australia.
1.4.2.1.2 Fetal heart rate monitoring and adverse clinical outcomes
Three systematic reviews have examined the effect of EFM, in comparison with intermittent
auscultation, on perinatal death rates (Grant, 1989; Vintzileos et al., 1995b; Thacker et al.,
2001) and none found a significant reduction in the perinatal death rates with EFM. When
analysed separately, none of the trials included in these reviews demonstrated a reduction in
either intrapartum or neonatal deaths. The addition of fetal blood sampling to EFM did not
improve the ability of EFM to reduce perinatal deaths.
In the systematic review of Vintzileos et al. (1995b) a subgroup analysis for perinatal death
was undertaken. Deaths in each trial were classified as due to either hypoxia or other causes.
A significant reduction in the odds of perinatal death due to hypoxia with the use of EFM
46
was found; however, this was a post-hoc analysis and is therefore prone to subjective
selection bias and thus the subgroup analysis result should be treated with caution. It should
be noted that the prevalence of perinatal mortality of intrapartum origin has been reported as
0.8 per 1000 live births (MCHRC, 2000) and for a clinical trial to show a 50% reduction in
the prevalence of this end point with 80% power it would require 64,000 women in each
arm.
In addition to reduction in perinatal death, the three systematic reviews comparing EFM to
intermittent auscultation found no significant differences in 1-minute Apgar scores below
four or seven, rates of admission to neonatal intensive care unit, or cerebral palsy. The use of
EFM was associated with an increase in the rate of caesarean delivery (RR 1.41, 95% CI 1.23-
1.61) and operative vaginal delivery (RR 1.20, 95% CI 1.11-1.30).
There have been three studies investigating cerebral palsy rates in the cohorts of babies
included in three of the randomised controlled trials comparing electronic FHR monitoring
with intermittent auscultation (Langendoerfer et al., 1980; Grant et al., 1989; Shy et al., 1990).
One of these studies found an increase in rates of cerebral palsy in babies monitored by EFM
compared with intermittent auscultation (19.5% versus 7.7%; relative risk (RR) 2.54; 95%
confidence interval (CI) 1.10-5.86; Shy et al, 1990). These results should be interpreted with
caution as the cohort studied included only preterm babies who weighted less than 1750gm
at birth. Furthermore, in that study, management subsequent to the detection of FHR
abnormalities was not consistent in both arms of the study, with a significantly longer delay
between onset of FHR abnormality and delivery in the EFM group (105min vs. 45 minutes).
The remaining two cohort studies found no significant difference in cerebral palsy rates
between the two groups at the end of the followup period (Langendoerfer et al., 1980; Grant
et al., 1989).
In two of the larger case-control studies investigating the relationship between EFM and
cerebral palsy, a significant association has been identified between abnormal
cardiotocograph (CTG) findings and cerebral palsy (Gaffney et al., 1994b; Nelson et al., 1996).
In the study of Nelson et al. (1996) the two fetal heart rate patterns found to be associated
with an increased risk of cerebral palsy were multiple late decelerations (OR 3.9; 95%CI 1.7-
9.3) and decreased FHR variability (OR 2.7; 95%CI 1.1-5.8); however, only 0.19% of
singletons with these patterns develop cerebral palsy, giving a false positive rate of 99.8%.
No other FHR patterns were found to have significant associations with the diagnosis of
cerebral palsy.
47
Three case control studies have examined whether abnormal EFM traces predict subsequent
development of neonatal encephalopathy (Gaffney et al., 1994a; Socol et al., 1994; Adamson et
al., 1995). In all three studies there were significant increases in the odds of developing
neonatal encephalopathy in the presence of abnormal intrapartum FHR patterns (odds ratios
ranging from 1.98 to 10.2). Unfortunately all of the studies used different FHR pattern
classification systems and none investigated individual patterns of FHR abnormality. In
addition, in one of the studies, if a different FHR pattern scoring system was used, no
association with neonatal encephalopathy was seen (Spencer et al., 1997). As all these studies
were case-control studies, caution is needed in ascribing a causal relationship to the observed
effect.
The use of continuous EFM rather than intermittent auscultation has been found to be
associated with a significant reduction in neonatal seizures in two systematic reviews (0.24%
vs. 0.5%; RR 0.51; 95%CI 0.32-0.82; number needed to treat (NNT) 384; Grant, 1989;
Thacker et al, 2001). The protective effect for neonatal seizures was only evident in studies
with high-quality scores in the systematic review by Thacker et al. (2001) and it was only
present in the studies which combined EFM with fetal blood sampling in the systematic
review by Grant et al. (1989).
In only one of the thirteen randomised clinical trials of EFM versus intermittent auscultation
has the relationship between convulsions and subsequent neurodevelopmental disability been
examined (MacDonald et al., 1985). In this study, the reduced convulsion rate seen in the
EFM arm in the original trial did not translate into a significant reduction in the rate of
cerebral palsy in the group on follow-up. Of the six babies from this cohort who
subsequently developed cerebral palsy, five were thought to be attributable to antepartum
factors (Grant et al., 1989). These results are dissimilar to other studies investigating the
outcome of children with neonatal seizures, which suggest that neonatal seizures are a good
predictor of later neurologic damage, including higher rates of cerebral palsy, specific learning
disabilities and poor social adjustment (Freeman, 1985; Temple et al., 1995; Dixon et al.,
2002). It is important to note that in the original Dublin trial of EFM there were only nine
neonates in the EFM arm and 21 neonates in the intermittent auscultation arm of the study
who had neonatal seizures (MacDonald et al., 1985). Given that the highest rate of
subsequent cerebral palsy that has been reported in neonates with encephalopathy and
seizures is 16% (Dixon et al., 2002), the followup study by Grant et al. had only 14% power to
detect a difference in cerebral palsy rates in neonates with seizures.
48
Only one randomised controlled trial has found that EFM was significantly more sensitive
than intermittent auscultation for detecting both respiratory and metabolic acidosis
(Vintzileos et al., 1995a). Despite having better sensitivity for detecting acidosis at birth, the
specificity for EFM was poor (detection of all acidosis: EFM: sensitivity 97%, specificity
84%; intermittent auscultation: sensitivity 34%, specificity 94%).
A number of recent studies have revisited EFM and the prediction of outcome. Most of
these case-control studies have investigated the ability of EFM to predict intervention at
delivery, umbilical artery acidaemia or neonatal intensive care admission (Spong et al., 1998;
Dellinger et al., 2000; Hadar et al., 2001; Low et al., 2001; Sheiner et al., 2001; Low et al., 2002).
None has investigated the ability to directly predict fetal neurologic injury. All of these recent
studies suggest that abnormalities in EFM are associated with an increased risk of adverse
clinical outcomes at birth; however, as all these studies were case-control studies, caution is
needed in ascribing a causal relationship to the observed effect
The last two decades of research into FHR monitoring have been best summarised by Low
(1998) who commented that EFM is a useful screening test for the prediction of intrapartum
fetal asphyxia in preterm and term pregnancy. Although fetal heart rate patterns will not
discriminate all asphyxial exposures, continuous fetal heart rate monitoring supplemented by
fetal blood gas and acid-base assessment can be a useful fetal assessment paradigm for
intrapartum fetal asphyxia. Such an assessment paradigm will not prevent all cases of
moderate or severe fetal asphyxia. However, prediction and diagnosis with intervention and
delivery during the first or second stage of labour may prevent the progression of mild
asphyxia to moderate or severe asphyxia in some cases (Low et al., 2001; Low et al., 2002).
1.4.2.1.3 Limitations of fetal heart rate monitoring
There are a number of important limitations in FHR monitoring which may partially explain
why its widespread use has not been matched by improvements in neonatal outcomes. First,
there is a lack of standardisation of interpretation of FHR patterns and there is disagreement
regarding algorithms for intervention for specific FHR patterns (NICH, 1997; Parer & King,
2000). This is particularly important when reviewing the results of the systematic reviews of
EFM versus intermittent auscultation where the definitions for those FHR patterns requiring
obstetric intervention were not the same in each study (Thacker et al., 2001). Recent
publications by professional societies have addressed some of the issues of standardisation of
definitions (ACOG, 1995; NICH, 1997; RCOG, 2001; Liston et al., 2002a; Liston et al.,
2002b; RANZCOG, 2002).
49
Second, amplifying the problem of varying definitions is the fact that inter-observer and
intra-observer reliability for interpreting FHR patterns is relatively poor. All the studies
assessing inter-observer agreement in interpretation of FHR patterns have found marked
variability in interpretation of both normal and abnormal tracings by obstetricians,
experienced perinatologists, midwives and residents with inter-observer agreement
commonly reported to be approximately 30% (Peck, 1980; Beaulieu et al., 1982; Helfand et al.,
1985; Nielsen et al., 1987; Bernardes et al., 1997; Blix et al., 2003). The proportion of
agreement is generally better for FHR patterns with normal baseline FHR, variability and
accelerations (range 56% to 71%) whereas for abnormal FHR patterns, the proportion of
agreement has been reported to be as low as 14% (Bernardes et al., 1997). Intra-observer
agreement is equally poor. This is illustrated by three studies (Beaulieu et al., 1982; Nielsen et
al., 1987; Borgatta et al., 1988) which repeatedly showed the same FHR records to
experienced clinicians on a number of occasions. The changed interpretation rate in these
studies varied from 16% to 28%.
Third, intrapartum FHR monitoring performs poorly as a screening test. It was originally
anticipated that EFM would solve two problems; first, that it would be a screening test for
severe asphyxia, and second, it would allow recognition of early asphyxia so timely
intervention could avoid asphyxia-induced brain damage or death. Unfortunately, if all
women had continuous EFM during labour, between 30% and 79% of them would have
some abnormality in their FHR patterns which may in some clinicians’ minds be ominous or
at best suspicious and therefore trigger intervention (Nielsen et al., 1987; Parer & King, 2000).
A screening test for uncommon outcomes, which is considered abnormal in such a high
percentage of the tested population, is destined to have poor specificity and a very high false
positive rate.
The fourth limitation of intrapartum FHR monitoring relates to fetal physiology. It has been
thought for many years that abnormal FHR patterns would progressively evolve as the fetal
biochemical and cardiovascular systems decompensate with progressive asphyxia. Both
human (Low et al., 2001) and animal studies of asphyxia (de Haan et al., 1997a) have shown
that this does not always occur. This is illustrated in the study by de Haan et al. (1997a) which
investigated FHR changes during repetitive brief umbilical cord occlusions in the sheep fetus.
They found that the changes in FHR pattern with ongoing hypoxia were variable and, even
though fetal arterial pressure fell after approximately 15 minutes of repeated brief cord
occlusion, the pattern of the FHR decelerations remained relatively consistent throughout the
two hours of the experiment. Despite the fact that fetuses had a severe metabolic acidosis
50
and hypotension, fewer than 30% of them had a lengthening of the time needed for the fetal
heart rate to recover to baseline after the final occlusion (de Haan et al., 1997a). It would
therefore appear that there may also be physiologic limitations in the ability to identify all
cases of moderate or severe asphyxia from FHR patterns alone.
1.4.2.2 Fetal scalp blood gas analysis
Intrapartum fetal scalp blood sampling (FBS) was introduced into clinical practice by Saling
in 1962 as the first invasive fetal monitoring tool (Saling, 1962). It allowed greater
understanding of the effect of labour on blood gas homeostasis in the human fetus and it
aided in defining the importance of FHR patterns revealed with continuous EFM which was
introduced soon after (Kubli et al., 1969; Beard et al., 1971).
Fetal blood sampling is limited to the skin covering the fetal presenting part and it requires
ruptured membranes and the cervix to be at least 2 to 3-cm dilated. Initially only fetal pH
was measured but with improvements in micro sample analysis technology it became
possible to measure pH, and oxygen and carbon dioxide tensions from the fetal scalp in
labour. The addition of carbon dioxide measurement allowed the calculation of base deficit
to quantify the metabolic component to an acidosis.
It is widely accepted that a normal FHR pattern in labour is associated with a good outcome
and, conversely, a persistent bradycardia is associated with a poor outcome. Unfortunately,
between these extremes are various patterns that do not provide reassurance, and as
described previously, there is poor inter- and intra-observer variability in the description of
these patterns. Even once a particular pattern is identified, there is difficulty in obtaining
agreement about the most appropriate management algorithm for all but a few patterns
(NICH, 1997). Heart rate decelerations occur in 50% of babies monitored with continuous
EFM (Ingemarsson, 1981). As at least 50% of labours are monitored by EFM in the
developed world, the high incidence of FHR decelerations has the potential to initiate a
cascade of events frequently resulting in intervention in a large number of women.
Intrapartum fetal scalp blood sampling is traditionally used to clarify fetal acid-base status
when a non-reassuring FHR pattern is present in labour. Although there is an association
between low cord pH (Krebs et al., 1979) and increased base deficit (van den Berg et al., 1987)
with increasingly abnormal FHR patterns in labour and their duration (Fleischer et al., 1982),
an ominous FHR pattern such as one which is tachycardic with late decelerations and
51
decreased baseline variability has only a 50% incidence of low pH (Beard et al., 1971). The
percentage of fetuses with fetal scalp pH less than 7.25 for particular FHR patterns is
presented in Figure 1.4.
1.4.2.2.1 Physiologic basis for fetal scalp blood gas analysis
Prior to labour, fetal acid-base status is largely determined by the acid-base status of the
mother. During labour, fetal acid-base status is determined by both the mother’s acid-base
status and placental function. When intra-uterine pressure exceeds 30mmHg, the arteries
supplying the intervillous space of the placenta are constricted so that there is temporarily
little blood flow and little exchange of oxygen (O2) and carbon dioxide (CO2). This can be
accommodated by a healthy feto-placental unit; however, if the placenta is compromised or
contractions occur too frequently or last too long, there will be a progressive accumulation of
CO2 leading to hypercapnia and hypoxaemia. Elevated levels of CO2 in the fetus lead to a
respiratory acidosis as the CO2 combines with water to form carbonic acid. Prolonged
hypoxia can lead to a decrease in oxygen delivery to fetal tissues which prompts a switch to
anaerobic metabolism and produces a metabolic acidosis (Figure 1.5A). The fetus usually
develops a respiratory acidosis before a metabolic acidosis with the former being less
significant as it may not be associated with tissue oxygen lack. With prolonged impaired
placental blood flow the fetus develops a mixed acidosis.
Any factor that affects placental blood flow and gas exchange in labour affects fetal acid-base
status. These factors include cord compression, hypertonic uterine contractions, maternal
supine hypotension, cord prolapse or placental abruption. Any decrease in FHR results in a
decreased cardiac output, decrease CO2 transfer, and CO2 accumulation so that prolonged or
repetitive bradycardias result in a respiratory acidosis; this sequence commonly occurs in the
second stage of labour (Greene, 1999). The rate of development of metabolic acidosis
depends on the particular insult and the individual fetal reserve and ability to compensate.
The fetus normally derives its cellular energy primarily from glucose. Under normal
conditions with sufficient oxygen supply to the fetus, aerobic metabolism occurs when
glucose (or glycogen) is broken down via the glycolytic pathway and citric acid cycle to
produce water, carbon dioxide and 38 units of ATP for each glucose molecule (Figure 1.5B).
52
Normal
FHR patte
rn
Accele
ration
s
Early D
ecele
ration
s
Baseli
ne Tac
hyca
rdia
Baseli
ne B
radyc
ardia
Variab
le Dec
elerat
ions
Compli
cated
Vari
able
Decel
Decrea
sed F
HR varia
bility
Decrea
sed v
ariab
ility +
other
0
10
20
30
40
50
60Fe
tal s
calp
blo
odpH
<7.2
5 (%
)
Figure 1.4 Fetal acidosis with abnormal fetal heart rate patterns. Fetal scalp samples were
collected during the first stage of labour in 281 fetuses and their FHR patterns were
classified. A normal FHR pattern was defined as one with a baseline between 120 and
160bpm with baseline variability between 5 and 15bpm. Tachycardia was defined as a FHR
greater than 160bpm and bradycardia less than 110bpm. Complicated variable decelerations
were defined as FHR patterns with variable decelerations and either late components or a
biphasic pattern. Decreased FHR variability was defined as less than 5bpm. Decreased FHR
variability + other included FHR patterns with both decreased FHR variability and either
tachycardia and/or FHR decelerations.
(Adapted from Beard et al. 1971)
52
Figure 1.5 Fetal aerobic and anaerobic metabolism. Figure A illustrates the pathophysiology
of respiratory and metabolic acidosis both of which increase the fetal concentration of
hydrogen ions. It is only by assessing base deficit that the type of acidosis can be determined.
Figure B schematically represents fetal cellular glucose metabolism. Glucose is broken down
along the glycolytic pathway to form pyruvate. Under aerobic conditions large amounts of
energy are produced in the citric acid cycle whereas anaerobic glycolysis renders less energy
and lactic acidosis. When there is adequate oxygen supply to the fetal tissues nicotinamide
adenine dinucleotide (NAD+) accepts a hydrogen ion to produce NADH. With the catabolic
reaction of NADH, oxygen is directly consumed producing NAD+ and water. This reaction
occurs at the inner membrane of the mitochondrion with the release of large amounts of
energy to drive oxidative phosphorylation producing ATP and water. H:- = hydride ion,
containing an extra unpaired electron.
(Adapted from Nordstom and Arulkumaran, review, 1998)
DecreasedPlacental Blood
Flow
OxygenSaturation
OxygenDelivery
AnaerobicMetabolism
MetabolicAcidosis
(Lactate + H+)
H+
PCO2
RespiratoryAcidosis
CO2 + H20 H2CO3 H+ + HCO3
A
B Glucose
2 Pyruvate 2 Lactate- + 2H+
CitricAcidCycle
CO2
2 NAD+
O2
2 NADH
4H+ + O2 + 4e-
2 ADP
2 ATP
4NAD+4NADH36 ADP
36 ATP
36 ATP
CO2
2 H20
Cytosol
2H:-
4e-
Mitochondrion
54
The fetus is able to adjust to short episodes of hypoxaemia and maintain aerobic metabolism
by initiating a synchronised response that involves behavioural, cardiovascular, metabolic and
hormonal adjustments (Greene & Rosen, 1995). When these compensatory mechanisms fail
to maintain an adequate oxygen supply to the central organs, aerobic metabolism is
supplemented by anaerobic metabolism of glucose and glycogen as far as lactic acid in the
Kreb cycle, with the generation of two units of ATP for each molecule of glucose.
Anaerobic metabolism is much less efficient than aerobic metabolism but it is an important
survival mechanism particularly to maintain cardiac and brain activity during hypoxaemia and
asphyxia. This form of anaerobic metabolism is largely dependent on the pre-asphyxial
glycogen content of the myocardium and liver which are depleted in this process (Dawes et
al., 1959).
1.4.2.2.2 Fetal blood sampling and adverse clinical outcomes
As previously described, there have been 13 randomised trials comparing EFM with
intermittent auscultation. In three of these trials intermittent auscultation was compared with
EFM without the use of FBS for pH (Haverkamp et al., 1976; Kelso et al., 1978; Haverkamp
et al., 1979) and there was a four fold increase in the odds of caesarean section for fetal
distress (OR 4.14, 95%CI 2.29-7.51) without improvement in neonatal outcome (neonatal
seizures OR 0.79, 95%CI 0.21-2.97). In contrast, EFM with the option of FBS (Renou et al.,
1976; Haverkamp et al., 1979; MacDonald et al., 1985; Neldam et al., 1986; Luthy et al., 1987)
showed a less marked increase in caesarean section for fetal distress (OR 1.98, 95%CI 1.33-
2.94) and a decrease in neonatal seizures (OR 0.49, 95%CI 0.29-0.82). It should be noted
that the reduction in caesarean section for fetal distress with the addition of FBS was
primarily through postponing intervention to the second stage of labour.
Although FBS has been used in clinical practice for 40 years there is limited information
about the predictive value of the results. A review of seven studies including 1547 patients
published in 1979 concluded that scalp blood pH was a poor predictor of Apgar score of less
than seven at one minute (Banta & Thacker, 1979). They report sensitivities ranging from
28% to 62% and specificities from 70-92%. No long term follow-up data were available. A
more recent prospective cohort of 1709 patients who underwent scalp sampling for fetal pH
assessment reported similar findings with scalp pH a poor predictor of cord pH<7.0, base
deficit >16mmol/L, low Apgar scores at 1 and 5 minutes and hypoxic-ischaemic
encephalopathy (Kruger et al., 1999). The area under the receiver operator curves (ROC) and
the sensitivities and specificities for pH at predicting these poor outcomes is summarised in
55
Table 1.10. It can be seen from this table that the areas under the ROC are only marginally
better than 0.5 which reflects chance alone and the sensitivities are universally poor.
Table 1.10 Scalp pH and the prediction of neonatal outcome
Outcome Variable Area under
ROC (95%CI)Sensitivity* Specificity*
Umbilical artery
pH < 7.0 0.64 (0.62-0.70) 35.7% 83.8%
Base deficit > 16mmol/L 0.69 (0.66-0.71) 54.2% 85.4%
Apgar score
<7 at 1 min 0.63 (0.60-0.65) 32.1% 85.4%
<7 at 5 min 0.63 (0.61-0.66) 23.5% 82.9%
<4 at 6 min 0.55 (0.52-0.57) 22.2% 82.4%
Hypoxic-ischaemic encephalopathy
Mild 0.57 (0.54-0.60) 33.3% 82.7%
Moderate or severe 0.66 (0.63-0.69) 50.0% 80.2% *Sensitivity and specificity are presented for scalp pH values at the 25th percentile (<7.21) (Adapted from Kruger et al. 1999)
1.4.2.2.3 Limitations of fetal blood sampling
There are many limitations associated with fetal scalp blood sampling. Although the use of
FBS has been widely advocated to improve the specificity of FHR monitoring, it remains an
unpopular procedure (Clark & Paul, 1985) because it is inconvenient for both the clinician
and mother and invasive for the mother and fetus. Consequently, the frequency of its use
varies dramatically between Europe, North America and Australia, from unit to unit, and
even within the same unit, depending on the attitude of individual clinicians (Greene, 1999).
In addition to being unpopular amongst some clinicians there are a number of important
practical limitations to the use of FBS. First, to perform the testing each obstetric unit
requires a blood gas analyser capable of analysing micro samples, preferably located in the
delivery suite. These machines cost from AUS$20,000 to AUS$80,000 depending on
whether they measure pH alone or pH, oxygen and carbon dioxide tensions, and they have
ongoing maintenance costs. This alone limits the utilisation of this method of testing to
larger units.
56
Second, to determine pH and base deficit, which is required to identify metabolic acidosis,
requires between 35 and 130µL of blood depending on the analysis technique. Obtaining
these volumes of blood can be technically challenging from a distant fetal scalp as was found
in one study evaluating scalp pH, where adequate samples (35µL) could be collected only in
80% of cases where assessment was attempted (Westgren et al., 1998). If inadequate sample
volumes are collected one is limited to either measuring pH alone or opting for intervention
to expedite delivery which may be unnecessary.
The third practical limitation to FBS for pH assessment relates to specimen contamination.
If the sample is contaminated with air bubbles, meconium or amniotic fluid, invalid results
will be obtained during analysis. Air bubbles can confound the analysis of gas tensions which
are required for the calculation of base deficit. Amniotic fluid has a pH ranging from 7.0 to
7.5 at term (Brace, 1999) and therefore contamination can yield both false positive and
negative results.
The final limitation to FBS for pH assessment relates to the relationship between scalp pH
and central arterial pH. A number of studies have investigated this relationship in animals
(Adamsons et al., 1970; Shier & Dilts, 1972) or humans (Stephens et al., 1969; Bowe et al.,
1970; Yoshioka & Roux, 1970; Sturbois et al., 1977) and the conclusion reported by most is
that there is a good correlation between fetal scalp pH values and central pH status (Greene,
1999). Detailed review of these studies demonstrates that the relationship between central
and peripheral acid-base status is not constant and is affected by many things including
hypoxia and local tissue conditions.
The original studies performed in monkeys during labour reported no difference between
exteriorised scalp and carotid arterial pH, whereas there was a significant difference between
transvaginal acquired scalp pH and central pH (Adamsons et al., 1970). However, when
samples were collected transvaginally, scalp pH was lower than carotid arterial pH by an
average of 0.047 pH units, with the difference becoming greater at lower pH values
(Adamsons et al., 1970). A similar discrepancy was reported in an ovine model of asphyxia
where scalp pH levels and carotid arterial and jugular venous pH levels were assessed under
both normoxic and hypoxic conditions in partially exteriorised fetuses (Shier & Dilts, 1972).
During normoxia scalp pH was, on average, 0.02 pH units lower than arterial pH whereas
during hypoxia this difference became greater until the mean difference was 0.07 pH units.
Furthermore, in this study scalp carbon dioxide tensions (PCO2) did not correlate with either
arterial or venous levels, which were consistently higher (Shier & Dilts, 1972). Similarly, scalp
base deficit levels were not representative of values in either arterial or venous blood. This
57
inaccuracy is probably explained by the poor PCO2 correlation, as base deficit is calculated
from pH and PCO2. Human studies comparing scalp pH and central pH reported no
significant differences between these two variables; however, the difference between them
became greater in “distressed fetuses” (Bowe et al., 1970; Yoshioka & Roux, 1970). The lack
of statistical significance of the difference identified in the study of Yoshioka et al. (1970) may
relate to the small sample size (non-distressed fetuses n=9, distressed fetuses n=5).
It has been noted by a number of authors that there are several situations in which scalp pH
measurements are inaccurate (Bowe et al., 1970; Parer & Livingston, 1990; Greene, 1999) .
These include scalp oedema and caput succedaneum which can result in scalp pH levels
being 0.026 to 0.06 pH units lower than carotid arterial or umbilical arterial pH. This can
confound interpretation of scalp pH results in as many as 10-20% of samples (Parer &
Livingston, 1990). Apparent inconsistencies between peripheral and central pH that may
occur with hypoxia and the presence of scalp oedema or caput succedaneum in labour may
partially explain the poor predictive ability of scalp pH with regard to the important neonatal
outcomes of severe metabolic acidosis and hypoxic-ischaemic encephalopathy.
1.4.2.2.4 Scalp stimulation as an alternative to fetal blood sampling
There is good evidence to suggest that stimulating the fetal scalp during a vaginal
examination during labour will reduce the need for fetal scalp sampling (Clark et al., 1982,
1984; Elimian et al., 1997; Porter & Clark, 1999; Skupski & Eglinton, 2002). A total of 512
studies have been performed in this area and a recent meta-analysis of the 11 high quality
randomized controlled trials reported 97% sensitivity and 50% specificity for a stimulated
FHR acceleration predicting normal fetal acid-base status in the fetus (Skupski & Eglinton,
2002). Acceleration in the FHR after scalp stimulation is reassuring and negates the need to
perform FBS. The absence of FHR acceleration is less helpful in clinical decision-making,
with 27% of these fetuses being acidotic on FBS. The utilisation of scalp stimulation is also
not assisted by FHR patterns with frequent decelerations or fluctuating baseline FHR where
identification of the required stimulated acceleration is sometimes difficult (Dixon et al,
1999).
58
1.4.2.3 Umbilical cord blood gas analysis
1.4.2.3.1 Physiological basis
As discussed in previous sections of this chapter, umbilical cord acid-base assessment at
delivery has a sound physiologic basis and provides a sensitive reflection of birth asphyxia
(Low, 1998; MacLennan, 1999). Eleven studies have reported umbilical cord arterial blood
gas values encompassing more than 35,000 deliveries (Huisjes & Aarnoudse, 1979; Sykes et
al., 1982; Eskes et al., 1983; Yeomans et al., 1985; Low, 1988; Ruth & Raivio, 1988; Ramin et
al., 1989; Thorp et al., 1989; Riley & Johnson, 1993; Nagel et al., 1995; Helwig et al., 1996).
These studies suggest that the reference ranges for all of the components of blood gas
analysis are wide and the lower limit for normal cord arterial pH should be between 7.04 and
7.10 and the lower limit of venous pH should be 7.14 to 7.20. Ideally paired arterial and
venous samples should be collected from the umbilical cord immediately after delivery
because a difference in the partial pressure of carbon dioxide of more than 25mmHg
suggests acute rather than chronic acidosis (Belai et al., 1998).
1.4.2.3.2 Prediction of adverse outcomes
Metabolic acidaemia at birth (pH<7.0, base deficit >12mmol/L) is relatively common
occurring in 2% of all births (Ruth & Raivio, 1988; Low et al., 1994). Severe metabolic
acidaemia with a base deficit greater than 16mmol/L is less common occurring in 0.05% of
all births (Low et al., 1994) and it has been suggested that this is a realistic cut off point for
defining pathological fetal acidaemia that correlates with an increasing risk of neurological
deficit (Goldaber et al., 1991; Sehdev et al., 1997). There is growing evidence that base deficit
values in cord arterial blood are significantly more useful than umbilical cord pH values
because base excess does not change significantly with respiratory acidosis, and demonstrates
a linear, rather than logarithmic correlation to the degree of metabolic acidosis (Low, 1998;
Ross & Gala, 2002).
As described in detail in Section 1.4.1.4, babies born with severe metabolic acidosis in cord
arterial blood are significantly more likely to suffer short term neonatal complications than
babies with normal acid-base status (Gilstrap et al., 1989; Low et al., 1995; van den Berg et al.,
1996). The outcomes for babies born with severe respiratory acidosis are no different from
babies born with normal umbilical arterial blood gas results (Low et al., 1994). In long term
follow-up studies, the association between metabolic acidaemia at birth and
neurodevelopmental outcome is poor with all but one study reporting no significant
59
association between acidaemia and neurodevelopmental disability (Low et al., 1983, 1988;
Ruth & Raivio, 1988; Socol et al., 1994; Ingemarsson et al., 1997).
1.4.2.3.3 Limitations of umbilical cord blood gas analysis
Umbilical cord blood gas assessment at birth is a sensitive marker of asphyxia, but its use is
somewhat limited because traditional markers of fetal acid-base status reflect fetal metabolic
adjustments that are not directly related to fetal damage unless the values are extreme
(Gilstrap et al., 1989; Goldaber et al., 1991; Winkler et al., 1991). Even a pH less than 7.0 is
poorly predictive of neurological deficit because a severely acidotic infant will usually have a
normal outcome (Winkler et al., 1991; Socol et al., 1994; Yudkin et al., 1994).
Other limitations of this technique relate to the cost of machines which perform blood gas
analysis and the issues of accurate collection of samples. To reliably perform blood gas
analysis on all deliveries, each delivery suite would require a blood gas machine on site. The
cost of this equipment precludes many smaller units from establishing this technique.
Second, for these results to be clinically useful the collection technique is critical and despite
staff eduction this has proven difficult in a number of units which report that 25% of their
results are invalid (Westgate et al., 1994).
1.4.3 Summary
Each of the three techniques that are widely used for the assessment of fetal wellbeing in
labour have a number of limitations.
Electronic fetal heart rate monitoring is associated with a significant increase in obstetric
intervention. It is only with the addition of fetal scalp blood sampling for acid-base
assessment that there is any improvement in neonatal outcome. Such combined monitoring
results in a 50% reduction in the number of babies with neonatal seizures. No studies of
EFM or FBS have shown a reduction in neonatal encephalopathy or cerebral palsy and even
though multiple late decelerations and decreased FHR variability have been shown to be
associated with the subsequent development of cerebral palsy, only 0.19% of singletons with
these patterns develop cerebral palsy giving a false positive rate of 99.8%.
Intrapartum fetal blood sampling, the one technique that has been shown to limit the
increase in intervention associated with EFM and reduce neonatal seizures, is unpopular and
the cost of the equipment required to utilise this technique limits its use primarily to tertiary
obstetric centres. Similarly, although umbilical cord blood gas assessment at birth is a
60
sensitive marker of asphyxia, the machines to perform this routinely are expensive. Babies
born with severe metabolic acidosis, as identified by umbilical cord gases, usually have
normal outcomes.
In order to reduce the burden of birth asphyxia on families, health care systems and the
community, new techniques that are inexpensive and widely available are required to allow
recognition of early intrapartum asphyxia so that timely obstetric intervention can avoid
asphyxia-induced brain damage or death in the newborn.
61
1.5 Lactate and fetal surveillance
My thesis describes studies designed to explore the role of lactate measurement in
intrapartum fetal surveillance. The preceding sections of this chapter have outlined the
importance of anaerobic metabolism and metabolic acidosis in perinatal asphyxia and brain
injury. Lactate is the end-product of anaerobic metabolism and it is the main fixed organic
acid in metabolic acidosis and the main contributor to base deficit (Nordstrom, 2001).
The first study investigating lactate measurement for intrapartum assessment of wellbeing
was performed in 1970 when it was noted that scalp lactate levels were higher in “distressed”
babies than those who were “not distressed” even though there was no difference in pH
(Yoshioka & Roux, 1970). The authors of this study concluded that scalp lactate may be an
earlier indicator of actual or impending fetal distress than scalp pH alone and the
measurement of scalp lactate was proposed as a test to discriminate when in doubt about the
fetal condition in labour. Despite this and other studies in the 1970’s and 1980’s which
suggested that lactate was at least as effective as pH at identifying fetal distress (Yoshioka &
Roux, 1970; Gardmark, 1974; Eguiluz et al., 1983; Smith et al., 1983; Schmidt, 1988), lactate
assessment did not become established in clinical practice. In all likelihood, this is because of
the amount of blood required for analysis. A blood sample volume of 150µL or more was
required, followed by haemolysing or spinning the blood sample and immediate
transportation of the sample to a biochemistry department for analysis (Soutter et al., 1978).
It has only been with the development of micro-volume test strip lactate meters that
sampling and analysis have been adapted to clinical obstetric practice (Gambke & Muller,
1993; Shimojo et al., 1993; Nordstrom et al., 1998a; Cobbaert et al., 1999; Pennell & Tracy,
1999; Sinn et al., 2001).
1.5.1 Lactate physiology
1.5.1.1 Lactate production
Lactate production and its effects on the mother and fetus are more complex than originally
thought. Lactate can be produced both under hypoxic and non-hypoxic conditions.
1.5.1.1.1 Hypoxic conditions
Lactate is the end-product of anaerobic metabolism. During anaerobic glycolysis, glucose is
broken down to pyruvate and converted to lactate and H+ ions. As described previously and
illustrated in Figure 1.5, the glycolytic process is dependent on the intracellular concentration
62
of NADH. NAD+ is the most important hydrogen carrier in catabolic reactions. It accepts a
hydrogen atom together with an additional electron, a hydride ion (H:-), forming NADH.
The conversion of NAD+ to NADH occurs during the reactions converting glucose to
pyruvate. The NADH molecule is unstable and the electron is easily transferred to other
molecules. To maintain glycolysis, NADH is oxidised to produce NAD+ which is required
for the further conversion of glucose to pyruvate. The hydride ion is used in the conversion
of pyruvate to lactate and H+. Energy in the form of 2 ATP molecules is produced, as
compared with 36 ATP molecules when pyruvate enters the citric acid cycle.
Fetal lactate concentrations increase during hypoxic conditions and it is likely that this lactate
is derived from the fetus (Milley, 1988); however, the exact source of lactate production is
difficult to identify. As previously discussed, the fetal response to hypoxia is to prioritise its
circulation to the heart, brain and adrenals whereas splanchnic and soft tissue circulations are
reduced. This hypothesis is supported by recent animal studies which showed that ATP
depletion was most severe in the kidneys compared with the brain and heart during graded
perinatal asphyxia (Seidl et al., 2000).
There is a steady state relationship between pyruvate and lactate under normal conditions
(1:10); however, the ratio increases with hypoxia (Marx & Greene, 1964; Low et al., 1979).
The value of this ratio does not appear to predict oxygen deprivation better that determining
the lactate level (Daniels, 1966).
1.5.1.1.2 Non-hypoxic conditions
Lactate production usually takes place in hypoxic tissue, but it can also be produced under
aerobic conditions. Lactate production can be induced by glycogenolysis and thus lactate
levels correlated with that of glucose. There is a steady state relationship between pyruvate
and lactate levels meaning that hyperglycaemia can produce lactic acidaemia even if oxygen is
available.
The experimental induction of fetal hyperglycaemia by high dose glucose infusions
(Gardmark, 1974; Kenepp et al., 1982; Philipson et al., 1987) or beta-mimetic drugs (Lunell et
al., 1977) induces glycogenolysis and may increase lactate levels even under aerobic
conditions. Maternal glucose infusions which are sometimes used in labour (5% Dextrose,
180mL/hr) do increase maternal and fetal glucose concentrations but do not alter maternal
or fetal lactate levels or acid-base status (Nordstrom et al., 1995a).
63
The “stress of being born” induces high levels of catecholamines in the course of normal
labour and delivery (Hagnevik et al., 1984; Lagercrantz & Slotkin, 1986). This catecholamine
surge leads to glycogenolysis in the fetal liver with concomitant hyperglycaemia; however, no
related lactate increase has been found, either in normal (Nordstrom et al., 1996b) or
complicated deliveries (Nordstrom et al., 1998b).
Maternal hyperventilation can also influence lactate levels. Decreasing maternal CO2 results
in bicarbonate depletion through the carbonic anhydrase buffering system. Maternal pH will
then increase as respiratory alkalosis develops. To maintain electroneutrality during the
depletion of the negatively charged bicarbonate ions, lactate serves as a supplementary source
of anions. Clinically, maternal hyperventilation can result in both maternal and fetal lactate
levels increasing by up to 1mmol/L (Huch, 1986).
1.5.1.2 Fetal lactate levels during pregnancy and delivery
Unlike pH, PO2, PCO2 and base excess, which all change with advancing gestation, the
concentration of lactate in umbilical blood samples obtained by cordocentesis in
normoxaemic healthy human fetuses does not change over the gestational age range of 17 to
38 weeks (Nicolaides et al., 1989). The levels obtained at cordocentesis are also not different
from the levels obtained at elective caesarean section at term (Nicolaides et al., 1989).
Animal and human studies suggest that there is a fetal consumption of lactate that probably
originates from uteroplacental metabolism of glucose (Burd et al., 1975; Char & Creasy, 1976;
Schneider et al., 1981; Nicolaides et al., 1989). In humans before labour, lactate in umbilical
venous blood is 7% higher than in umbilical arterial blood. In fetal lambs, lactate is
aerobically produced in the placenta and there is net transfer to the maternal and fetal
circulations (Battaglia & Meschia, 1986).
Lactate is likely to serve as a source of fuel for the fetus. In the human fetus, oxidative
metabolism of lactate represents 10-25% of the overall metabolic activity (Fowden, 1994). In
the fetal lamb, the heart uses lactate as a substrate for one third of myocardial oxygen
consumption (Bartelds et al., 2000).
Lactate equilibrates slowly over the placenta and the maternal lactate level has limited effect
on the fetal level, except during severe maternal hyperventilation or prolonged second stage
of labour (Kalinkov et al., 1980). Transfer of lactate between the maternal and fetal
circulations occurs in both directions although at a much slower rate than for transfer of
carbon dioxide (Daniels, 1966). A lactate carrier mechanism has been demonstrated on both
64
the maternal and fetal surfaces of the trophoblast (Carstensen et al., 1983) and the transfer
mechanism is probably through facilitated diffusion coupled with proton transfer (Piquard et
al., 1990).
Several early studies suggested that fetal lactate levels increased during labour (Jacobsen &
Rooth, 1971; Suidan et al., 1984b), but recent studies have found no correlation between
cervical dilatation and fetal lactate levels, nor have they found any change in fetal lactate
levels during the first stage of labour (Smith et al., 1983; Nordstrom et al., 1994; Nordstrom et
al., 1995b). There are increases in both maternal and fetal lactate levels during the active
component of second stage labour (Kalinkov et al., 1980; Nordstrom et al., 2001) which may
account for the results found by the earlier investigators who may not have differentiated
first and second stages of labour. The increase in lactate during the second stage of labour is
partly explained by transplacental injection of maternal lactate and partly due to increasing
duration of intermittent fetal hypoxia during maternal pushing (Nordstrom et al., 1996a;
Nordstrom et al., 2001).
It is well established that during fetal distress, the increase in fetal lactate level is derived from
the fetus (Nordstrom et al., 1996a). Higher scalp blood lactate and umbilical arterial lactate
levels were found in babies with non-reassuring FHR patterns, when compared with babies
with reassuring FHR patterns (Nordstrom et al., 1995b). Fetal hypoxia may also result in an
increase of the umbilical cord arterio-venous lactate gradient (Suidan et al., 1984a). In animal
studies, the net transfer of lactate to the fetus during experimental hypoxia is reduced to zero
(Milley, 1988). Thus endogenous fetal lactate production occurs in response to hypoxia.
1.5.1.3 Negative effects of lactate
From studies in hypoxic monkeys, Myers suggested that lactate was toxic for brain tissue
causing oedema and necrosis in the late gestation fetus (Myers, 1977). In-vivo magnetic
resonance spectroscopic (MRS) studies of fetal lambs undergoing experimental hypoxia have
shown that lactate concentrations in the fetal brain begin to increase, due to anaerobic
metabolism, at an arterial pH of less than 7.28 or a base deficit over 8mmol/L (van
Cappellen et al., 1999).
1.5.1.4 Positive effects of lactate
Lactate has traditionally been thought of as the “dead end” product of anaerobic metabolism
but recent studies suggest that it can also have positive effects. Lactate can dilate human
placental arteries in the chorionic plate, a mechanism that might be of importance in fetal
adaptation to hypoxia (Omar et al., 1993). Lactate may also have a role in attenuation of
65
brain injury. When L-lactate was infused into rats prior to experimentally-induced brain
injury, the cortical injury was smaller in size (Ros et al., 2001) and there were fewer cognitive
deficits two weeks later (Rice et al., 2002). The positive effects of lactate on the brain may be
explained by the finding that neurons are capable of utilising lactate as an energy source. In
vitro studies have demonstrated the ability of neurons to carry out synaptic function with
lactate as the only available carbon source (Schurr et al., 1997).
1.5.2 Fetal scalp lactate
There are few studies which have investigated the relationship between lactate concentrations
in blood from the fetal scalp lactate and central circulation. This paucity of data is probably
due to the volume of blood that was required for lactate measurement and the wet chemistry
techniques which were necessary prior to the development of micro volume test strip lactate
meters.
The relationship between fetal scalp lactate concentrations and umbilical artery and vein
lactate concentrations have been investigated in two human studies (Yoshioka & Roux, 1970;
Kruger et al., 1998). The first of these studies compared lactate concentrations from these
three sampling sites in 14 human fetuses, six who were delivered with forceps vaginally and 8
who were delivered by caesarean section (Yoshioka & Roux, 1970). The scalp blood samples
were collected one to three minutes prior to delivery of the fetus and collection of the
samples from the umbilical cord. There were no significant differences in the mean lactate
concentrations from the three sampling sites in either the fetuses delivered vaginally or those
delivered by caesarean section. Of note, the lactate concentrations in blood from the fetal
scalp and umbilical artery were identical in samples collected at caesarean section whereas
scalp lactate concentration was lower than arterial lactate concentration for fetuses delivered
vaginally. These differences were not statistically significant; however, only six blood samples
were analysed. In a subsequent study, 103 scalp lactate concentrations were compared with
umbilical artery and vein lactate concentrations sampled within 60 minutes of delivery.
Lactate concentration in fetal scalp blood before delivery showed a significant correlation
with lactate levels in the umbilical artery (r=0.65, p<0.001) and venous blood (r=0.62,
p<0.001) (Kruger et al., 1998). In both these human studies no paired statistical analysis was
performed for blood collected from the same individual but different sampling sites. Unlike
scalp pH assessment, there have been no studies in animal models comparing fetal scalp
lactate concentration with carotid arterial and jugular venous blood lactate concentration.
There have also not been any studies which have investigated the relationship between
66
different sampling sites for lactate concentration and fetal hypoxaemia or acidaemia, two
known confounders of scalp pH assessment.
Normal lactate levels derived for human fetuses in the first stage of labour with reactive FHR
patterns prior to their FBS are presented in Table 1.11. The variation between studies almost
certainly relates to the different sample types and measuring techniques used in the each
study; some analysed plasma whilst others analysed whole blood or haemolysed blood. The
highest levels were measured in plasma, which is to be expected because lactate is unevenly
distributed in plasma and erythrocytes with the concentrations in erythrocytes only 50-60%
of that in plasma (Devadatta, 1934; Decker & Rosenbaum, 1942; Sahla et al., 1964). The
higher levels in the haemolysed blood may relate to production of lactate by erythrocytes,
which can occur during storage of blood in a test tube prior to analysis. Issues relating to
delay in sample analysis are abolished by the new bedside micro volume test strip methods.
Table 1.11 Normal ranges for scalp lactate in first stage of labour
Authors Mean lactate
(mmol/L)
95th percentile
Sample size (N) Sample type
Jacobsen & Rooth, 1971 3.0 5.0 45 Plasma
Schmidt, 1973 2.9 3.2 20 Haemolysed blood
Eguiluz et al., 1983 1.0 NA 52 Whole blood
Smith et al., 1983 1.8 2.9 97 Haemolysed blood
Nordstrom et al., 1994 1.7 3.3 66 Whole blood
Nordstrom et al., 1995b 1.2 2.9 64 Whole blood NA = not available There have been a number of observational studies which have shown that scalp lactate
performs as well as pH in predicting low Apgar scores at delivery (Eguiluz et al., 1983; Smith
et al., 1983; Nordstrom et al., 1995b). There has been one randomised clinical trial of 341
parturients, comparing fetal scalp blood lactate and pH in the diagnosis of intrapartum fetal
distress (Westgren et al., 1998). No difference was identified between the two groups with
respect to mode of delivery, intervention rates, neonatal outcome or umbilical artery acid-
base status or lactate. The positive findings of the trial were that scalp lactate assessment was
quicker, required significantly fewer scalp incisions, and had a lower blood sampling failure
rate than scalp pH assessment. Lactate analysis required 5µL of blood whereas pH required
35µL. Sampling failure rates were 1.2% and 20.6% respectively (OR 16.1, 95% CI 5.8-44.7).
67
There has been one large retrospective observational study investigating the predictive values
of 1709 scalp lactate and pH samples as markers of neurological disability (Kruger et al.,
1999). The pH results have previously been discussed in section 1.4.2.2.2 and are presented
in Table 1.10. Scalp lactate was a significantly better predictor than scalp pH of Apgar score
less than 4 at 5 minutes (area under ROC: lactate 0.79, pH 0.55, p=0.033) and, more
importantly, moderate and severe hypoxic-ischaemic encephalopathy (area under ROC:
lactate 0.95, pH 0.64, p=0.015). The sensitivity and specificity for lactate to predict severe
metabolic acidaemia in cord arterial blood, low Apgar scores and hypoxic-ischaemic
encephalopathy are summarised in Table 1.12. Comparing Tables 1.10 and 1.12 it can be
seen that scalp lactate assessment has higher sensitivity for predicting these adverse outcomes
than scalp pH with no significant loss in specificity. There were six cases in this series that
had hypoxic brain injuries and five had both scalp lactate and pH measurements performed.
Four of the five had elevated levels of scalp lactate; only two of the five had low levels of
scalp blood pH. There are a number of possible explanations for the discrepancy between
the hyperlactaemic and acidaemic fetuses. First, it has been suggested by animal studies of
asphyxia that subcutaneous lactate increases earlier than the pH decrease, and precedes the
brain tissue lactate increase (Engidawork et al., 1997). Second, the difference may relate to
the slower time to normalise lactate compared to pH after hypoxic insults (Ikeda et al., 1998a;
Keunen et al., 1999; Kaneko et al., 2003). This has been illustrated by a recent study, in which
repeated cord occlusion of four minutes every 90 minutes for a total duration of six hours
was performed in fetal sheep. Significant reductions in pH occurred in response to each cord
occlusion; however, by the time of next cord occlusion pH had normalised. The pH before
the first cord occlusion was 7.33±0.01 and before the last occlusion it was 7.30±0.02. The
pH response was very different from the lactate response in this study; there was a
progressive rise in lactate with each cord occlusion such that before the first occlusion lactate
was 1.21±0.21mmol/L and before the last occlusion it had increased to 6.1mmol/L (Kaneko
et al., 2003). It appears from these data that lactate is a better marker than pH for
intermittent acute insults because pH can be normalised by both the placenta and buffering
systems whereas the slow clearance of lactate by the placenta affords it a greater ability to act
as an indirect marker of these insults.
68
Table 1.12 Scalp lactate and prediction of neonatal outcome
Outcome Variable Area under ROC (95%CI) Sensitivity* Specificity*
Umbilical artery
pH < 7.0 0.72 (0.69-0.75) 78.6% 74.4%
Base deficit > 16mmol/L 0.78 (0.75-0.81) 70.8% 75.5%
Apgar score
< 7 at 1 min 0.68 (0.65-0.71) 49.1% 76.2%
< 7 at 5 min 0.68 (0.65-0.71) 52.9% 73.5%
< 4 at 6 min 0.71 (0.68-0.74) 77.8% 73.5%
Hypoxic-ischaemic encephalopathy
Mild 0.70 (0.66-0.72) 66.7% 73.0%
Moderate or severe 0.83 (0.80-0.85) 100% 72.8% *Sensitivity and specificity for scalp lactate values at the 75th percentile (>4.8mmol/L). (Adapted from Kruger et al. 1999)
1.5.3 Umbilical cord lactate levels at delivery
There are several reasons for performing routine cord blood acid-base balance or lactate
measurements at delivery: a quality assessment of obstetric practice; a diagnostic test for
hypoxia in cases of depressed newborns; and for the prediction of neonatal complications
and long-term outcome. There have been several reports of routine cord blood lactate
measurements (Westgren et al., 1995; Chanrachakul et al., 1999; Pennell & Tracy, 1999).
Umbilical artery lactate measurement has been shown to be equally as good as pH and base
excess at predicting low Apgar scores at 1 and 5 minutes (Westgren et al., 1995). Umbilical
artery lactate levels are significantly better predictors of hypoxic-ischaemic encephalopathy
than pH (Chou et al., 1998; Kruger et al., 1999).
1.5.4 Lactate levels in the newborn
There have been a number of studies that have investigated lactate levels in the newborn.
Early postnatal magnetic resonance spectroscopy (MRS) examinations of lactate levels have
shown a significant correlation with poor outcome (death or neurologic deficit) (Leth et al.,
1996). Urinary lactate-creatinine ratio at 6 hours of age assessed by proton nuclear MRS have
also been shown to have both high sensitivity (94%) and specificity (100%) for predicting
hypoxic-ischaemic encephalopathy in a group of babies with perinatal asphyxia (Huang et al.,
1999).
69
1.6 Thesis outline
1.6.1 Aim
The aim of this thesis is to further evaluate the role of lactate in intrapartum fetal
surveillance.
1.6.2 Studies performed
1.6.2.1 Evaluation of a new method for rapid assessment of lactate in fetal and
neonatal blood
It has only been with the development of micro-volume test strip lactate meters that
sampling and analysis of lactate have been adapted to clinical obstetric practice. When
commencing my candidature no micro volume lactate meter that had been evaluated in fetal
and neonatal blood was available in Australia. Therefore the aim of my first series of studies
was to determine the accuracy and precision of the commercially available Accutrend®
handheld reflectance photometer for measuring whole blood lactate levels in umbilical cord
blood and neonatal blood micro samples across the gestational age range of 26 to 42 weeks.
1.6.2.2 The relationship between neuropathologic damage and biochemical and
physiologic markers of asphyxia induced by graded cord occlusion in
chronically instrumented ovine fetuses.
The aim of my second series of experiments was to use an ovine model of repeated umbilical
cord occlusion (UCO) to evaluate the relationship between fetal lactate responses and fetal
brain injury. To address this aim I have utilised an UCO protocol, designed to mimic events
which may occur in labour, to compare the relationship between:
1. fetal acid-base status and neuropathologic damage,
2. fetal lactate and neuropathologic damage,
3. fetal heart rate patterns (FHR) and neuropathologic damage, and
4. fetal arterial pressure and neuropathologic damage.
70
1.6.2.3 The relationship between fetal scalp lactate levels, central lactate levels and
oxygen free radical activity in the fetal brain during asphyxia induced by
graded cord occlusion in the ovine fetus
Fetal scalp blood sampling is the principal technique utilised to collect blood for biochemical
assessment of fetal wellbeing during labour. Prior to the potential introduction of a new
biochemical marker of fetal wellbeing into clinical practice, two important questions require
investigation. First, what is the relationship between fetal scalp blood levels and central
blood levels of the new biochemical marker and is this relationship altered by asphyxia?
Second, do fetal scalp levels rather than central levels of the new marker predict fetal brain
injury? The purpose of the series of studies presented in this chapter was to address these
two important questions. Prior to exploring the peripheral-central lactate relationships, I
have compared and contrasted the acutely instrumented model used in this study with the
chronically instrumented model described in the previous chapter. To investigate the
relationship between fetal scalp lactate and brain injury I have investigated the relationship
between fetal lactate levels and oxygen free radical activity in the fetal brain. This approach
was chosen because there is growing evidence at a molecular level that oxygen free radicals
are critically involved in the mechanisms of hypoxic-ischaemic brain injury (Maulik, 1998;
Volpe, 2001b; du Plessis & Volpe, 2002; Gitto et al., 2002; Inder et al., 2002) and because of
the terminal nature of the experiments. Oxygen free radical activity was assessed both
during and after the asphyxial insult and related to scalp lactate and other biochemical
markers of fetal wellbeing.
1.6.2.4 Fetal lactate responses in growth restricted ovine fetuses during asphyxia
induced by graded cord occlusion
There is growing evidence that an adverse intrauterine environment alters fetal physiologic
responses during asphyxia (Gardner et al., 2002; Gardner et al., 2003). Fetal growth restriction
secondary to placental insufficiency is associated with an adverse intrauterine environment
which includes hypoxaemia, hypercapnia, hyperlacticaemia, and acidaemia (Nicolaides et al.,
1989; Marconi et al., 1990). Growth restricted fetuses have also been shown to have altered
lactate metabolism (Marconi et al., 1990), glucose metabolism (Jansson et al., 1993; Marconi et
al., 1996; Pardi et al., 2002), amino acid metabolism (Cetin et al., 1996; Marconi et al., 1999)
and fatty acid metabolism (Cetin et al., 2002) when compared to appropriately grown
controls. Human growth restricted fetuses are at increased risk of non-reassuring FHR
patterns during labour (Low et al., 1976; Dashe et al., 2000), fetal acidosis in fetal scalp blood
samples collected during labour (Nieto et al., 1994), metabolic acidosis in umbilical arterial
blood samples collected at delivery (Low et al., 1981) and neonatal encephalopathy secondary
71
to acute intrapartum hypoxic events (Bukowski et al., 2003). The purpose of the series of
studies presented in this chapter was to explore fetal lactate responses and central-peripheral
lactate relationships in growth restricted ovine fetuses during asphyxia.
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C h a p t e r 2
EVALUATION OF A NEW METHOD FOR RAPID ASSESSMENT
OF LACTATE IN FETAL AND NEONATAL BLOOD
The best preparation for tomorrow is to do today’s work superbly well.
Sir William Osler (1892)
2.1 Introduction
Fetal scalp blood lactate measurement for the assessment of fetal wellbeing has been
investigated since the 1970’s (Yoshioka & Roux, 1970; Gardmark, 1974; Eguiluz et al., 1983;
Smith et al., 1983; Schmidt, 1988). Despite initial studies suggesting that lactate was at least as
effective as pH at identifying fetal distress as reflected by low Apgar scores (Smith et al.,
1983), lactate assessment has not become established in clinical practice. In all likelihood, the
reluctance to adopt this technique probably related to the amount of blood required for
analysis. Until the recent development of micro-volume test strip methods (Gambke &
Muller, 1993; Shimojo et al., 1993; Nordstrom et al., 1998a; Cobbaert et al., 1999; Sinn et al.,
2001), at least 150µl of blood has been required for analysis (Soutter et al., 1978) which is far
greater than the 20 to 50µl volume routinely available when collecting fetal scalp blood
samples.
Lactate concentrations vary in different body compartments. This is important because
different methods of lactate measurement sample different body compartments. Lactate is
unevenly distributed between plasma and erythrocytes with the concentration of lactate in
erythrocytes being 50-60% of the plasma concentration (Devadatta, 1934; Decker &
Rosenbaum, 1942; Sahla et al., 1964). Thus within an individual, plasma has the highest
lactate concentration with lower concentrations in haemolysed blood and the lowest
concentration in whole blood with intact erythrocytes.
Until 1986, whenever blood was to be analysed for lactate levels, samples required
stabilisation for transport to the laboratory and wet chemistry techniques involving
precipitation with perchloric acid in order to obtain lactate concentrations from both
erythrocytes and plasma. In recent years micro volume (5-25µl) test strip based analysis
73
methods have been developed which use capillary whole blood with intact erythrocytes (Weil
et al., 1986; Shimojo et al., 1989; Wandrup et al., 1989; Gambke & Muller, 1993; Shimojo et al.,
1993). These newer meters are easy to use and provide both accurate and precise
measurement in adult, fetal and neonatal blood. Unfortunately, when commencing my
candidature, none of these meters were commercially available in Australia.
During the 1990’s lactate electrodes were developed for micro sample blood-gas analysers.
The electrodes measure lactate with amperometric techniques where the current generated
from a series of biochemical reactions is proportional to the lactate concentration. The lactate
electrodes are both accurate and precise (Toffaletti et al., 1992); however, these machines are
expensive, ranging in price from AUS $50,000 -$80,000. These machines require biomedical
staff for maintenance and sample stabilisation if delays are encountered between sampling
and testing.
For lactate to be a practical alternative to the current biochemical techniques for the
assessment of fetal wellbeing during labour, the measurement system needs to meet a
number of criteria. First, the system should be inexpensive, easy to use, and suitable for
primary, secondary and tertiary obstetric units. Second, the measurement system should
analyse small samples of fetal blood (approximately 20µl), preferably at the bedside, and
require minimal ongoing maintenance. Third, the measurement system should be accurate
and precise across a range of ages from 24 to 42 weeks gestation for samples from fetal scalp,
umbilical cord and neonatal sampling sites. Finally, the measurement system should be
accurate across a range of haematocrit concentrations. This is particularly important as the
haematocrit of fetal and neonatal blood is influenced by gestational age, fetal growth
restriction, sampling location and labour (Jacobsen & Rooth, 1971; Soothil et al., 1987;
Economides & Nicolaides, 1990; Weiner et al., 1991; Brace, 1992). Errors due to varying
haematocrit levels are a flaw of many lactate analysers including those which use
amperometric techniques (Toffaletti et al., 1992) and test strip methods (Nordstrom et al.,
1998a).
The Accutrend® lactate meter (Roche Diagnostics, Mannheim, Germany) is an inexpensive
handheld unit that costs approximately AUS$900 and analysis of each sample costs AUS$1.
The unit measures 115mm x 62mm x 18.5mm and analyses sample volumes as small as 15µl
with a measurement time of 60 seconds. The meter uses reflectance photometry to measure
lactate from 0.8 to 22mmol/L for blood and 0.7 to 26mmol/L for plasma. It performs at a
temperature range of 5-35°C for blood lactate values less than 8mmol/L and 15-35°C for
blood lactate values greater than 8mmol/L
74
The Accutrend® meter has been compared with many laboratory lactate assessment
techniques and found to be accurate when measuring lactate in adult capillary blood samples
from 0.8 to 22mmol/L (Gambke & Muller, 1993). Comparisons with wet chemistry
techniques for lactate determination in horse plasma have also been performed and the
results from the two techniques were highly correlated (r=0.928; Williamson et al, 1996).
Based on its specifications, the Accutrend® lactate meter is ideally suited to point of sample
analysis in the labour ward and the nursery; however, prior to using this meter for both
research and clinical management, it requires evaluation for accuracy and precision in
neonatal blood across the gestational ages from 24 to 42 weeks with a range of haematocrit
concentrations.
2.1.1 Aim
To determine the accuracy and precision of the commercially available Accutrend® handheld
reflectance photometer in measuring whole blood lactate levels in umbilical cord blood and
neonatal blood micro samples across the gestational age range of 26 to 42 weeks.
75
2.2 Methods
All experimental procedures were approved by the Wentworth Area Health Service Scientific
Advisory Panel and Ethics Committee, New South Wales, Australia. Informed maternal
consent was obtained prior to the collection of cord and neonatal blood.
2.2.1 Measuring principle
2.2.1.1 Accutrend® meter
The Accutrend® meter is a handheld reflectance photometer (Figure 2.1) based on dry
chemistry and requires 15-50µL of blood for analysis. Whole blood is applied to a multilayer
test strip and a thin glass fibre carpet separates red blood cells from whole blood (Figure 2.2).
Plasma is drawn into a plasma reservoir via capillary force to reach a reaction layer where
enzymatic determination of lactate commences using lactate dehydrogenase (Figure 2.3A). A
colour reaction is measured using an optical system through a transparent film, with the lactate
concentration being calculated from the intensity of reflected light (Figure 2.4). The
Accutrend® meter is accurate in adult blood for lactate concentration ranging from 0.7-
26mmol/L (Gambke & Muller, 1993).
2.2.1.2 Reference method
The reference method for this study was the Radiometer ABL 625® blood gas machine
(Radiometer Medical Pty Ltd, Copenhagen, Denmark) which measures lactate with an
amperometric electrode and an enzyme membrane utilising lactate oxidase. A two-step
reaction occurs and the current generated is proportional to the lactate concentration in the
sample (Figure 2.3B). The ABL625® lactate electrode has been validated over a range of 0 to
30mmol/L (Buch-Rasmussen, 1990; Cobbaert et al., 1999; Sinn et al., 2001).
2.2.2 Assessment of precision
The precision of a clinical test is the degree of refinement with which an operation is
performed or a measurement stated (Gennaro et al., 1984). To assess the precision of the
Accutrend® handheld lactate meter, repeated analysis of neonatal plasma samples (n=20) at
six known lactate concentrations were performed on both the Accutrend® meter and the
reference method by a single operator. Plasma was harvested from a single neonate during an
exchange transfusion for septic shock. The baseline serum lactate concentration was
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Figure 2.1 Accutrend® handheld lactate meter
Figure 2.2 Design of lactate test strip for Accutrend® handheld lactate meter
Glass Fibre Reaction Layer Detection Film
Side View
Bottom View
Calibration Strip
77
A
Lactate + Mediator form1 → Pyruvate + Mediator reduced
Mediatorreduced + 2,18-Phosphomolybdate → Molybdenum Blue + Mediatorform 2
B
Lactate + O2 + H2O → Pyruvate + H2O2
H2O2 → 2H+ + O2 + 2e-
Figure 2.3 Reaction principles used for lactate measurement by the Accutrend® and
ABL625®. Figure A is the reaction principle of the Accutrend® handheld lactate meter using
lactate dehydrogenase for step one of the reaction. Figure B is the measuring principle of the
comparison method, the Radiometer ABL625® blood gas machine. Step one of this reaction
uses lactate oxidase and the generated current from step 2 is proportional to the lactate
concentration in the sample.
78
Figure 2.4 Analysis technique for Accutrend® handheld lactate meter. Plasma is drawn into a
plasma reservoir by capillary force to reach a reaction layer where the enzymatic determination
of lactate commences using lactate dehydrogenase. A colour reaction is measured using an
optical system with the lactate concentration being calculated from the intensity of reflected
light.
LED(660nm)
Detector
Plasma Reservoir
5°
40°
79
5.3mmol/L. D-lactate was added to produce higher concentrations of 9.6mmol/L,
15.4mmol/L and 19.5mmol/L and dilution of the baseline serum with saline produced lower
concentrations of 2.0mmol/L and 1.0mmol/L. The coefficients of variation were calculated
for both techniques at each of the six concentrations assessed.
2.2.3 Assessment of accuracy
The accuracy of a test is the degree of conformity of a measure to a standard or true value
(Gennaro et al., 1984). To assess the accuracy of Accutrend® handheld lactate meter, neonatal
plasma and blood, and umbilical arterial blood were tested simultaneously on the Accutrend®
handheld lactate meter and the Radiometer ABL625 blood gas machine (reference method).
2.2.3.1 Neonatal plasma samples
The initial studies of accuracy were performed on the 120 neonatal plasma samples used for
the analysis of precision (see Section 2.2.2).
2.2.3.2 Umbilical cord blood samples
Umbilical artery blood was collected at the Nepean Hospital Delivery Suite from 160
consecutive deliveries with gestational ages between 26 and 43 weeks. Each umbilical cord
was clamped at delivery, before the first breath by the neonate, and arterial samples were
collected in heparinised syringes which were capped and kept on ice until analysis could be
performed simultaneously using the Accutrend® meter and the reference method in the
neonatal intensive care unit. Clinical data from the mother and baby were recorded together
with the full blood-gas analysis performed on the Radiometer ABL 625®.
2.2.3.3 Neonatal arterial blood samples
Point-of-sample testing is where blood samples are analysed at the same location as their
collection. To test the Accutrend® meter as a true point-of-sample test, 110 neonatal arterial
blood samples were collected from umbilical artery catheters from babies in the Nepean
Hospital Neonatal Intensive Care Unit (gestational age from 26-41 weeks) and immediately
analysed simultaneously with both techniques.
2.2.3.4 Effect of temperature on accuracy
To assess the effect of temperature on the accuracy of the Accutrend® meter, we undertook
repeated measurement of neonatal blood at temperatures from 0.5°C to 37°C performed at
5°C increments on both the Accutrend® meter and the reference method. Blood was
80
collected from an exchange transfusion and samples were stored in capped heparinised
syringes (Radiometer Medical Pty Ltd, Nunawading, Victoria, Australia). The samples were
maintained at the appropriate temperature in a water bath while repeated measurements were
made on each machine. To confirm the discrepancy that became apparent in chilled samples,
blood at two known lactate concentrations (4.8mmol/L and 9.4mmol/L) was held in a water
bath at 0.5°C and measurement was repeated 20 times using both methods.
2.2.4 Statistical methods
Coefficients of variation (CV), defined as the standard deviation divided by the mean and
expressed as a percent, were calculated for the repeated sampling at known concentrations.
Accuracy between the two techniques was assessed using Passing Bablok (PB) regression
analysis (Passing & Bablok, 1983; Bablok et al., 1988) and modified Altman-Bland plots (Bland
& Altman, 1986; Pollock et al., 1992; NCCLS, 1995).
Passing Bablok regression is a technique widely used in clinical chemistry for formally testing
for agreement between two clinical methods. This regression technique is useful when
imprecision occurs in both variables and unlike other techniques, the imprecision does not
need to be normally distributed. Passing Bablok regression does not assume the variance to
be constant over the range of measurement and the regression line is not excessively biased by
extreme values (Passing & Bablok, 1983).
Altman-Bland plots illustrate graphically the agreement (or disagreement) between two clinical
methods. Two methods can be compared for both bias and agreement, and the relationship
between imprecision and concentration is presented graphically. Various options are available
for comparing the methods, depending on the best estimation of the true values. In Altman-
Bland plots, the difference between the methods is plotted against the mean of the methods.
The mean of the methods is treated as the best estimation of the true values (Bland & Altman,
1986). In the NCCLS EP9-A modification of Altman-Bland plots, the difference between
methods is plotted against the reference method or gold standard which is known to have
greater precision and therefore is the best estimate of the true value (NCCLS, 1995). In this
study NCCLS EP9-A plots have been used.
Descriptive data regarding the cord samples used in the analysis are presented as median, inter
quartile range (IQR) and range. The relationships between haematocrit, operator, and delay in
analysis and the accuracy of the Accutrend® meter were investigated using regression
modelling. Changes in accuracy (test method – reference method) at different temperatures
81
were analysed using analysis of variance (ANOVA) and paired t tests for the repeated sampling
at 4.8 and 9.4mmol/L. Statistical analyses were carried out using the Statistical Package for
Social Sciences (SPSS) for Windows, version 11.0 (SPSS Inc., Chicago, IL, USA) and Analyse-
It for Excel, version 1.63 (Leeds, England, UK).
82
2.3 Results
2.3.1 Analysis of precision
The precision of plasma lactate analysis for the Accutrend® handheld lactate meter and the
reference method are summarised in Tables 2.1 and 2.2. The coefficients of variation were
between 1.23% and 5.53% over the clinically significant lactate range of 2.2 to 19.3mmol/L
for the Accutrend® handheld lactate meter and between 1.93% and 6.74% for the reference
method. The Accutrend® meter had a higher coefficient of variation for lactate
concentrations of 1.1mmol/L; however, the range was only 1.0 to 1.2mmol/L.
Table 2.1 Precision of the Accutrend® Reflectance Photometer
Test Sample No. 1 2 3 4 5 6
Replicates (n) 20 20 20 20 20 20
Mean 1.01 2.23 6.16 9.50 16.39 19.05
SD* 0.079 0.091 0.11 0.35 0.91 0.23
CV† (%) 7.32 4.09 1.78 3.64 5.53 1.23
Range 1.0-1.2 2.1-2.4 6.0-6.4 8.8-10.0 15.3-18.3 18.5-19.7
*SD = standard deviation; † CV = coefficient of variation
Table 2.2 Precision of the Radiometer ABL 625® blood-gas machine (reference method)
Test Sample No. 1 2 3 4 5 6
Replicates (n) 20 20 20 20 20 20
Mean 1.05 2.04 5.48 9.57 15.39 19.49
SD* 0.079 .091 0.11 0.49 1.04 0.78
CV† (%) 4.89 2.93 1.93 5.12 6.74 4.03
Range 1.0-1.1 2.0-2.2 5.4-5.7 8.9-10.4 14.3-17.0 18.3-21.2
*SD = standard deviation; † CV = coefficient of variation
83
2.3.2 Analysis of accuracy using neonatal plasma samples
The accuracy of the Accutrend® lactate meter for measuring plasma lactate was assessed by
comparing the results of simultaneous analysis with the Accutrend® meter and the reference
method. This assessment was performed over the clinically significant range of lactate
concentrations of 1-20mmol/L. The Passing Bablok regression equation for this comparison
was y = 0.200 + 1.000x with 95% confidence intervals for slope of 0.989 to 1.018 and for y
intercept of 0.080 to 0.222 indicating a high degree of accuracy. Both the Passing Bablok
regression and the Altman-Bland plots demonstrate a constant bias, with the Accutrend®
meter overestimating the lactate concentration as measured by the reference method by
0.228mmol/L (Figure 2.5).
2.3.3 Analysis of accuracy using umbilical cord blood samples
The characteristics of umbilical arterial samples from the 160 consecutive deliveries used for
this analysis are summarised in Table 2.3. Gestational ages ranged from 26 to 43 weeks and
haematocrits ranged from 41.5 to 63.4%.
Table 2.3 Characteristics of 160 cord samples utilised in accuracy analysis.
Median Inter Quartile Range (IQR) Range
Gestation (weeks) 40 39 – 40 26 - 43
Haematocrit (%) 51.3 48.8 – 55.1 41.5 – 63.4
Haemoglobin (g/L) 16.8 15.9 – 18.1 13.3 – 20.8
pH 7.28 7.23 – 7.34 7.00 – 7.48
Base Excess -4.2 -7.0 to -2.2 -14.4 to - 0.50
PO2 (mmHg) 18.5 14.1 – 25.3 3.5 – 48.7
PCO2 (mmHg) 49.3 41.2 – 59.0 27.2 – 91.6
Lactate (mmol/L) 3.7 2.7 – 5.1 1.3 – 9.8
Analysis delay* (min) 11 5 - 25 2 - 132
*time between collection and analysis
83
Figure 2.5. Accuracy of the Accutrend® lactate meter for measurement of lactate in neonatal
plasma. Figure A is an X-Y plot of lactate levels obtained from the Accutrend® lactate
meter and the reference method for 120 neonatal plasma samples, showing the Passing
Bablok regression line (y = 0.200 + 1.000x) with 95% confidence intervals, slope 0.989 to
1.018 and intercept 0.080 to 0.222. The identity line, which demonstrates the relatinship if
the two test methods were to report identical lactate levels, is plotted in blue in Figure A.
Figures B, C and D are Altman-Bland plots displaying the difference in each pair of analyses
against the reference method in this study. Figure B is a scatterplot of the difference between
the two methods plotted against the reference method. Figure C is a frequency distribution
of the data presented in Figure B. Figure D is a scatterplot of the difference between the two
methods as a percentage of the reference lactate concentration plotted against the reference
method.
84
Identity lineY = X
y = x + 0.2
0
5
10
15
20
25
0 5 10 15 20 25Radiometer ABL625 Lactate (mmol/L)
Accu
tren
d La
ctat
e (m
mol
/L)
Zero bias
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(m
mol
/L)
0 20 40 60 80
-2.0
-1.4
-0.7
-0.1
0.6
1.2
1.9
Diff
eren
ce b
etw
een
met
hods
(m
mol
/L)
Frequency (%)
Zero bias
-10
-5
0
5
10
15
20
25
0 5 10 15 20 25
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(%)
A
D
B C
85
The accuracy of the Accutrend® handheld lactate meter for measurement of lactate in
umbilical artery blood from 160 consecutive deliveries is presented in Figure 2.6. The
Passing Bablok regression line was very similar to that obtained with plasma; however, the
95% confidence intervals were slightly wider (y = 0.008 + 0.923x with 95% confidence
intervals for slope of 0.857 to 1.000 and intercept of -0.300 to 0.214). The Altman-Bland
plots suggest that the Accutrend® meter has a bias of -0.380mmol/L to underestimate the
reference method lactate concentration when analysing umbilical arterial blood. This bias
applied consistently across the test range of 1.3mmol/L to 9.8mmol/L.
The accuracy of the Accutrend® meter was not affected by gestational age (p=0.237) or
operator (p=0.399). Similarly, haematocrit from 41.5 to 62% did not affect the accuracy of
the meter (r=0.09, p= 0.220; Figure 2.7). Four of the 160 cord samples had haematocrits
higher than 62%, and for these samples, the Accutrend® meter tended to underestimate the
lactate level by 1.3 to 1.8mmol/L. A weak correlation was identified between haemoglobin
concentration in the blood sample and the accuracy of the meter (r=0.199, 95% CI 0.044 to
0.346, p=0.012). For each increase in haemoglobin of 3g/L, the Accutrend® meter
underestimates lactate level by 0.2mmol/L. A weak correlation was also identified between
time delays in analysis and accuracy of the meter (r= 0.185, 95% CI 0.027 to 0.335, p=0.019),
with greater delays in analysis being associated with a small reduction in the accuracy.
2.3.4 Analysis of accuracy using neonatal arterial samples
The analysis of umbilical arterial blood samples suggested that the Accutrend® lactate meter
was less accurate with blood samples than with plasma samples. The reduced accuracy may
have been due to the presence of red cells or the delay in analysis of the cord samples. The
reference method blood gas machine was located in the neonatal intensive care unit that was
one floor above the delivery suite, and the Accutrend® meter was located in delivery suite.
Samples were kept in foam cups filled with crushed ice whilst awaiting analysis.
To further investigate these issues, 110 neonatal arterial blood samples were collected from
umbilical artery catheters of babies (gestational age from 26 to 41 weeks) in the nursery and
were immediately analysed simultaneously with both techniques (point-of-care analysis). The
accuracy of the Accutrend® meter measuring lactate from these neonatal blood samples is
presented in Figure 2.8. The Passing Bablok regression equation was y=0.011 + 0.916x with
85
Figure 2.6. Accuracy of the Accutrend® lactate meter for measurement of lactate in umbilical
arterial blood. Figure A is an X-Y plot of lactate levels obtained from the Accutrend® lactate
meter and the reference method for 160 umbilical arterial samples, showing the Passing
Bablok regression line (y = 0.008 + 0.923x) with 95% confidence intervals, slope 0.857 to
1.000 and intercept -0.300 to 0.214. The identity line, which demonstrates the relationship if
the two test methods were to report identical lactate levels, is plotted in blue in Figure A.
Figures B, C and D are Altman-Bland plots displaying the difference in each pair of analyses
against the reference method. Figure B is a scatterplot of the difference between the two
methods plotted against the reference method. Figure C is a frequency distribution of the
data presented in Figure B. Figure D is a scatterplot of the difference between the two
methods as a percentage of the reference lactate concentration plotted against the reference
method.
86
Identity lineY = X
y = 0.9231x + 0.0077
0
2
4
6
8
10
0 2 4 6 8 10Radiometer ABL625 Lactate (mmol/L)
Accu
tren
d La
ctat
e (m
mol
/L)
Zero bias
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
0 2 4 6 8 10
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(m
mol
/L)
0 20 40 60
-2.5
-2.1
-1.6
-1.2
-0.8
-0.3
0.1
0.6
Diff
eren
ce b
etw
een
met
hods
(m
mol
/L)
Frequency (%)
Zero bias
-60
-50
-40
-30
-20
-10
0
10
20
30
0 2 4 6 8 10
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(%)
B C
A
D
87
40 45 50 55 60-1
0
1
2
3
Haematocrit (%)
∆La
ctat
e (m
mol
/L)
Figure 2.7 The effect of haematocrit on accuracy of the Accutrend lactate meter. No
relationship was identified between haematocrit and the accuracy of the meter for
haematocrits between 41.5 and 62%. The Pearson correlation coefficient r = 0.09 (95% CI
-0.06 - 0.25, p=0.219).
87
Figure 2.8 Accuracy of the Accutrend® lactate meter for measurement of lactate in neonatal
arterial blood. Figure A is an X-Y plot of lactate levels obtained from the Accutrend® lactate
meter and the reference method for 110 umbilical arterial samples collected in the nursery
showing the Passing Bablok regression line (y = 0.011 + 0.916x) with 95% confidence
intervals, slope 0.889 to 0.949 and intercept -0.146 to 0.111. Figures B, C and D are Altman-
Bland plots displaying the difference in each pair of analyses against the reference method.
Figure B is a scatterplot of the difference between the two methods plotted against the
reference method. Figure C is a frequency distribution of the data presented in Figure B.
Figure D is a scatterplot of the difference between the two methods as a percentage of the
reference lactate concentration plotted against the reference method.
88
y = 0.9157x + 0.0107
0
2
4
6
8
10
12
14
16
18
20
0 5 10 15 20Radiometer ABL625 Lactate (mmol/L)
Acc
utre
nd L
acta
te (m
mol
/L)
Zero bias
-3
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
0 5 10 15 20 25
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(m
mol
/L)
0 20 40 60
-3.0
-2.4
-1.9
-1.3
-0.7
-0.1
0.4
Diffe
renc
e be
twee
n m
etho
ds
(mm
ol/L
)
Frequency (%)
Zero bias
-60
-40
-20
0
20
40
0 5 10 15 20 25
Radiometer ABL625 Lactate (mmol/L)
Diff
eren
ce b
etw
een
met
hods
(%)
B C
A
D
89
95% confidence intervals for the slope of 0.889 to 0.949 and for the intercept from -0.146 to
0.111. The confidence intervals were narrower than those found for the analysis of umbilical
arterial samples. A fixed bias was identified of -0.367mmol/L with lactate concentrations
reported by the Accutrend® meter being consistently lower that those reported by the
reference method.
2.3.5 Effect of temperature on accuracy
The accuracy in the Accutrend® meter improved when it was used for point-of-care
assessment rather than delayed assessment. The absence of any interaction with haematocrit
suggests that the inaccuracies that occurred in the cord blood analyses (Section 2.3.3) were
related to the delays in analysis that occurred (median delay 11 minutes, inter quartile range
(IQR) 5-25, range 2-132 minutes). During these delays, the samples were kept in foam cups
filled with crushed ice.
To assess the effect of temperature on accuracy, blood with a known lactate concentration
(4.8mmol/L) was assessed simultaneously on both meters, 10 times each, over a temperature
range of 37°C to 0.5°C. The differences between each pair of results are plotted in Figure
2.9A. Over the duration of the repeated analysis (160 minutes), the lactate concentration (as
assessed by the reference methodology) slowly increased from 4.84±0.02 (mean±SEM)
during the 37°C sampling to 5.26±0.02 during the 0.5°C sampling (p<0.001). Over the range
of temperature studies, there was a significant change in the accuracy of the Accutrend®
meter (test method minus reference method; p<0.001). When the sample temperature was
30°C or 37°C, the Accutrend® meter significantly overestimated the lactate concentration
(p<0.004). Over the temperature range 15°C to 25°C inclusive, both methods were in
agreement (p=0.07 to 0.371). When the temperature was decreased to 10°C or below, the
Accutrend® meter significantly underestimated the lactate concentration (p<0.016).
To complete the assessment of the effect of temperature, blood with known lactate
concentrations of 4.8mmol/L and 9.4mmol/L was stored in heparinised syringes in ice slurry
with a temperature of 0.5°C and 20 repeated measurements were performed on both the
Accutrend® meter and the reference method (Figure 2.9B). The chilled samples had
significantly lower lactate levels with the Accutrend® meter than the reference method at
both 4.8mmol/L (mean difference in lactate 1.10mmol/L, p<0.001) and 9.4mmol/L (mean
difference in lactate 2.12mmol/L, p<0.001). The variance in the Accutrend® meter was
significantly greater at 0.5°C than that of the reference method (p<0.001 for both
concentrations).
90
0.5 5 10 15 20 25 30 37-2
-1
0
1
2
* * *
* *
Temperature (°C)
Diff
eren
ce in
Lac
tate
†
(mm
ol/l)
AB
L625
Acc
utre
nd
AB
L625
Acc
utre
nd
0.0
2.5
5.0
7.5
10.04.8 mmol/L 9.4 mmol/L
*
*
Lact
ate
(mm
ol/L
)
A
B
Figure 2.9 The effect of temperature on the accuracy of the Accutrend® lactate meter.
Figure A demonstrates the differences in lactate concentration measured by the Accutrend®
lactate meter and the reference method as a box and whisker plot over the 8 temperatures
assessed († test method minus reference method). Figure B shows the 20 paired analyses at
4.8mmol/L and 9.4mmol/L for both test methods. The asterisk represent p-values <0.05
for repeated measures ANOVA in Figure A and paired t tests for Figure B.
91
2.4 Discussion
The Accutrend® handheld lactate meter measures lactate accurately and precisely in neonatal
plasma and neonatal blood micro samples, providing the sample is analysed at room
temperature. It is significantly cheaper than the reference method (AUS$900 vs.
AUS$80,000) is portable, and requires minimal maintenance; it is ideally suited to point-of-
care analysis of fetal and neonatal blood in both the delivery suite and in the neonatal
intensive care unit.
2.4.1 Reference method precision and accuracy
The reference method used in this study, the Radiometer ABL625® has been found to be
accurate to within ±0.8mmol/L in 95% of samples (Sinn et al., 2001) and it has published
coefficients of variation (CV) that range from 12.2% at 0.9mmol/L, 2.6% at 4.3mmol/L and
2.3% at 12.8mmol/L (Radiometer, 1997). Similar CV’s were found for the Radiometer
ABL625® in this study, however it performed less well at lactate concentrations
approximating 10mmol/L where the CV was found to be 5.1%.
2.4.2 Accutrend® handheld lactate meter precision and accuracy
The Accutrend® meter’s precision compares favourably with the more expensive reference
method and is well within the guidelines defined by the Royal College of Pathologists of
Australia (RCPA) and the Australasian Association of Clinical Biochemists (AACB). The
Altman-Bland difference plots in my study show that 95% of the Accutrend® lactate results
were within ±0.9mmol/L when blood was analysed at the point-of-care. This finding
compares very favourably with the AUS$80,000 Radiometer ABL 625® which when
compared to another gold standard “reference” methods was found to be accurate to within
±0.8mmol/L in 95% of samples (Sinn et al., 2001). The RCPA-AACB quality assurance
program quotes acceptable limits for performance for lactate assessment by any method as
±1.0mmol/L if less than 10mmol/L and 10% if greater than 10mmol/L (RCPA-AACB,
2002).
The Accutrend® handheld lactate meter is significantly more precise than other handheld
lactate meters previously reported which have coefficients of variation ranging from 5.5% to
45.9% over the same range of lactate concentrations used in this study (Nordstrom, 1995). A
new handheld lactate meter (Lactate Pro™) has become commercially available in Australia
92
in 2003. This meter has been compared with the Accutrend® meter (Nordstrom et al.,
1998a) and has coefficients of variation similar to those obtained in this study, however it
analyses samples as small as 5µL. In comparison, the Accutrend® meter is most accurate
with samples of 15-50µL and when presented with smaller samples it provides a result which
significantly underestimates the true lactate levels (Nordstrom et al., 1998a).
Under ideal circumstances, whole blood should be used to assess the precision of the two
lactate testing methods. In this study precision was assessed using plasma samples as the
additives required to inhibit lactate production can damage the electrode used in the
reference technique. Both in this study and the study by Sinn et al. (2001), lactate
concentrations increase when samples are stored at room temperature without additives to
inhibit lactate production. In my study during the temperature analysis, the lactate
concentration increased from 4.84mmol/L to 5.26mmol/L over 160 minutes in partially
chilled samples. Sinn et al. (2001) found that lactate concentrations increased at
0.012mmol/min at room temperature and were stable for 20 minutes with samples kept in
ice slurry. After 20 minutes, lactate levels of samples kept in ice slurry also increased (Sinn et
al., 2001).
2.4.3 Temperature and lactate measurement
The Accutrend® lactate meter provided accurate lactate measurement in neonatal plasma
and neonatal blood providing the sample was analysed at room temperature and the
haematocrit was less than 62%. Similar accuracy was reported by Nordstrom et al. when they
compared the Accutrend® meter to another test-strip based lactate meter (Nordstrom et al.,
1998a).
The reduced accuracy identified in the cord blood study when compared to the neonatal
blood point-of-care study is probably secondary to the effect of temperature as all samples in
the cord study were kept in ice for between 2 to 132 minutes before analysis. Ice was
required because the reference method was in the neonatal intensive care unit, not labour
ward or the operating theatre. The Radiometer ABL 625® blood-gas machine is not subject
to inaccuracies associated with temperature because all samples are warmed within the
machine to 37°C prior to analysis.
The issues of sample temperature is critical for the assessment of both fetal acid-base status
and lactate because deliveries often occur in operating theatres rather than delivery suites, or
the events that are occurring at delivery preclude immediate analysis of cord blood. In both
93
of these situations, samples are typically maintained on ice to minimise changes in gas
tensions, pH and lactate. If one is to use the Accutrend® lactate meter both clinically or for
research, it is optimal that the samples are warmed to at least 10°C prior to analysis if they
have been kept on ice for greater than 5 minutes. This can be easily achieved by warming the
sample in one’s hands for one minute prior to analysis.
There are a number of reasons why cooling of blood may impair the accuracy of the
Accutrend® meter. Cooling of blood increases its viscosity which may interfere with the
process of plasma separation in the lactate test strip. Cooling may also slow diffusion into
the reaction layer of the test strip or slow the enzymatic process that occurs in the reaction
layer of the strip. These possibilities are supported by evidence that the meter tends to
overestimate the lactate concentration if the sample is analysed at temperatures greater than
35°C where the elevated temperature may accelerate the analysis processes in the multilayer
strip.
My study has demonstrated that the ideal utilisation of the Accutrend® lactate meter is at the
point of sample. Such utilisation avoids the effects of temperature and clotting and it affords
optimal performance of the meter. This is supported by the 95% confidence intervals of the
Passing Bablok regression lines assessing the accuracy of the Accutrend® lactate meter. The
confidence intervals were narrower when blood samples were analysed at the point of
sampling rather than when the analysis was delayed. Wider confidence intervals imply greater
inaccuracy for each measurement.
A number of different methods have been used in clinical chemistry to compare a new
method with an established one prior to changing to a new analysis technique (Pollock et al.,
1992). These methods include simple least squares regression analysis which assumes that
the reference method (x variable) has no error, Deming’s regression method which allows for
variance in both variables (Payne, 1985) and graphical difference plots (Bland & Altman,
1986). In this study, in addition to the difference plots, the Passing Bablok regression
method has been utilised as it is a more reliable method of comparability than simple linear
regression or Deming’s regression. Passing Bablok regression is similar to Deming’s
regression where variance is present in both variables. Unlike Deming’s regression, Passing
Bablok regression assumes constant variance over the range of measurements and the
regression line is not excessively biased by extreme values (Passing & Bablok, 1983; Bablok et
al., 1988).
94
2.4.4 Haematocrit and lactate measurement
There are a number of factors which alter fetal haematocrit during pregnancy. First, with
advancing gestational age, fetal red cell count increases whereas white cell count and platelet
count do not change (Forrestier, 1987). This results in the fetal haematocrit increasing with
advancing gestational age from 34% (95%CI 31-37%) at 20 weeks gestation to 41% (95%CI
38-44%) at 40 weeks (Weiner et al., 1991). Second, growth restricted fetuses, which are at
increased risk of adverse events during labour (Low et al., 1976; Nieto et al., 1994; Dashe et al.,
2000; Bukowski et al., 2003), may experience chronic hypoxaemia in utero and they often
develop haemoconcentration with a resulting increase in their haematocrit (Soothil et al.,
1987; Economides & Nicolaides, 1990). Third, in the course of normal labour, haemo-
concentration has been described in fetal scalp blood (Jacobsen & Rooth, 1971). This effect
is probably due to loss of water from the fetal scalp with the average haemoglobin
concentration measured in scalp blood being 24g/L (Jacobsen & Rooth, 1971). Owing to
plasma reduction, fetal blood volume is reduced by 14% during labour when compared with
pre-labour values (Brace, 1992). It is unknown if this effect is more pronounced in
prolonged labour although it is doubtful whether the increase would be great enough to
significantly affect lactate measurement. Therefore, as seen in my study, there can be a wide
range of haematocrits in blood samples collected around delivery and when considering a
device to measure fetal lactate levels during the perinatal period, it is important to confirm
that accuracy is not affected by changes in haematocrit.
In this study the accuracy of the Accutrend® lactate meter was maintained across a
haematocrit range of 41.5 to 62% in umbilical cord blood. In contrast, other authors have
previously reported a deleterious effect of haematocrit on the accuracy of different handheld
lactate meters to the one assessed in this study (Shimojo et al., 1993; Nordstrom et al., 1998a).
They found that the meters they assessed reported lower lactate concentrations than the
reference method when haematocrit values were greater than 50%. This would limit the
clinical value of these meters as a significant proportion of blood samples in fetal and
neonatal blood (63% in this series) have haematocrit values greater than 50%. As with the
effect of temperature, it has been suggested the reduced accuracy at high haematocrits may
be due to slower diffusion into the reaction layer of the test strip (Nordstrom, 1995). The
Accutrend® lactate meter may be less affected by haematocrit than other meters due to the
different structure of the test strip that has a thin glass fibre carpet to separate plasma from
red cells. In this study, the accuracy of the Accutrend® lactate meter did not change until
95
the haematocrit was greater than 62%, which occurred in 2.5% of the consecutive arterial
samples collected from umbilical cords at delivery across a gestational age from 26 to 43
weeks.
2.4.5 Lactate measurement bias
In this study the Accutrend® meter consistently underestimated lactate levels by
0.367mmol/L relative to the reference method. This bias however was constant across the
range of lactate concentrations assessed. Similar bias has been identified for other methods
of lactate measurement (Nordstrom et al., 1998a; Sinn et al., 2001). It is imperative that if
using the Accutrend® meter clinically, appropriate reference ranges are utilised, preferably
determined with the same testing methodology. The published normal reference ranges for
umbilical arterial lactate after labour vary widely depending on the type of sample analysed,
the method used to measure each sample and the number of cases in each series (range 14 to
3932; Table 2.4). Many of the published reference ranges are for whole blood, which is
expected to yield values 40-50% lower than those obtained by analysing plasma from the
same specimen.
Table 2.4 Normal reference range for umbilical arterial lactate after labour
Lactate (mmol/L)
(mean)
+2SD (95th centile)
Sample type Reference
2.8 N.A. N.A. Rooth & Nilsson, 1964
2.7 5.1 N.A. Jouppila & Hollmen, 1976
3.3 6.1 Haemolysed blood Low et al., 1974
3.5 7.3 Haemolysed blood Smith et al., 1983
2.6 5.0 Haemolysed blood Suidan & Young, 1984
2.9 5.4 Haemolysed blood Ruth & Raivio, 1988
3.7 6.1 Whole blood Nordstrom et al. 1994
1.9 3.8 Whole blood Westgren et al., 1995
96
2.5 Conclusion
The Accutrend® handheld lactate meter provides an affordable, commercially available
means of measuring plasma lactate levels in only 60 seconds at the point-of-care. It requires
only 15µl of blood and is significantly cheaper than other assay methods.
The precision of the Accutrend® handheld lactate meter compares favourably with more
expensive machines and is well within the guidelines prescribed by the Royal College of
Pathologists of Australasia and the Australasian Association of Clinical Biochemists. I have
shown that the accuracy of the Accutrend® handheld lactate is acceptable for clinical and
research use providing that samples are analysed at room temperature and that the
haematocrit is less than 62%.
The Accutrend® handheld lactate meter is well suited to assess lactic acidaemia in small
samples to help quantify hypoxic stress in the perinatal period.
97
C h a p t e r 3
THE RELATIONSHIP BETWEEN NEUROPATHOLOGIC
DAMAGE AND BIOCHEMICAL AND PHYSIOLOGIC MARKERS
OF ASPHYXIA INDUCED BY GRADED CORD OCCLUSION IN
CHRONICALLY CANNULATED OVINE FETUSES
In seeking absolute truth we aim at the unattainable,
and must be content with finding broken portions.
Sir William Osler (1889)
3.1 Introduction
Until recently, hypoxic-ischaemic events in the perinatal period were assumed to be the main
cause of early neonatal encephalopathy and seizures. They were also thought to be the major
cause of brain damage in infants with a clinical diagnosis of cerebral palsy. These theories
were challenged during the last decade by epidemiological studies showing that there was
often an absence of evidence of severe intrapartum asphyxia in infants with neonatal
encephalopathy, and conversely, many infants who do have signs of fetal distress and birth
asphyxia do not develop neurologic sequelae (Blair & Stanley, 1988; Nelson & Leviton, 1991;
Yudkin et al., 1994; Badawi et al., 1998a; Nelson & Grether, 1998). These observations,
together with epidemiological evidence that there is a higher incidence of maternal illness,
antenatal complications, and adverse social factors in infants with neonatal encephalopathy,
seizures or both, have led to the view that the main causes of such conditions occur before
birth (Badawi et al., 1998b). These epidemiological studies suggest that in about 90% of cases
intrapartum hypoxia could not be the cause of cerebral palsy and that in the remaining 10%,
intrapartum signs compatible with damaging hypoxia may have had antenatal or intrapartum
origins (Nelson, 1988; Yudkin et al., 1995).
A number of recent studies however, provide support to the original hypothesis that events
in the immediate perinatal period are the most important in neonatal brain injury in the term
infant. Hagberg et al. studied the prevalence, cause and timing of cerebral palsy in a
population based study in Sweden and reported a perinatal origin for cerebral palsy in 36% of
98
affected term infants (Hagberg et al., 2001). Similarly in a study of 351 term infants with
neonatal encephalopathy, seizures or both, magnetic resonance imaging found evidence of an
acute insult without established injury or atrophy in 80% of infants with neonatal
encephalopathy and 69% of infants with seizures (Cowan et al., 2003). When these infants
were followed up for a minimum of 18 months, 19% (66/351) and 24% (85/351) had
evidence of cerebral palsy (Cowan et al., 2003).
The goal of intrapartum fetal surveillance is to reduce the incidence of fetal asphyxial
exposure and to prevent moderate and severe asphyxia. The three techniques which are
currently widely used for this surveillance are electronic fetal heart rate monitoring (EFM),
scalp blood gas analysis and umbilical cord blood gas analysis after delivery.
Continuous EFM has been adopted widely since its development in 1968. Thirteen
randomized clinical trials have addressed the efficacy and safety of EFM comparing EFM to
intermittent auscultation (Haverkamp et al., 1976; Renou et al., 1976; Kelso et al., 1978;
Haverkamp et al., 1979; Langendoerfer et al., 1980; Wood et al., 1981; MacDonald et al., 1985;
Neldam et al., 1986; Luthy et al., 1987; Shy et al., 1990; Vintzileos et al., 1993; Herbst &
Ingemarsson, 1994; Mahomed et al., 1994). Meta-analysis of nine of these trials found a 50%
reduction in neonatal seizures in the continuous EFM group but no significant difference in
rate of admission to neonatal intensive care units, cerebral palsy or perinatal death (Thacker et
al., 2001). Late decelerations are the only fetal heart rate (FHR) pattern that has been noted
to correlate with neonatal neurologic examination in the first week of life (Ellison et al., 1991).
Fetal blood sampling for pH measurement was introduced into clinical practice by Saling in
1962 as the first invasive fetal monitoring tool (Saling, 1962). Although it has been widely
advocated to improve the specificity of FHR monitoring (Clark & Paul, 1985; Low et al.,
2001; Low et al., 2002) its utilisation varies widely, partly due to the restricted availability of
the equipment required to perform the test and also because of its invasive nature.
Based on current published literature, umbilical cord acid-base assessment at delivery
provides the most sensitive reflection of birth asphyxia (MacLennan, 1999). There are
several reasons for performing routine cord blood acid-base balance at delivery: a quality
assessment of obstetric practice; a diagnostic test for hypoxia in cases with depressed
newborns; and for the prediction of neonatal complications and long-term outcome.
Although the presence of metabolic acidaemia does not define the timing of onset of birth
asphyxia, its absence excludes an acute intrapartum hypoxic event (MacLennan, 1999).
99
Two percent of fetuses experience an asphyxial exposure during labour and delivery as
demonstrated by an umbilical artery base deficit greater than 12 mmol/L (Low et al., 1994).
Most of these events are mild with little short or long-term significance (Gilstrap et al., 1989;
Low, 1997). More severe asphyxial exposures are associated with a range of outcomes.
Pelowski and Finer (1992), in a review of five studies of term newborns, reported a good
outcome for newborns with minor encephalopathy. The risk of death and severe handicap
in moderate encephalopathy was 5.6% and 20% respectively, and in severe neonatal
encephalopathy, 60% and 70% respectively. A recent study of 276 term newborns with
moderate or severe encephalopathy reported similar findings with 25% and 62% respectively
having poor outcomes (Table 3.1, adapted from Dixon et al., 2002).
Table 3.1 Outcome after term neonatal encephalopathy
Outcome Moderate Neonatal Encephalopathy
Severe Neonatal Encephalopathy
Died 3% 31%
Cerebral palsy then died 3% 2%
Cerebral palsy surviving 7% 14%
Abnormal cognitive impairment or developmental delay 12% 15%
Normal 75% 38%
Adapted from Dixon et al, 2002
Our principal biochemical markers of perinatal asphyxia, fetal scalp and umbilical cord acid-
base status, are limited because they reflect fetal metabolic adjustments that are not directly
related to fetal damage except if values are extreme (Gilstrap et al., 1989; Goldaber et al., 1991;
Winkler et al., 1991). Even a pH of less than 7.0 is poorly predictive of neurological deficit
because a severely acidotic infant will usually have a normal outcome (Winkler et al., 1991;
Socol et al., 1994; Yudkin et al., 1994). The presence of acidosis, reflected by a fall in pH, may
reflect the physiological switch to anaerobic metabolism which is a healthy response to
hypoxia, and paradoxically it may be the fetuses that are unable to mount this response and
100
therefore have “normal” umbilical gases in the face of hypoxia who suffer long-term
neurological sequelae (Dennis et al., 1989; Westgren et al., 1999).
There is growing evidence that lactate, the end product of anaerobic metabolism of glucose,
has a role in the assessment of the fetus in labour and the neonatal period. In human studies,
intrapartum scalp lactate assessment has been shown to be a more sensitive diagnostic tool
than the measurement of scalp pH values for predicting either an Apgar score <4 at five
minutes of age or moderate to severe neonatal encephalopathy (Kruger et al., 1999).
Similarly, urinary lactate-creatinine ratios at six hours of age assessed by proton nuclear
magnetic resonance spectroscopy (MRS) have both high sensitivity (94%) and specificity
(100%) for predicting hypoxic-ischaemic encephalopathy in a group of babies with perinatal
asphyxia (Huang et al., 1999).
3.1.1 Aim
The aim of this study was to use an ovine model of repeated umbilical cord occlusion (UCO)
to evaluate the relationship between fetal lactate responses and fetal brain injury. To address
this aim I have utilized an UCO protocol designed to mimic events which may occur in
labour to compare the relationship between:
1. fetal acid-base status and neuropathologic damage,
2. fetal lactate and neuropathologic damage,
3. fetal heart rate patterns (FHR) and neuropathologic damage, and
4. fetal arterial pressure and neuropathologic damage.
3.1.2 Hypothesis
Fetal lactate is a better predictor of neuropathologic injury after repeated UCO than fetal
heart rate patterns and fetal acid-base status.
101
3.2 Methods
All experimental procedures were approved by the Wentworth Area Health Service Scientific
Advisory Panel and Ethics Committee and the Westmead Hospital Animal Ethics
Committee, New South Wales, Australia. Merino ewes with singleton pregnancies were
transported to the research facility at 120 days of pregnancy to allow for acclimatization.
The timeline for the protocol utilised in this study is presented in Figure 3.1
3.2.1 Surgical procedure
Sixteen ewes underwent surgery at 126±3 days (mean±SEM) of pregnancy. Anaesthesia was
induced with Thiopentone 4mg/kg in 15mL saline solution (Abbott Australasia Pty Ltd,
Cronulla, NSW, Australia), ewes were intubated, and anaesthesia was maintained with 0.75-
1.5% Isoflurane in 100% oxygen delivered by positive pressure ventilation with the ventilator
settings selected to maintain maternal blood gases in the normal range.
The uterus was exposed through a paramedian abdominal incision and a hysterotomy was
performed to deliver the fetal head, neck and upper thorax through a small incision with care
to minimize amniotic fluid leakage. Vascular catheters were inserted into the left fetal carotid
artery and jugular vein and an amniotic catheter was inserted. Fetal electrocardiograph
(ECG) leads were sutured to the fetal chest and a vascular snare was sutured around the
umbilical cord to enable intermittent complete cord occlusion. A circular three crystal
ultrasound cuff (10MHz Triton Technology Inc., USA) was loosely applied around the
umbilical vessels. After replacement of the fetus into the uterus, Benacillin (1mL procaine
penicillin, 150mg/mL; benzathine penicillin, 150mg/mL; procaine hydrochloride, 20mg/mL;
Troy Laboratories, Smithfield, Australia) was injected into the amniotic fluid. The uterus was
closed and catheters were tunnelled subcutaneously to the ewe’s flank. The maternal
abdomen was closed and 3-mL intramuscular Benacillin was given to the ewe at completion
of the surgery. After surgery, sheep were held in individual pens with access to food and
water. Two hours after surgery, fetal blood samples (3.5mL) were collected for baseline
measurement of arterial gas tensions and pH (Radiometer ABL 300, Radiometer Pty Ltd,
Copenhagen, Denmark), lactate (Accutrend® Lactate, Roche Diagnostics GmbH,
Mannheim, Germany), glucose, cortisol, haemoglobin, and catecholamine concentrations.
Two days after surgery, fetal blood was also collected (0.3mL) for measurement of arterial gas
tensions, pH and lactate measurement. After all fetal blood samples were withdrawn sterile
heparinised saline was infused to replace the volume removed for analysis.
102
Figure 3.1 Experimental timeline for the entire study period and below, the cord occlusion protocol
during the experimental phase of the study. Red bars during the experimental phase indicate both the
timing and the duration of the cord occlusions. The first nine occlusions were for 0.5 minutes
duration every 3 minutes; the second nine occlusions were 1.0 minutes duration every 3 minutes; and
the third nine occlusions were for 1.5 minutes duration every 3 minutes. This figure demonstrates the
progressive shortening of the interval between occlusions.
ExperimentalPhase
0 1 2 3 4 5 6 7
SurgeryCord
Occlusion Delivery
SampleSamplesSample Sample
Days
0 3 6 9 12 15 18 21 24 27
Sample SampleSample
27 30 33 36 39 42 45 48 51 54
Sample SampleSample
54 57 60 63 66 69 72 75 78 81
Sample SampleSample
Minutes
Study Period
103
3.2.2 Cord occlusion protocol
After a four day recovery period (130±3 days gestation) the experimental phase of the study
commenced. After a 60-minute control period, baseline fetal blood samples (3.5mL) were
collected for measurement of gas tensions, pH, lactate, glucose, cortisol and catecholamine
concentrations. Twelve fetuses were then randomly allocated to the cord occlusion protocol
which was designed to mimic events that may occur in labour (Figure 3.1). The remaining 4
fetuses served as controls with blood samples and other measurements taken as scheduled
for the experimental group but without UCO. All studies were performed with the ewe
unrestrained with free access to food and water.
For those fetuses that underwent the UCO protocol the umbilical cord was completely
occluded every 3 minutes for a total of 81 minutes. The first nine occlusions each lasted for
0.5 minutes every three minutes; the second series of nine occlusions were for 1.0 minutes
every 3 minutes; and the final series of nine occlusions were for 1.5 minutes every three
minutes. Fetal carotid arterial blood samples (0.3mL) were collected for measurement of gas
tensions, pH, and lactate levels every nine minutes over the 81 minute experimental phase.
Additional arterial samples (1.0mL) were collected at 27 and 54 minutes to measure fetal
glucose. Arterial blood samples (3.5mL) were collected from all fetuses at the end of the
occlusion phase for the assessment of fetal catecholamine, cortisol and glucose
concentrations. The cord occlusion protocol was terminated early, prior to 81 minutes, if any
of the following were observed; arterial pH levels less than 6.9; base excess levels less than
-20mmol/L; persistent fetal hypotension; or prolonged fetal bradycardia.
3.2.3 Fetal heart rate assessment
Fetal ECG was recorded continuously onto a computer at 1 KHz after amplification (Bio
amp, PowerLab 8e, ADInstruments, Sydney, Australia) during the cord occlusion protocol.
Fetal heart rate (FHR) was calculated by measuring every RR interval on the fetal ECG.
Over the 60 second period immediately before each cord occlusion, three measures of FHR
variation were assessed; mean minute range (MMR), short term variation (STV) and long
term variability (LTV). Both MMR and STV were calculated as previously described (Dawes
et al., 1991; Street et al., 1991). Mean minute range was calculated as the difference between
the maximum and minimum R-R intervals for each minute and short term variation was
calculated as the average of successive epochal R-R interval differences (Epoch = 1/16th-
minute {3.75 second}). Long term FHR variation was defined as the difference in the
maximum and minimum fetal heart rate during each 60 second block (NICH, 1997; RCOG,
104
2001). I also visually confirmed the FHR variability, a method which has previously been
shown to relate directly to mathematical quantitation (Knopf et al., 1991). FHR traces were
visually assessed to classify the fetal heart rate pattern during each of the 27 cord occlusions.
All measurements were made blind to the treatment groups. The baseline FHR during the
experimental phase was the mean FHR over the one minute prior to the cord occlusion.
Accelerations were defined as an abrupt increase in FHR above the baseline of more than 15
beats and lasting longer than 15 seconds (NICH, 1997). Variable decelerations were defined
as an abrupt decrease in FHR below the baseline, which was ≥15 beats/min, lasting ≥15
seconds, followed by an abrupt return to baseline (NICH, 1997; RCOG, 2001). Severe
variable decelerations were defined as variable decelerations with depth greater than 60 beats
per min below the baseline FHR. Given that there were no uterine contractions in this study,
the traditional definition of a late deceleration was not appropriate in this context. A late
component to the decelerations was therefore defined as FHR decelerations that had not
returned to baseline within 15 seconds of the cessation of cord occlusion. Overshoot
instability was defined as a rebound increase in FHR immediately after the release of the cord
occlusion. These were at least 15 beats above the baseline FHR and their duration was >15
seconds, after which the FHR returned to the baseline or another deceleration. Increased
FHR variability was defined as long term FHR variability which was greater than 25 beats per
min.
3.2.4 Fetal arterial pressure assessment
Fetal arterial pressure was recorded continuously onto a computer at 1 KHz after
amplification (BP transducer MLT380, bridge amplifier ML110, PowerLab 8e,
ADInstruments, Sydney, Australia) during the cord occlusion protocol. Fetal arterial pressure
was normalised for amniotic fluid pressure and responses to repeated UCO were assessed
during and between cord occlusions. For assessment during the resting phase between
occlusions, systolic, diastolic and mean arterial pressure were measured for each heart beat
and averaged for the 1 minute preceding each cord occlusion.
3.2.5 Umbilical blood flow
The ultrasound cuff around the umbilical vessels was linked to a vascular spectrum analyser
(Model SP 25 by Vasculab, Meda Sonics, USA) which enabled continuous monitoring of the
umbilical artery Doppler waveforms to confirm normal umbilical blood flow between
occlusions and complete obstruction of umbilical blood flow during cord occlusion.
105
3.2.6 Biochemical assessment
Plasma samples for glucose, cortisol and catecholamine measurement were recovered after
centrifugation at 1500g for 10 minutes at 4°C and were stored at -80°C until analysed.
Plasma adrenaline, noradrenaline and dopamine were measured by electrochemical detection
after high performance liquid chromatography (HPLC) fractionation. Fetal cortisol
concentrations were measured by solid-phase chemi-luminescent immunoassay (DPC
Immulite System; Diagnostic Products Corporation, Los Angeles, USA). The intra-assay
coefficient of variation was 6.8% and inter-assay 9.9%. This immunoassay has been validated
in human, dog, cat, horse, sheep and dolphin plasma (Singh et al., 1997; Ma, 1999; Beetson,
2001). Catecholamine measurements were performed by the renal research laboratory of
King George V Hospital, Camperdown, Sydney. The HPLC technique utilised was the same
as that previously described and validated in fetal sheep plasma by others (Murotsuki et al.,
1997b).
3.2.7 Delivery and neuropathologic examination
Seventy two hours after the experimental phase of the study, fetal blood samples (3.5mL)
were collected for measurement of arterial gas tensions, pH and lactate, glucose, cortisol,
catecholamine and haemoglobin concentrations. Ewes were then anaesthetised (as above)
and the fetus was delivered and injected with a lethal dose of phenobarbitone. Brains were
perfused with ice-cold heparinized saline solution through newly inserted catheters in both
carotid arteries, and then with 10% buffered formalin. After perfusion the fetal brain was
carefully removed, sectioned and blocked at multiple levels including representative blocks of
all cortical regions of the cerebrum, basal ganglia, thalamus, brain stem and cerebellum.
Blocks were then embedded in paraffin and 6 to 8µm sections were cut and stained with
hematoxylin and eosin, Luxol fast blue-cresyl violet, Bielschowsky, glial fibrillary acidic
protein, myelin basic protein, HAM-56, or vimentin. The severity of histologic changes was
classified into five grades for 9 areas on each side of the fetal brain using a classification
system previously described (Ikeda et al., 1998a) and summarized in Table 3.2.
All neuroanatomical assessments were performed by a neuropathologist (Dr Thomas Ng)
who was blinded to experimental group and biochemical data. The grade attributed to each
hemisphere was the highest grade found in any of the nine areas studied.
106
Table 3.2 Grading of histopathologic changes in fetal brains Grade 0 Intact white matter
Intact gray matter, including that of hippocampus
Grade 1 Random or focal vacuolisation and loss of myelin or increase in cellularity (macrophages, astrocytes) primarily in white matter at its junction with cerebral cortex
Intact gray matter, including that of hippocampus
Grade 2 More widespread involvement of subcortical white matter with increase in cellularity with loss of myelin sheaths
Intact gray matter, including that of hippocampus
Grade 3 Focal necrosis of subependymal white matter with or without cystic change (random)
Random or focal scattered ischaemic neuronal change in cerebral cortex (minimal)
Grade 4 More extensive change within white matter, including that of basal ganglia, thalamus, and other gray matter structures (increase in cellularity, loss of myelin)
Well-defined infarcts of gray and white matter (random)
Grade 5 Extensive necrosis involving both gray and white matter with marked macrophagic and astroglial reaction
(Adapted from Ikeda et al., 1998a)
3.2.8 Statistical methods
Descriptive data are presented as mean and standard error of the mean (SEM) unless
otherwise stated. Univariate analyses of categorical data were performed with Fisher’s exact
test and continuous data were analysed with t-tests or the non-parametric equivalent as
appropriate.
Correlations between the three measures of FHR variability were assessed using regression
modelling considering experimental group and time as fixed effects, and individual animals as
random effects repeated over time. Correlations between the grade of neuronal injury and
the duration of FHR patterns, arterial pressure responses and biochemical markers were
performed using Spearman rank correlation test.
Changes in fetal biochemical variables and cardiovascular parameters were analysed using
multivariate regression modelling with occlusion and time modelled as a fixed effects and
individual animals as random effects repeated over time.
107
Receiver operator curve (ROC) analysis has been used, both in this chapter and subsequent
chapters, to assess the ability of biochemical measures to predict categorical outcomes. The
ROC is a technique to visualise and quantify the effectiveness of a procedure by graphing the
true positive rate (sensitivity) against the false positive rate (1-specificity). The ROC curve
depicts the relationship between sensitivity and specificity for all allowable cut-off points
(Harding et al., 1995). The incorrectly classified non-diseased cases (1-specificity) are plotted
as the x-coordinate and sensitivity as the y-coordinate. The point (0,1) is called an ideal point
of the test as it reflects perfect classification while the line through points (0,0) and (1,1)
reflects a random classification rule (no discrimination line) (Chmura Kraemer, 1992). A
diagnostic test no better than chance alone would have a ROC curve positioned close to the
no discrimination line. Curves above and to the left of this random line reflect better
discrimination ability that is usually assess by the area under the curve. Generally areas under
the curve of 90-100% are considered excellent; those between 80-90% are considered good,
while those of approximately 50% indicate chance alone.
In this chapter, sensitivity and specificity were calculated for the ability of specific FHR
patterns to predict each pattern of fetal arterial pressure response during UCO. Receiver
operator curve analysis was used to assess the ability of biochemical measures to predict fetal
arterial pressure responses during UCO (Hanley & McNeil, 1983; Beck & Shultz, 1986;
DeLong et al., 1988). Optimum sensitivity and specificity were chosen from the ROC tables
to maximize the total of these two measures of diagnostic accuracy (Hanley & McNeil, 1983).
Accuracy is reported for biochemical prediction of arterial pressure responses during UCO
and is defined as the proportion of correctly classified cases (true positives + true negatives)
using the biochemical cut-off point selected to maximise the sensitivity and specificity (Zweig
& Campbell, 1993).
SAS statistical software was used for data analysis (Statistical Analysis Systems Institute Inc.
1999, Version 8.2., Cary, NC). P-values <0.05 were considered statistically significant, and p-
values <0.10 were considered marginally significant. A conservative significance level of 0.10
is often used while performing tests on small sample sizes which are often associated with
low power to detect differences. This allows the identification of potential effects which may
be worthwhile investigating in larger studies (Woodward, 1999).
108
3.3 Results
For consistency in the description of results, the total seven day period of this study will be
referred to as the “study period” and the two hours of intensive fetal assessment, when 12 of
the fetuses underwent UCO and four fetuses were studied as controls, will be referred to as
the “experimental phase” (Figure 3.1).
3.3.1 Biochemical responses to repeated cord occlusion
3.3.1.1 Fetal arterial blood gas and lactate responses during umbilical cord
occlusion
In controls there were no significant changes in fetal arterial pH, PCO2, or base excess over
the 7 days of the experimental protocol (all p>0.778; Figure 3.2). Fetal PO2 did not change
during the 81 minutes of the experimental phase in controls (p=0.369); however there was a
significant fall in PO2 in controls three days after the experiment (experimental phase PO2
22.9±0.7mmHg; day 7, 11.7±2.8mmHg, p=0.026).
Fetal lactate levels were stable during the experimental protocol in controls (1.5±0.1mmol/L,
p=0.108); however, at the time of catheter implantation (day 0) and at delivery (day 7), lactate
concentrations were slightly increased compared with the levels during the experimental
phase (day 0, 4.4±0.4mmol/L; day 7, 3.4±0.9mmol/L; p<0.001 for both). A significant fall
in fetal glucose concentrations occurred between the time of catheter implantation and
commencement of the experimental phase (day 0, 1.72±0.30mmol/L; day 4, 0.95±0.13
mmol/L, p<0.001) but after this time there were no significant changes in fetal glucose
concentrations (p=0.875).
Nine of the twelve fetuses that underwent the repetitive UCO protocol completed the 81
minutes of intermittent occlusion and these have arbitrarily been called regular responders.
In three of the twelve fetuses that underwent UCO protocol was terminated at 54 (n=1) or
57 minutes (n=2) respectively when physiologic variables crossed the predetermined end-
points for terminating the protocol. These fetuses have arbitrarily been classified as fast
responders. Despite ceasing the cord occlusion protocol early, none of the fast responders
was alive at the time of delivery 72 hours later whereas all but one of the regular responders
and all controls were alive at delivery.
109
-4-26.8
7.0
7.2
7.4
7.6
Control
0 27 54 81 100
Regular(p=0.004)
Fast(p<0.001)
3
pH
Days Minutes Days
Arte
rial p
H
-4-20
10
20
30
40
0 27 54 81 100
Control
Regular(p=0.142)
Fast(p<0.001)
3
PO2
Days Minutes Days
Arte
rial P
O2
(mm
Hg)
-4-20
25
50
75
100
125
0 27 54 81 100
Control
Regular(p=0.171)
Fast(p<0.001)
3
PCO2
Days Minutes Days
Arte
rial P
CO
2 (m
mH
g)
-4-2-30
-20
-10
0
10
0 27 54 81 100
Control
Regular(p<0.001)
Fast(p<0.001)
3
BE
Days Minutes DaysAr
teria
l Bas
e Ex
cess
-4-20
5
10
15
20
0 27 54 81 100
Control
Regular(p<0.001)
Fast(p=0.016)
3
Lact
Days Minutes Days
Arte
rial L
acta
te(m
mol
/L)
-4-20
1
2
3
0 27 54 81 100
Control
Regular(p=0.003)
Fast(p=0.002)
3
Gluc
Days Minutes Days
Arte
rial G
luco
se(m
mol
/L)
Figure 3.2 Fetal arterial gas tensions, pH, base excess, lactate and glucose (mean± SEM) for
control (open circles, n=4), regular responders (closed circles, n=9) and fast responders
(closed triangles, dashed line, n=3) over the seven day study period. P values were calculated
using repeated measures ANOVA comparing the occlusion groups to the control group.
Significant changes were identified for changes in fetal arterial gas tensions, pH, base excess,
lactate, and glucose for the fast responders and for pH, base excess, lactate and glucose for
the regular responders.
110
Regular responders had significant falls in fetal pH (7.40±0.01 to 7.08±0.03, p<0.001) and
base excess (-0.59±1.35 to -14.73±1.30, p<0.001) and significant increases in fetal lactate
(1.48±0.25 to 10.40±1.47mmol/L, p<0.001) and glucose (0.76±0.09 to 1.34±0.29mmol/L,
p=0.003) during repeated cord occlusion relative to controls (Figure 3.2). Fetal PO2 fell and
PCO2 increased during the experimental phase but these differences did not reach statistical
significance (p=0.142 and p=0.171 respectively).
Fast responders had significant changes in pH, PO2, PCO2, base excess, lactate and glucose
when compared to controls (all p<0.016). When compared with regular responders, fast
responders had significantly greater falls in fetal pH (p<0.001) and base excess (p=0.011).
Lactate, glucose and PCO2 were higher in fast responders than regular responders but these
differences did not reach statistical significance (all p>0.200).
3.3.1.2 Fetal glucose, cortisol and catecholamine responses during umbilical cord
occlusion
There were no significant changes in fetal cortisol, noradrenaline, adrenaline or dopamine
levels in the control fetuses over the seven day study period (all p >0.442; Figure 3.3).
Regular responders had significantly higher cortisol concentrations than controls (Figure 3.3)
after repeated cord occlusion (regular responders cortisol 24.79±4.472ng/mL; vs. control
12.75±1.58ng/mL, p=0.001). Fetal noradrenaline, adrenaline and dopamine concentrations
at the end of the experimental phase were higher than in controls but these differences did
not reach statistical significance (noradrenaline regular responders 114.21±31.39ng/L vs.
control 1.80±0.90ng/L, p=0.055; adrenaline regular responders 88.39±42.71ng/L vs. control
1.10±0.35ng/L, p=0.082; dopamine regular responders 1.03±0.92ng/L vs. 0.00±0ng/L,
p=0.170). In contrast, fast responders had significantly higher cortisol, noradrenaline,
adrenaline and dopamine concentrations than controls by 57 minutes when their UCO
protocol was discontinued (all p<0.030).
3.3.1.3 Fetal haemoglobin response to experimental protocol
During the seven days of the experimental protocol, a total of 19.0mL of fetal blood was
sampled for analysis. Each time the fetus was sampled, an equal volume of normal saline was
replaced. No significant difference was found between baseline fetal haemoglobin (day 0)
and haemoglobin collected prior to delivery (day 7; mean difference 0.27 g/L, 95%CI -0.57
to 1.10, p=0.495). Paired haemoglobin concentrations are presented in Figure 3.4.
111
-4-20
10
20
30
40
Control
0 27 54 81 100
Regular(p=0.001)
3
Fast(p<0.001)
Days Minutes Days
Cortisol
Cor
tisol
(ng/
mL)
-4-20
100
200
300
Control
0 27 54 81 100
Regular(p=0.055)
Fast(p=0.033)
3
Days Minutes Days
Noradrenaline
Nor
adre
nalin
e (n
g/L)
-4-20
25
50
75
100
125
Fast(p<0.001)
0 27 54 81 100
Regular(p=0.082)
Control
3
Days Minutes Days
Adrenaline
Adre
nalin
e (n
g/L)
-4-20
1
2
3
4
5
Fast(p<0.001)
0 27 54 81 100
Regular(p=0.170)
Control
3
Days Minutes Days
Dopamine
Dop
amin
e (n
g/L)
Figure 3.3 Fetal cortisol, noradrenaline, adrenaline and dopamine concentrations (mean±
SEM) for control (open circles, n=4), regular responders (closed circles, n=9) and fast
responders (closed triangles, dashed line, n=3) over the seven day study period. P values
were calculated using repeated measures ANOVA comparing the occlusion groups to the
control group. Regular responders had significantly increased cortisol concentrations
(p=0.001) and non-significant increases in noradrenaline (p=0.055), adrenaline (p=0.082) and
dopamine (p=0.170) after 81 minutes of cord occlusion. The fast responders had significant
increases in cortisol and all catecholamines after 54 minutes of cord occlusion (p<0.033).
112
0.0
2.5
5.0
7.5
10.0
12.5
Day 0 Day 7
p=0.495
Hae
mog
lobi
n (g
/L)
Figure 3.4 Fetal haemoglobin responses to experimental protocol. Paired haemoglobin
concentrations are presented on day 0 of the experimental protocol (collected after
instrumentation) and day 7 of the experimental protocol (collected prior to delivery). Paired
analysis found no significant decrease in haemoglobin over the duration of the protocol.
113
3.3.1.4 Biochemical prediction of fetal response to umbilical cord occlusion
Arterial gas tensions, pH, cortisol and catecholamine concentrations collected over the four
days prior to the experimental phase were not different between regular and fast responders
(p=0.233 to p=0.960); however, there were differences in fetal lactate and glucose levels
between groups (Figure 3.5). Fast responders had significantly higher lactate concentrations
(fast responders lactate 6.81±1.19mmol/L vs. regular 2.29±0.82mmol/L, p=0.011), and
higher glucose concentrations (fast responder glucose 1.38±0.19mmol/L vs. regular
0.78±0.14mmol/L, group effect p=0.094) than regular responders over the four days prior to
the experimental phase.
3.3.2 Fetal heart rate responses to repeated cord occlusion
3.3.2.1 Baseline FHR response to repeated cord occlusion
Baseline FHR did not change over the duration of the experimental phase in controls
(182.5±9.7 beats per minute (bpm), p=0.816; Figure 3.6A). In the fetuses that underwent
UCO the baseline FHR varied during the occlusion protocol (p=0.055) and was significantly
different from controls (p=0.003). There was a significant decrease in baseline FHR after
commencement of UCO (p=0.043) followed by a progressive increase in baseline FHR to
levels higher than before the commencement of the UCO (Figure 3.6B). Unlike the
biochemical markers of fetal condition, there was no difference in the baseline FHR response
to UCO between regular and fast responders (p=0.943; Figure 3.6C)
3.3.2.2 Fetal heart rate variability response to repeated cord occlusion
The three measures of FHR variability did not change significantly during the experimental
phase in controls (MMR 46.67±1.67 msec, p=0.999; STV 3.19±1.17msec, p=0.999; LTV
21.67±2.19bpm, p=0.287; Figure 3.7). In fetuses that underwent UCO there was a
significant increase in all three measures of FHR variability during the experimental phase
when compared to controls (MMR, p=0.028; STV, p=0.050; and LTV p<0.001). Statistically
significant increases in MMR occurred after 42 minutes of UCO (p<0.05) and significant
increases in STV and LTV occurred after 45 minutes (p<0.05) and 36 minutes respectively
(p<0.05). Fetal heart rate variability as indicated by MMR and LTV appeared to decrease at
the end of the experimental phase in the fetuses that were subject to UCO with the
differences between occluded and control fetuses not reaching statistical significance.
114
Pre-occlusion Lactate
Control Regular Fast0.0
2.5
5.0
7.5
10.0 p=0.011
Lact
ate
(mm
ol/L
)
Pre-occlusion Glucose
Control Regular Fast0.0
0.5
1.0
1.5
2.0 p=0.064
Glu
cose
(mm
ol/L
)
Figure 3.5 Biochemical prediction of response to repeated cord occlusion. Baseline fetal
lactate and glucose concentrations for the controls (n=4), regular responders (n=9) and the
fast responders (n=3) are presented as the adjusted mean ± standard error for lactate and
glucose concentration over the 4 days prior to the experimental phase considering both
sheep and study group in repeated measures ANOVA model. Fast responders had
significantly higher lactate concentrations (p=0.011) and glucose concentrations (p=0.064)
prior to repeated cord occlusion protocol than regular responders.
115
100
125
150
175
200
225
Control
p=0.816FH
R (b
pm)
0 9 18 27 36 45 54 63 72 81100
125
150
175
200
225
Fast
Regular
p=0.943
Time (minutes)
FHR
(bpm
)
100
125
150
175
200
225
Occluded
Control
p=0.003
FHR
(bpm
)
A
B
C
Figure 3.6 Baseline fetal heart rate during the experimental phase. FHR (mean± SEM) over
the one minute prior to each cord occlusion for controls (open circles, n=4), occluded
fetuses (regular and fast responders combined; closed diamonds, n=12), regular responders
(closed circles, n=9), and fast responders (closed triangles, dashed line, n=3). There was no
change in the baseline FHR in the control fetuses over the duration of the experimental
phase (p=0.816) and in the fetuses that underwent UCO the baseline FHR was significantly
different from the controls (p=0.003). Baseline FHR was not significantly different between
fast and regular responders (p=0.943).
116
0 9 18 27 36 45 54 63 72 810
20406080
100120140160
D
Control
Time (minutes)
FHR
MM
R (m
sec)
Fast
Regular
0 9 18 27 36 45 54 63 72 810
20406080
100120140160
A
Control
Occluded
p=0.028
Time (minutes)
FHR
MM
R (m
sec)
0 9 18 27 36 45 54 63 72 810
3
6
9
12
15E
Fast
Control
Regular
Time (minutes)FH
R S
TV (m
sec)
0 9 18 27 36 45 54 63 72 810
3
6
9
12
15B
Control
Occluded
Time (minutes)
FHR
STV
(mse
c) p=0.050
0 9 18 27 36 45 54 63 72 810
10203040506070
F
Control
Regular
Fast
Time (Minutes)
FHR
LTV
(bpm
)
0 9 18 27 36 45 54 63 72 810
10203040506070
C p<0.001
Occluded
Control
Time (Minutes)
FHR
LTV
(bpm
)
Figure 3.7 Fetal heart rate variability during the experimental phase. Figures A, B and C
present data from control and occluded fetuses (both the regular and fast responders). P
values are for the comparison between groups, controlling for sheep and time. A significant
increase in MMR (p=0.028), STV (p=0.050) and LTV (p<0.001) occurred in the occluded
fetuses. Figures D, E and F present data from the three study groups. MMR, STV and LTV
were not significantly different between regular and fast responders (MMR, p=0.167; STV,
p=0.925; LTV, p=0.436).
117
Fetal heart rate variability was not different between fast and regular responders (MMR,
p=0.167; STV, p=0.925; and LTV, p=0.436; Figure 3.7D-F). After controlling for
experimental group, sheep and time, there was a significant increase in all three measures of
FHR variability (LTV vs. STV r=0.838, p<0.001; LTV vs. MMR r=0.858, p<0.001; MMR vs.
STV r=0.862, p<0.001).
3.3.2.3 Fetal heart rate patterns during repeated cord occlusion
No FHR accelerations or decelerations were present during the 81 minutes of the
experimental phase in controls (Figures 3.8 – 3.12). One control fetus had a single episode of
increased FHR variability after fetal blood sampling. No fetal heart rate information is
available for one regular responder due to difficulties collecting the fetal ECG. Due to the
small sample size, no formal statistical comparison was made between the FHR patterns of
the regular and fast responders during the experimental phase. Descriptive data relating to
these patterns is presented as median and range.
3.3.2.3.1 Variable fetal heart rate decelerations
Variable FHR decelerations occurred in all but two of the 275 umbilical cord occlusions for
which data is available. All fetuses that underwent UCO had severe variable decelerations
with their first UCO. During the experimental phase, seven of the 11 occluded fetuses had
intermittent variable decelerations which did not meet the criteria for severe variable
decelerations (decrease in FHR by >60 bpm during cord occlusion). The pattern of variable
decelerations is presented graphically in Figure 3.8 which demonstrates that there is no
obvious difference in the pattern of variable decelerations between regular and fast
responders.
3.3.2.3.2 Overshoot after variable fetal heart rate decelerations
Fetal heart rate overshoot began at a median of 30 minutes (range 9-57 minutes) into the
experimental phase in nine of the 11 fetuses subjected to UCO. The timing of FHR
overshoot for each fetus is presented graphically in Figure 3.9. No overshoot was present in
two fetuses who were both fast responders.
3.3.2.3.3 Late components to variable fetal heart rate decelerations
With their first UCO, all fetuses subjected to UCO had late components associated with their
severe variable decelerations (Figure 3.10). These continued throughout the experimental
phase in two of the three fast responders and one of the eight regular responders. In the
remainder of the fetuses subjected to UCO, late components to variable decelerations were
117
Figure 3.8 Variable fetal heart rate decelerations. The blue segments of the bar chart
represent no variable decelerations during the cord occlusion. The yellow segments
represent variable FHR decelerations and the orange segments represent the severe variable
FHR decelerations. No decelerations were present in control fetuses. All of the fetuses that
underwent UCO had severe variable decelerations in response to their first umbilical cord
occlusion.
118
9908
9918
9922
9925
9917
9913
9912
9911
9906
9920
9919
9905
0 9 18 27 36 45 54 63 72 81Time (minutes)
9915
9914
9921
9918
9908
9922
9925
9919
9906
9905
9920
9913
9917
9912
9911
9921
9914
9915
Con
trols
Reg
ular
Res
pond
ers
Fast
Res
pond
ers
Variable Decelerations
SheepNumber
118
118
Figure 3.9 Overshoot after fetal heart rate decelerations. The blue segments of the bar chart
represent no FHR overshoot after the decelerations during the cord occlusion and the purple
segments represent the presence of overshoot.
119
9908
9918
9922
9925
9917
9913
9912
9911
9906
9920
9919
9905
0 9 18 27 36 45 54 63 72 81Time (minutes)
9915
9914
9921
9918
9908
9922
9925
9919
9906
9905
9920
9913
9917
9912
9911
9921
9914
9915
Con
trols
Reg
ular
Res
pond
ers
Fast
Res
pond
ers
Overshoot
SheepNumber
119
119
Figure 3.10 Late components to fetal heart rate decelerations. The blue segments of the bar
chart represent no late component to the variable decelerations during the cord occlusion
and the red segments represent the presence of late components.
120
9908
9918
9922
9925
9917
9913
9912
9911
9906
9920
9919
9905
0 9 18 27 36 45 54 63 72 81Time (minutes)
9915
9914
9921
9918
9908
9922
9925
9919
9906
9905
9920
9913
9917
9912
9911
9921
9914
9915
Con
trols
Reg
ular
Res
pond
ers
Fast
Res
pond
ers
Late Components to Decelerations
SheepNumber
120
120
Figure 3.11 Increased FHR variability between cord occlusions. The blue segments of the bar
chart represent no increased FHR variability and the green segments represent the presence
of increased variability between the cord occlusions.
121
9908
9918
9922
9925
9917
9913
9912
9911
9906
9920
9919
9905
0 9 18 27 36 45 54 63 72 81Time (minutes)
9915
9914
9921
9918
9908
9922
9925
9919
9906
9905
9920
9913
9917
9912
9911
9921
9914
9915
Con
trols
Reg
ular
Res
pond
ers
Fast
Res
pond
ers
Increased Variability
SheepNumber
121
121
Figure 3.12 Complex variable fetal heart rate decelerations. The blue segments of the bar
chart represent no FHR deceleration. The yellow segments represent simple variable
decelerations and the orange segments represent simple severe variable decelerations. The
red segments represent variable decelerations with late components to their recovery and the
purple segments represent variable decelerations with overshoot. The dull cherry segments
of the bar chart represent variable decelerations with both late components to their recovery
and overshoot after the deceleration.
122
9908
9918
9922
9925
9917
9913
9912
9911
9906
9920
9919
9905
0 9 18 27 36 45 54 63 72 81Time (minutes)
9915
9914
9921
9918
9908
9922
9925
9919
9906
9905
9920
9913
9917
9912
9911
9921
9914
9915
Con
trols
Reg
ular
Res
pond
ers
Fast
Res
pond
ers
Complex Variable Decelerations
SheepNumber
122
123
not present in the latter half of the experimental phase (median of 25.5 min, range 6-57 min)
usually having been replaced by FHR overshoot. Comparing the figures illustrating late
components to variable FHR decelerations and overshoot after variable FHR decelerations
(Figures 3.9 and 3.10) it appears that in the majority of cases, fetuses have only one of these
patterns at each time point, with the combined pattern occurring in only 30 of the 275 (11%)
cord occlusions for which data are available for analysis.
3.3.2.3.4 Increased fetal heart rate variability between cord occlusions
Increased FHR variability was present in all fetuses that underwent UCO at some period of
the experimental phase (Figure 3.11). It was present intermittently during the first 36
minutes of cord occlusions after which it was present for the majority of the time (85%) in 10
of the 11 occluded fetuses. Regular and fast responders displayed similar patterns of
increased FHR variability.
3.3.2.3.5 Complex variable fetal heart rate decelerations
Figure 3.12 graphically presents the combinations of variable FHR decelerations with both
late components to the decelerations and FHR overshoot. In two of the three fast
responders fetal heart rate overshoot did not occur and late components to the variable FHR
decelerations were present with each UCO. In all of the regular responders, combined
patterns were present.
3.3.3 Fetal arterial pressure responses to repeated cord occlusion
3.3.3.1 Fetal arterial pressure between cord occlusions
There were no significant changes in systolic pressure (76.0±5.7mmHg, p=0.967) or diastolic
pressure (53.5±3.9mmHg, p=0.967) during the experimental phase in controls (Figure 3.13).
Systolic pressure increased significantly during the experimental phase in fetuses that
underwent UCO (regular + fast responders) compared to controls (p=0.049). The systolic
pressure progressively increased during the first 36 minutes of UCO and remained
significantly elevated between 39 and 51 minutes (p<0.05) after which it decreased again
towards baseline levels. Diastolic pressures were not significantly different between fetuses
subjected to UCO and controls (p=0.999).
124
0 9 18 27 36 45 54 63 72 8160708090
100110120
Occluded
Control
p=0.049
Time (minutes)
Syst
olic
Pre
ssur
e(m
mH
g)0 9 18 27 36 45 54 63 72 81
30405060708090
Control
Occluded
p=0.967
Time (minutes)
Dia
stol
ic P
ress
ure
(mm
Hg)
0 9 18 27 36 45 54 63 72 8160708090
100110120
Control
Regular
p=0.244
Time (minutes)
Syst
olic
Pre
ssur
e(m
mH
g)
0 9 18 27 36 45 54 63 72 8130405060708090
Control
Regular
p=0.653
Time (minutes)
Dia
stol
ic P
ress
ure
(mm
Hg)
0 9 18 27 36 45 54 63 72 8160708090
100110120
Fast
Control
p=0.024
Time (minutes)
Syst
olic
Pre
ssur
e(m
mH
g)
0 9 18 27 36 45 54 63 72 8130405060708090
Fast
Control
p=0.024
Time (minutes)
Dia
stol
ic P
ress
ure
(mm
Hg)
A
B
C
D
E
F
Figure 3.13 Fetal arterial pressure responses between repeated cord occlusion. Fetal systolic
pressures are presented in Figures A, B and C and diastolic pressures in D, E and F. The p
values on each graph represent the comparison of the occluded group or subgroup to the
control group. There were no significant changes in the systolic pressure (p=0.967) or the
diastolic pressure (p=0.999) in controls.
125
Systolic (p=0.244) and diastolic (p=0.159) pressures were not significantly different between
regular and fast responders; however, the magnitude of the increase in systolic and diastolic
pressure was greater in the fast than the regular responders with only fast responders having
significantly higher pressures than controls (p=0.024).
3.3.3.2 Fetal arterial pressure during cord occlusion
Fetal arterial pressure responses to UCO progressed through several distinct patterns during
the experimental phase. These have been arbitrarily classified as the “hypertensive response”,
the “decreasing hypertensive response”, the “biphasic systolic response” and the “biphasic
diastolic response”. Representative arterial pressure and heart rate recordings, illustrating
each of these different patterns of response, are shown in Figure 3.14.
Initially during UCO the fetal arterial pressure was elevated and this was accompanied by a
deceleration in fetal heart rate. This has been arbitrarily classified as the “hypertensive
response”. The first sign of fetal cardiovascular decompensation was when the fetus could
no longer maintain hypertension during the UCO. This has been arbitrarily classified as the
“decreasing hypertensive response” and is illustrated by the second column of panels in
Figure 3.14. As progressive cardiovascular deterioration continued, the fetus first developed
systolic hypotension during cord occlusion (biphasic systolic response) and finally systolic
and diastolic hypotension during occlusion as the pre-terminal event (biphasic diastolic
response). The fall in systolic and diastolic pressures during cord occlusion represents the
most severe form of deterioration of fetal wellbeing preceding prolonged fetal bradycardia,
hypotension and death.
All regular responders progressed through these four stages of cardiovascular decompression
albeit at different rates. The time of onset of the biphasic diastolic response phase varied
from 12 to 81 minutes into the protocol with a median time of 46.5 minutes. One of the fast
responders displayed the preterminal biphasic diastolic response during UCO for the entire
protocol and another developed this response 12 minutes into the protocol.
3.3.4 Neuropathologic findings after repeated cord occlusion
Fetal brains were examined from all four control fetuses and from eight of the nine regular
responders to repeated cord occlusion. Considerable variation was present on macroscopic
brain examination. Some brains appeared normal with good topographic preservation and
clear gray-white matter demarcation. In other cases the brain had extensive white and gray
matter damage with pronounced softening and cavitation.
125
Figure 3.14 Fetal arterial pressure responses during repeated cord occlusion. The four
distinct patterns of arterial pressure response during cord occlusion are presented in this
figure which is taken directly from the digital recording of fetus 9921. The top row of panels
shows the arterial pressure signal in red. The systolic and diastolic arterial pressures are
shown in the middle two rows, and the fetal heart rate patterns are in blue in the lowest row
of panels with the red bars representing the cord occlusion.
126
FHR
BP Signal
Systolic Pressure
Diastolic Pressure
Biphasic Diastolic
Response
Hypertensive Response
Decreasing Hypertensive
Response
Biphasic Systolic
Response
126
127
The grades of histopathologic injury in right and left sides of the brain are summarised in
Table 3.3. Injury was present in the left side of all fetal brains, corresponding with the side of
vascular catheterization. There was no correlation between damage on the right and left side
of the fetal brains (r=0.032, p=0.922). All quantification of neurologic damage relating to
biochemical markers has been performed on the right side of the fetal brain.
There was no histopathologic damage in the right side of the brain in any of the controls. In
the fetuses exposed to UCO, four were found to have mild histopathologic damage (grade 1)
and the other four had damage ranging from widespread white matter injury (grade 2)
through to areas of necrosis and infarction in the gray and white matter (grade 4 and 5).
In controls the cortical neurons had abundant cytoplasm and vesicular nuclei (Figure 3.15A).
In the brains of four of the fetuses exposed to UCO there were subtle changes, with
vacuolisation and loss of myelin or an increase in cellularity with macrophages and astrocytes.
This occurred primarily in the parietal and frontal subcortical white matter at its junction with
the cerebral cortex. Four of the eight fetuses exposed to repeated cord occlusion had more
substantial neuropathologic damage (Figure 3.15B and C) varying from more widespread
white matter injury through to well defined infarcts and extensive necrosis of gray and white
matter with marked macrophagic and astroglial reaction.
3.3.5 Relationship between markers of fetal well-being during repeated cord
occlusion and fetal neuropathologic damage
3.3.5.1 Correlation of biochemical markers with fetal neuropathologic damage
Fetal arterial gas tensions, pH, base excess and lactate, glucose, cortisol, and catecholamine
concentrations at three time points were compared to the grade of fetal brain injury.
Correlation coefficients for these comparisons are presented in Table 3.4. Fetal base excess
(r=-0.743, p=0.009) was the only biochemical measure prior to repeated UCO with a
significant correlation with the grade of fetal brain injury assessed 72 hours after UCO.
Fetuses with lower base excess levels prior to umbilical cord occlusion had higher grades of
fetal brain injury.
Of the twelve biochemical markers collected immediately after repeated UCO, significant
correlations were found between the grade of fetal brain injury and lactate (r=0.759,
p=0.007), noradrenaline (r=0.640, p=0.046) and arterial PO2 (r=-0.637, p=0.035). The only
biochemical marker 72 hours after UCO with a significant correlation with the grade of fetal
128
Table 3.3 Histopathologic damage in right side (no vascular ligation) and left side (vascular ligation) of fetal brain
Sheep Number
Overall Right Grade
Parietal Frontal White Occipital Frontal
Gray Thalamus Striatum
(basal ganglia)
Hippo-campus Pons Cerebellum
9918* 0 9908* 0 9922* 0 9925* 0 9901 1 + 9912 1 + 9911 1 + + 9917 1 + + + 9920 2 + ++ 9905 2 ++ ++ ++ + ++ 9919 4 ++++ ++++ ++++ ++++ ++++ ++++ ++++
Right side of fetal brain
9906 5 +++++ ++++ +++++ ++++ ++++ +++ ++++ ++++
9918* 5 +++++ ++++ +++++ ++++ 9908* 5 +++++ +++ +++ +++ ++++ ++ 9922* 4 ++++ ++++ ++++ ++++ 9925* 4 ++++ ++++ ++++ ++++ 9901 2 + ++ ++ 9912 4 ++++ ++ ++++ ++ 9911 1 + + + + 9917 1 + + + 9920 4 ++ ++++ ++++ 9905 2 ++ ++ ++ ++ ++ 9919 4 ++++ ++++ ++++ ++++ ++++ ++++ ++++
Left side of fetal brain
9906 5 +++++ +++++ ++++ +++++ ++++ ++++ +++++ ++++ ++ * Denotes Control Fetuses
128
128
Figure 3.15 Range of histopathologic damage to fetal brains. A, B and C are photo-
micrographs (haematoxylin-eosin stain) of ovine fetal brain under high power showing the
range of neuronal injury produced by the intermittent cord occlusion protocol utilized in this
study. Figure A is a section of cortex (100x magnification) showing neurons with abundant
cytoplasm and vesicular nuclei (grade 0 changes). Figure B is a section of the frontal cortex
(40x magnification) showing scattered ischaemic neuronal changes in the cerebral cortex.
The neurons are shrunken, triangular, and eosinophilic with dark nuclei (grade 3 changes).
Figure C is a section of the parietal lobe (40x magnification) showing extensive necrosis
involving both grey and white matter with marked macrophagic and astroglial response
(grade 5 changes).
129
A
B
C
130
Table 3.4 Spearman’s Rho for fetal biochemical markers and the grade of fetal brain injury in the right side of brain
pH
PO2
PCO
2
Base
Exc
ess
Lact
ate
Glu
cose
Corti
sol
Nor
-adr
enali
ne
Adr
enali
ne
Dop
amin
e
r -0.421 -0.249 -0.508 -0.743 -0.481 -0.291 -0.356 -0.334 0.072 0.216 Pre-Occlusion
p-value 0.197 0.460 0.111 0.009 0.134 0.386 0.313 0.380 0.854 0.681
r -0.432 -0.637 0.388 -0.478 0.759 0.560 0.234 0.640 0.611 0.390 Post-Occlusion
p-value 0.184 0.035 0.238 0.137 0.007 0.136 0.515 0.046 0.060 0.265
r -0.350 -0.541 0.363 -0.226 0.322 -0.847 -0.046 0.178 0.352 0.306 Delivery (3 days later)
p-value 0.322 0.160 0.302 0.530 0.364 0.008 0.907 0.647 0.353 0.390
130
131
brain injury was fetal glucose levels (r=-0.847, p=0.008) with the highest grades of injury
occurring in the fetuses with the lowest glucose concentrations.
3.3.5.2 Correlation of fetal heart rate patterns with fetal neuropathologic damage
The total duration of the UCO protocol in which specific FHR decelerative patterns were
present was calculated for each fetus and correlation coefficients for the comparison of these
durations with the grade of neuronal injury are presented in Table 3.5. The only two patterns
in which there was a significant correlation between the duration of the pattern and the grade
of fetal brain injury was increased FHR variability (r=0.671, p=0.039) and overshoot after
variable decelerations (r=0.705, p=0.027).
The correlation coefficients between the duration of compound FHR abnormalities (similar
to those assessed in the clinical environment) and the grade of fetal brain injury are presented
in Table 3.6. None of these coefficients reached statistical significance.
Table 3.5 Spearman’s Rho for the duration of specific FHR patterns and grade of fetal brain injury
Duration of Pattern (n=11) R p-value
Late component 0.470 0.166
Severe variable decelerations 0.566 0.096
Increased FHR variability 0.671 0.039
Overshoot 0.705 0.027
Table 3.6 Spearman’s Rho for the duration of compound FHR patterns and grade of fetal brain injury
Duration of Pattern (n=11) R p-value
Late component and overshoot 0.325 0.349
Increased variability and late component 0.336 0.349
Severe variable and late component 0.510 0.133
Severe variables and increased variability 0.568 0.088
Increased variability and overshoot 0.592 0.073
132
3.3.5.3 Correlation of fetal arterial pressure with fetal neuropathologic damage
3.3.5.3.1 Fetal arterial pressure between the umbilical cord occlusions
The mean fetal arterial pressures over the 10 minutes following the final cord occlusion of
the protocol were compared with the grade of neurologic injury. There was no significant
correlation between the grade of fetal brain injury and absolute arterial pressure or changes in
arterial pressure over the duration of the experimental phase (Table 3.7). Similarly, there was
no significant correlation between the grade of fetal brain injury and the percentage of the
experimental phase where the diastolic pressure was less than 30mmHg (r=0.142, p=0.802).
Table 3.7 Spearman’s Rho for arterial pressure at the completion of the cord occlusion protocol and grade of neuronal injury
(n=12) R p-value
Systolic pressure 0.104 0.840
Diastolic pressure 0.169 0.713
Change in systolic pressure 0.660 0.110
Change in systolic pressure 0.374 0.396
3.3.5.3.2 Fetal arterial pressure during the final three occlusions
Fetal arterial pressure during the final three occlusions in each fetus was assessed as an
estimate of the severity of arterial pressure decompensation during the experimental phase.
The following parameters were not significantly correlated with the grade of fetal brain injury
(p all >0.268):
- Mean decrease in systolic, diastolic and mean arterial pressure
- Maximum decrease in systolic, diastolic and mean arterial pressure
- Percentage of the UCO with hypotension
- Total duration of mean arterial pressure hypotension
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3.3.5.3.3 Duration of fetal arterial pressure responses during umbilical cord
occlusion
The duration of each of the fetal arterial pressure responses illustrated in Figure 3.14 were
compared to the grade of fetal brain injury (Table 3.8). There was a strong correlation with
the duration of the biphasic diastolic response during UCO and the grade of fetal brain injury
(r=0.808, p=0.022) and an even stronger correlation for the duration of any arterial pressure
instability (total of the three patterns of cardiovascular instability) during UCO (r=0.955,
p=0.001). The earlier the fetus lost the ability to maintain fetal hypertension during the
UCO, the greater the degree of neuronal injury with damage only occurring after more than
39 minutes of cardiovascular instability. Duration rather than the severity of cardiovascular
instability during the UCO correlated with the grade of fetal brain injury after repeated UCO
in the sheep fetus.
Table 3.8 Spearman’s Rho for the duration of arterial pressure instability during cord occlusion and grade of neuronal injury
Duration of Pattern (n=12) R p-value
Biphasic Diastolic Response 0.808 0.022
Biphasic Systolic Response* 0.602 0.120
Any arterial pressure instability** 0.955 0.001
* Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns
** Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases
3.3.6 Prediction of fetal arterial pressure responses during cord occlusion
Given the strong correlation between fetal responses during UCO and neuronal injury it
would be clinically useful to predict the presence of these patterns from the two techniques
available for intrapartum fetal assessment; fetal heart rate patterns and fetal scalp blood
biochemical assessment.
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3.3.6.1 Prediction of arterial pressure responses during umbilical cord occlusion
from fetal heart rate patterns
The sensitivity and specificity for fetal heart rate patterns to predict fetal arterial pressure
responses during cord occlusion are presented in Table 3.9. None of the FHR patterns had
both good sensitivity and good specificity for the prediction of fetal arterial pressure
responses during cord occlusion. The four patterns assessed had poor specificity for
detecting any arterial pressure instability and poor sensitivity for the biphasic diastolic
response during cord occlusion.
Table 3.9 Fetal heart rate prediction of arterial pressure responses during cord occlusion
Any Arterial Pressure Instability*
Biphasic Systolic Response**
Biphasic Diastolic Response FHR Pattern
Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity
Severe Variable Decelerations 82% 66% 70% 84% 59% 89%
Late component to decelerations 72% 20% 61% 39% 52% 51%
Increased variability 89% 53% 74% 63% 63% 72%
Overshoot 98% 39% 83% 51% 66% 57%
* Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ** Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns
3.3.6.2 Prediction of arterial pressure responses during umbilical cord occlusion
from fetal biochemical measures
Receiver operator curves (ROC) were used to assess the diagnostic utility of fetal biochemical
measures to assess fetal responses during cord occlusion. The area under the ROC and the
optimum sensitivity and specificity are summarised in Table 3.10.
For all three patterns of arterial pressure response during UCO, fetal lactate levels had
significantly greater area under the receiver operator curves than pH (any arterial pressure
instability, p=0.011; biphasic systolic response, p=0.037; biphasic diastolic response,
p=0.049; Figure 3.16). The area under the lactate ROC for any arterial pressure instability
was also significantly greater than the area under the PO2 and PCO2 receiver operator curves
(p<0.001) but was not significantly different from the area under the base excess curve
135
(p=0.730). Similarly, the areas under the lactate ROC for both the biphasic systolic response
and the biphasic diastolic response were significantly greater than those for base excess, PO2
and PCO2 (all p<0.031).
Table 3.10 Biochemical prediction of arterial pressure responses during cord occlusion
Any Arterial Pressure Instability*
Biphasic Systolic Response**
Biphasic Diastolic Response Measure
Area§ Sens† Spec‡ Area§ Sens† Spec‡ Area§ Sens† Spec‡
Lactate 0.931 86% 91% 0.950 86% 91% 0.924 86% 83%
pH 0.891 82% 95% 0.923 73% 95% 0.902 87% 84%
Base excess 0.925 85% 86% 0.919 68% 89% 0.887 77% 74%
PO2 0.781 87% 27% 0.780 88% 69% 0.769 91% 60%
PCO2 0.662 48% 89% 0.757 59% 91% 0.786 62% 88% * Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ** Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns §Area under receiver operator curve, † sensitivity, ‡ specificity
The accuracy for each of the biochemical measures in predicting arterial pressure patterns
during cord occlusion was calculated for optimum cut off points that maximised sensitivity
and specificity (Table 3.11). There was little or no difference in optimum cut off values for
the three patterns of arterial pressure during cord occlusion for pH, base excess, PO2 and
PCO2 whereas for lactate, the level progressively increased as the arterial pressure responses
during cord occlusion deteriorated. For all three measures of arterial pressure response
during UCO, lactate had greater accuracy detecting these patterns than the other markers of
acid-base status.
The ROC analysis suggests that lactate is a better indicator of arterial pressure changes during
UCO than other markers of fetal acid-base status. A progressive rise in lactate is associated
with deterioration in control of fetal arterial pressure during cord occlusion.
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Table 3.11 Accuracy of biochemical prediction of arterial pressure responses during UCO
Any Arterial Pressure Instability*
Biphasic Systolic Response**
Biphasic Diastolic Response Measure
Accuracy§ Optimum Cut Off Accuracy§ Optimum
Cut Off Accuracy§ Optimum Cut Off
Lactate (mmol/L) 90% 2.2 90% 2.7 87% 3.6
pH 88% 7.365 88% 7.360 85% 7.360
Base excess 85% -3.0 83% -3.0 79% -3.0
PO2 (mmHg) 79% 17.0 78% 17.0 73% 17.9
PCO2 (mmHg) 69% 44.6 79% 44.6 79% 44.3
* Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ** Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns § Accuracy is the proportion of correctly classified cases using the biochemical cut off point selected to maximise the sensitivity and specificity
137
Any Blood Pressure Instability
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity
Sens
itivi
ty
No discriminationLactpH
Biphasic SystolicResponse
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
1 - Specificity
Sens
itivi
ty
No discriminationLactpH
Diastolic BiphasicResponse
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity
Sens
itivi
ty
No discriminationLactpH
Figure 3.16 Receiver operator curves for biochemical prediction of arterial pressure patterns
during cord occlusion. Sensitivity is the proportion of true positives and 1-specificity is the
proportion of false positives. The area under each of these curves was significantly greater for
lactate than pH (p=0.011, p=0.037 and p=0.049).
138
3.4 Discussion
The purpose of this study was to relate a number of biochemical and physiologic parameters,
measured before, during or after the experimental period of UCO, to histopathologic
changes that occur after asphyxia. Recent evidence suggests that 80% of infants with
neonatal encephalopathy and 69% of infants with seizures at birth have evidence of an acute
brain insult (Cowan et al., 2003). Despite advances in perinatal care over the past three
decades, the incidence of cerebral palsy attributed to birth asphyxia has not changed (Volpe,
2001b). The ability to predict the fetal response to labour and subsequent neuronal injury
may allow both intrapartum and new postnatal interventions to prevent or reduce hypoxic-
ischaemic brain injury (Perlman, 1999).
3.4.1 Cord occlusion model
The cord occlusion model in this study was designed to replicate events which may occur in
labour. Other studies have focused on relatively prolonged episodes of asphyxia (10 minutes
to 4 hours) which, if they occur clinically, are easily detected and seldom present a therapeutic
dilemma. In labour, contractions typically begin at duration of 20-30 seconds and
progressively increase to be 1 or at most 2 minutes long. When uterine contractions
compromise placental blood flow or compress the umbilical cord, a likely outcome will be
one of brief repetitive asphyxia, rather than prolonged periods of reduced placental gas
exchange.
3.4.2 Histopathological changes induced by cord occlusion model
In chronic preparation models where fetal sheep are used, different protocols of umbilical
cord occlusion lead to various patterns of fetal damage. Chronic partial umbilical cord
occlusion leads to severe acidaemia and diffuse brain damage mainly in the white matter
(Ikeda et al., 1998a). Repeated short occlusions lead to striatal or white matter damage
(Penning et al., 1994; Mallard et al., 1995a; de Haan et al., 1996; Ohyu et al., 1999; Marumo et
al., 2001). Ten minutes of complete umbilical cord occlusion results in predominant
hippocampal damage (Mallard et al., 1992; Mallard et al., 1994; Fujii et al., 2003). The reason
for the dissimilar results is unclear but may relate to the different patterns of haemodynamic
response or the different distributions of regional blood flow in the brain during the hypoxic-
ischemic insults (Ball et al., 1994; Bennet et al., 1998; Kaneko et al., 2003).
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The neuropathologic changes identified in my study are consistent with changes identified in
other studies using similar cord occlusion protocols in fetal sheep (Clapp et al., 1988; de Haan
et al., 1996; Marumo et al., 2001). Using recurrent cord occlusion for 1 out of every 2.5
minutes and 2 out of every 5 minutes, De Haan et al. (1996) found the predominant
neuropathology was selective neuronal necrosis throughout the fetal brain; however, six of
the surviving 14 asphyxiated fetuses had focal infarcts in the parasaggital cortex, thalamus
and cerebellum. Similarly, Marumo et al. (2001) found three patterns of brain injury utilising
five three minute total cord occlusions at five minute intervals. One third of the fetuses had
no or minimal pathologic lesions, one third had dominant lesions in periventricular white
matter and one third had lesions in the cortex and thalamus.
The neuropathologic changes identified in this study are also consistent with those identified
using MRI in term human infants with neonatal encephalopathy (Cowan, 2000; Biagioni et al.,
2001; Volpe, 2001a; Cowan et al., 2003). In a study of 351 full term human infants with
neonatal encephalopathy, the lesions identified were mostly bilateral abnormalities in the
basal ganglia, thalami, cortex or white matter, although three percent had focal infarction
(Cowan et al., 2003).
3.4.3 Fetal acid-base status and neuropathologic damage
In this study I have demonstrated that traditional biochemical markers of fetal acid-base
status measured prior to cord occlusion did not predict the rate of cardiovascular or
biochemical deterioration in response to repeated cord occlusion. In addition, similar to
previous animal studies (Gunn et al., 1992; Ikeda et al., 1998a), fetal acid-base status did not
correlate with the grade of neuronal injury identified histologically 72 hours after cord
occlusion. A similar poor association between fetal acid-base status and neurologic outcome
has been identified in human studies where fetal umbilical arterial acidosis at delivery is a
relatively good measure of the severity or duration of intrauterine asphyxia (Winkler et al.,
1991). A realistic cut off point for defining pathological fetal acidaemia that correlates with
an increased risk of neurological deficit has been found to be a pH of less than 7.00 and a
base deficit of more than 16mmol/L (Goldaber et al., 1991; Sehdev et al., 1997; MacLennan,
1999). Despite defining a group of neonates at increased risk, fetal umbilical arterial acidosis
at delivery has a poor correlation with outcome as most term infants with severe acidaemia at
delivery have an uncomplicated neonatal course (Winkler et al., 1991). Even in newborns
with birth asphyxia, no differences have been shown in the first postnatal arterial pH, base
140
deficit or blood glucose in those newborns with encephalopathy compared with those
without this condition (Huang et al., 1999).
A lack of association between fetal acid-base status and the severity of neuronal injury has
consistently been reported in animal studies since 1972 in both models of global asphyxia and
partial asphyxia (Myers, 1972; Ting et al., 1983; Gunn et al., 1992; Mallard et al., 1992; de Haan
et al., 1993; Ikeda et al., 1998a). Using a global asphyxia model with three 10 minute cord
occlusions, Mallard et al. (1992) induced severe acidosis (pH 6.9, lactate 6mmol/L) and
hypotension in fetal sheep. At post-mortem, neuronal necrosis was identified in the
hippocampus, basal ganglia, and thalamus and the magnitude of neuronal necrosis correlated
with fetal hypotension but not with the severity of acidosis.
Similar results have also been identified using sheep and monkey models of partial asphyxia.
Myers (1972) exposed fetal monkeys to partial asphyxia for one to three hours and at post-
mortem found that some had evidence of neuropathology while others were normal. The
one to three hours of partial asphyxia induced focal neuronal necrosis principally in
parasaggital regions and the junctional zones between the occipital and parietal lobes. In
some fetuses there was also neuronal damage in the basal ganglia and thalamus. All of the
fetal monkeys had severe acidosis; however, it was only those with concurrent hypotension
that had neuropathology. Similarly, both mid- (Ting et al., 1983) and late- (Gunn et al., 1992)
gestation fetal lambs exposed to prolonged partial asphyxia for 60 to 120 minutes exhibited
neuronal damage only if moderate acidosis and hypotension occurred concurrently. Fetuses
that were acidaemic without hypotension showed no neuronal damage. In addition to the
concurrent requirement for acidosis and hypotension to produce neuronal injury, the severity
of hypotension has also been shown to correlate with the extent of neuropathology (Gunn et
al., 1992; Ikeda et al., 1998a).
Severe acidosis in the absence of fetal hypotension appears to induce minimal or no injury to
the fetal brain. This was illustrated in fetal sheep in a study by de Haan et al. (1993) in which
partial asphyxia for 120 minutes induced severe acidosis (pH<7.0) without hypotension.
Neuronal injury, principally in the cerebellum and to a lesser extent the cerebral cortex,
occurred in only a few fetuses. No injury was identified in the striatum or hippocampus of
any of the asphyxiated fetuses. Coagulative cell changes were found in the cortex of one
fetus, and the thalamus or medulla of two other asphyxiated fetuses. The remaining 27 of
the 34 areas of the fetal brains that were assessed had no coagulative cell changes.
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The lack of association between fetal acid-base status and the severity of neuronal injury
probably relates to the fetal response to the insult. The variability of outcome after a fetal
exposure to either partial asphyxia or repeated short episodes of global asphyxia can be
attributed to the concept emerging from laboratory studies (Figure 1.1; Low, 1998):
Fetal Exposure + Fetal Response = Outcome
Fetal exposure to asphyxia is associated with the development of an acidosis which provides
some information about the timing and severity of the exposure (Belai et al., 1998;
MacLennan, 1999). The fetal response to the exposure is variable with the fetal brain
protected for differing periods of time before the threshold of decompensation is reached.
Initially the fetal response to the insult is compensatory, consisting of increased blood flow to
the fetal brain, heart and adrenals (Cohn et al., 1974; Peeters et al., 1979; Field et al., 1990;
Jensen et al., 1991; Reid et al., 1991) with oxygen supply to these organs being sustained and
no cerebral dysfunction or damage occurring. This response is initiated by arterial
chemoreflexes and then augmented by an increase in plasma catecholamines, ACTH, cortisol
and possibly by arginine vasopressin and angiotensin (Rurak, 1978; Jones et al., 1988; Challis
et al., 1989; Bennet et al., 1994). With prolonged or severe insults the fetus may
decompensate with hypotension, decreased cerebral blood flow and decreased oxygen supply
to the fetal brain (Low, 1998). The resulting oxygen debt and anaerobic metabolism will
initiate a cascade of biochemical events leading to brain cell dysfunction and death.
The lack of association between fetal acid-base status and the severity of neuronal injury may
also relate to the mechanisms of fetal brain injury. There is a growing body of evidence to
suggest that changes in pH, PCO2 and base excess are not the main cause of fetal brain
damage, but instead are contributing factors. The primary initiator appears to be neuronal
energy failure which initiates a complex cascade of interacting events which include
generation of reactive oxygen species and its consequences (Maulik, 1998). This oxidative
stress is paradoxically prominent during reoxygenation after the initial insult (McCord, 1985).
The importance of the reoxygenation-reperfusion phase after injury has recently been
demonstrated in fetal sheep; the severity of ischaemia and also reperfusion had significant
correlations with the severity of neuronal damage after a ten minute asphyxial episode. No
correlation with the severity of neuronal injury was found with acidemic or hypoxic
parameters (Fujii et al., 2003).
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3.4.4 Fetal lactate and neuropathologic damage
In this study fetal lactate levels were found to be associated with both the pattern of fetal
response to repeated cord occlusion and with the severity of neuronal injury associated with
the asphyxial insult. The fetal lactate concentration at delivery accounted for 58% (r=0.759,
r2=0.576) of the variation in the grade of neuronal injury assessed 72 hours after repeated
UCO. This compares very favourably with the current markers of fetal wellbeing, namely,
umbilical artery pH and base excess, collected at delivery, which accounted for 19% and 23%
of the variability in neuronal injury respectively. Although accounting for more of the
variability in outcome than pH and base excess, fetal lactate levels after cord occlusion did
not perform as well as the duration of fetal arterial pressure instability during cord occlusion,
which accounted for 91% of the variability in outcome. Of all of the biochemical and
physiologic markers of fetal wellbeing assessed in this study, fetal arterial pressure was the
best predictor of the severity of neuronal injury. Unfortunately the assessment of fetal
cardiovascular responses expressed by cardiac output, systemic arterial pressure and blood
flow to the brain is not available in the clinical setting (Low, 1998).
There is now a growing body of evidence from animal and human studies that lactate levels
can predict neuronal injury. In this study a significant association was identified between
lactate collected immediately after cord occlusion and brain injury 72 hours later. Ikeda et al
found a similar significant correlation in fetal sheep between lactate concentrations 24 hours
after a 60 minute partial cord occlusion and brain injury assessed 72 hours later (Ikeda et al.,
1998a). In human studies, intrapartum scalp lactate assessment has been shown to be a more
sensitive diagnostic tool than the measurement of scalp pH values for predicting either an
Apgar score <4 at 5 minutes or moderate to severe neonatal encephalopathy (Kruger et al.,
1999). Urinary lactate-creatinine ratio at 6 hours of age assessed by proton nuclear magnetic
resonance spectroscopy (MRS) has been shown to have both high sensitivity (94%) and
specificity (100%) for predicting hypoxic-ischaemic encephalopathy in a group of babies with
perinatal asphyxia (Huang et al., 1999).
There have been a number of studies which have found elevated levels of lactate with proton
MRS in the fetal brain after birth asphyxia (Hanrahan et al., 1996; Hanrahan et al., 1998;
Amess et al., 1999; Barkovich et al., 1999; Malik et al., 2002). Elevated lactate has been shown
to be associated with poor outcome in the neonatal intensive care unit (Hanrahan et al.,
1996), at two months (Malik et al., 2002) and at 12 months of age (Amess et al., 1999). In
infants with birth asphyxia who had neurologic and developmental abnormalities present at
12 months of age, the most common findings on MRS performed at birth were elevated
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lactate and diminished N-acetylaspartate peaks (Amess et al., 1999). The maximum cerebral
peak-area ratio lactate:N-acetylaspartate accurately predicted outcome at 12 months of age
with a specificity of 93% and a positive predictive value of 92% whereas neurological
assessment had a tendency for false-positive predictions (Amess et al., 1999). Magnetic
resonance spectroscopy studies have also shown that infants exposed to birth asphyxia with
elevated levels of lactate on MRS at one month of age were more likely to have an abnormal
neurodevelopmental outcome than those whose lactate levels had returned to normal
(Hanrahan et al., 1998).
Lactate levels in the fetus and neonate may be an indirect marker of the severity of hypoxic-
ischaemic insults. Mitochondrial aerobic respiration can be disturbed by severe hypoxic-
ischaemic insults leading to heightened conversion to lactate, and increased lactate levels may
enhance the severity of damage to brain tissue (Wagner & Myers, 1979). Similarly, severe
asphyxial insults may result in a decrease in cerebral blood flow. It has been suggested that
hypotension plays a major role in the development of hypoxic-ischaemic encephalopathy
(Gunn et al., 1992; Volpe, 1995a; Volpe, 2001b) and it is likely that the hypoxia-related
increase in cerebral blood flow that occurs initially as a protective response to hypoxia cannot
be maintained to the same extent during associated hypotension (Yaffe et al., 1987).
Fetal lactate levels may be an indirect marker of fetal arterial pressure. High levels of lactate
have previously been shown to be associated with progressive compromise in cardiac
function and thus output (Richardson et al., 1996). This study provides evidence that fetal
lactate is an indirect measure of fetal arterial pressure responses during UCO. For all three
patterns of arterial pressure response during UCO, fetal lactate levels had significantly greater
area under the receiver operator curves than traditional markers of fetal acid-base status. In
addition, the fetal lactate levels identified as optimum cut off values for prediction of the
three stages of cardiovascular deterioration during UCO progressively increased as the blood
pressure control during UCO deteriorated. There was no change in optimum cut off values
for pH, base excess, PO2 or PCO2 with progressive cardiovascular deterioration during UCO.
It may be through the association with arterial pressure responses during asphyxia that lactate
collected at the end of the experimental phase predicted the severity of neuronal injury.
The rise in fetal lactate in response to repeated short episodes of global asphyxia may have a
role in attenuation of brain injury. In rats, when L-lactate is infused into the cortex with
glutamate, a significant reduction in the size of the cortical lesion was noted compared to
glutamate infusion alone. When L-lactate was replaced with D-lactate, the lesion size was
greater than when glutamate was infused alone (Ros et al., 2001). Similarly in rats, an
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intravenous infusion of lactate 30 minutes after a stimulus designed to produce a moderate
brain injury, resulted in significantly less cognitive deficits assessed 2 weeks later (Rice et al.,
2002). These results may be explained by the finding that neurons are capable of utilizing
lactate as an energy source (Schurr et al., 1997).
Rather than being a marker of the insult producing the injury, lactate may be a marker of the
endogenous response to protect the fetal brain from injury. Lactate has traditionally been
thought of as the “dead end” product of anaerobic metabolism. Recent in vitro studies have
demonstrated the ability of neurons to carry out synaptic function with lactate as the only
available carbon source (Schurr et al., 1997). When administered intravenously after neuronal
injury, lactate has been shown to be taken up at the injury site, which led to a preservation of
extracellular glucose levels after injury (Chen et al., 2000b). After brain injury, lactate levels
rise while glucose levels decline in the extracellular fluid, suggesting that lactate may be
preferentially produced in the injured tissue, as opposed to being a by-product of anaerobic
metabolism, as traditionally thought (Chen et al., 2000a). The beneficial effect of lactate may
be to provide neurons with an easily metabolised carbon source to more rapidly replenish
energy stores after injury when there is a large energy demand to re-establish ionic
homeostasis (Schousboe et al., 1997). Normally, a cell catabolises glucose to pyruvate using
two ATP molecules in turn generating four ATP molecules. If ATP levels are depleted, then
glucose may not be as readily metabolised. Thus, providing the neuron with lactate bypasses
this initial ATP requirement and may help it to recover more quickly.
3.4.5 Fetal glucose and neuropathologic damage
In this study fetal glucose concentrations prior to UCO were identified as predictors of the
fetal response to cord occlusion with high glucose levels predicting rapid biochemical and
cardiovascular deterioration. Glucose levels 72 hours after the cord occlusion protocol were
also found to strongly correlate with the grade of neuronal injury present at that time;
however, after the injury it was low glucose levels predicting higher grades of neuronal injury.
These results suggest that the fetuses with brain injury which survived the experimental phase
of this study utilised glucose as a source of energy during their recovery phase after the
repeated hypoxic-ischaemic insult.
The importance of glucose in hypoxic-ischaemic brain injury has been noted since the earliest
studies of asphyxia in monkeys (Myers et al., 1981). In that study it was noted that
supranormal levels of glucose may be associated with dangerously high levels of cerebral
lactate which may potentiate injury. Similarly, in piglets up to three days old, glucose worsens
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hypoxic-ischaemic brain injury (LeBlanc et al., 1997). The mechanism by which supranormal
levels of glucose increase brain injury is unknown. In the piglet model of hypoxic-ischaemic
brain injury, the provision of additional glucose has been shown to preserve ATP during the
hypoxic injury and did not change cerebrospinal fluid glutamate, suggesting that glucose
worsens outcome by elevating brain L-lactate levels above the threshold for cellular injury
(LeBlanc et al., 1997). The importance of this issue warrants further direct assessment in
human infants using techniques such as proton magnetic resonance spectroscopy (Volpe,
2001a).
3.4.6 Fetal heart rate patterns and neuropathologic damage
In this study the duration of increased FHR variability and FHR overshoot correlated with
the grade of subsequent neuronal injury. The duration of severe variable decelerations, late
components to decelerations and compound FHR patterns did not have significant
correlations with the grade of neuronal injury 72 hours after the occlusion protocol.
3.4.6.1 Increased fetal heart variability and neuropathologic damage
An increase in FHR variability has been shown previously in studies of inhalational hypoxia
in fetal sheep (Dalton et al., 1977; Parer et al., 1980; Lilja et al., 1984; Field et al., 1991; Kozuma
et al., 1997) and monkeys (Ikenoue et al., 1981), placental embolisation in fetal sheep (Gagnon
et al., 1996; Murotsuki et al., 1997a) and cord occlusion in the ovine fetus (Westgate et al.,
1999) and rat (Syutkina, 1988). It is also consistent with the increase in baseline variability
seen in humans in some cases of hypoxia (Pello et al., 1991). The mechanisms which underlie
the increase in FHR variability and indeed control normal FHR variation are not fully
understood but are likely to involve a combination of activation of the carotid
chemoreceptor reflex (Kozuma et al., 1997) and sympathetic stimulation (Dalton et al., 1983;
Rosen et al., 1986; Lindecrantz et al., 1993; Segar et al., 1994).
To my knowledge, mine is the first study to report the association between the duration of
increased FHR variability during repeated short episodes of global asphyxia and the severity
of the resultant neuronal injury. A relationship between FHR variability and neuronal injury
has previously been reported in near-term fetal lambs, however in that study it was only the
FHR variability after the 60 minute episode of partial asphyxia that related to the severity of
subsequent neuronal injury (Ikeda et al., 1998b). No differences in LTV during the
prolonged period of asphyxia were found between the fetuses with mild, moderate and
severe neuronal injury with all fetuses progressively decreasing their long term variability after
60 minutes of partial cord occlusion (pre-occlusion LTV 32.0±17.3bpm; 60 minutes
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occlusion LTV 4.0±12.6bpm) (Ikeda et al., 1998a). There were, however, differences in LTV
during the recovery phase with the fetuses with severe neuronal injury having slower recovery
in FHR variability to baseline levels than those with mild or moderate injury. The fetuses
with severe neuronal injury had a further secondary reduction in LTV 12 hours after the
insult with no FHR variability present at 24, 48 and 72 hours of recovery. In fetuses with
mild or moderate injury, the delayed secondary fall in LTV did not occur (Ikeda et al., 1998b).
The mechanism responsible for the association between the duration of increased FHR
variability and the severity of neuronal injury is unknown. There is reasonably good evidence
to suggest that brain injury after fetal asphyxia is associated with fetal hypotension (Ting et al.,
1983; Gunn et al., 1992; Ikeda et al., 1998a) however in the study by Ikeda et al. (1998b) fetal
blood pressure could not be reliably predicted from FHR variability with fetal arterial
hypotension occurring in only 34% (17/50) of the occasions when FHR variability was
reduced. Similarly in this study, increased FHR variability had both poor sensitivity and
specificity for predicting the three stages of arterial pressure decompensation during repeated
UCO (Table 3.9).
The association between increased FHR variability and neuronal injury may be indirect and
through the association of this FHR pattern with stimulation of the sympathetic nervous
system (Dalton et al., 1983; Rosen et al., 1986; Lindecrantz et al., 1993; Segar et al., 1994).
Stimulation of the sympathetic nervous system results in increases in FHR, peripheral
vascular resistance and catecholamine concentrations (Jensen & Lang, 1992). These changes
lead to a progressive increase in arterial blood pressure and rapid circulatory centralisation in
favour of the brain stem and heart at the expense of most of the peripheral organs. This
response is important for short-term adaptation and intact survival of asphyxia (Jensen &
Lang, 1992). It may be that the earlier the fetus requires this sympathetic response during
repeated UCO (which may also be responsible for the increase in FHR variability seen in this
study and that of Westgate et al, 1999), the earlier the fetus will cross the critical threshold for
cardiovascular decompensation. Once crossed, there is a fall of arterial pressure due to either
a decrease in cardiac output or a failure to maintain peripheral vascular resistance. The
combination of asphyxia and ischaemia due to hypotension results in a decrease in cerebral
oxygen consumption and, if sustained, brain damage (Low, 1998).
3.4.6.2 Fetal heart rate overshoot and neuropathologic damage
In this study and two retrospective human studies, FHR overshoot has been associated with
neurologically compromised fetuses (Goodlin & Lowe, 1974; Schifrin et al., 1994). This FHR
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pattern, occurring when there is an acceleration immediately following a variable deceleration,
has been reported previously in ovine cord occlusion models (Westgate et al., 1999; Westgate
et al., 2001). A number of authors have attempted to ascribe clinical significance to this
pattern. Goodlin and Lowe (1974) reported that a deceleration-overshoot pattern was
associated with newborns requiring resuscitation and suggested that the pattern might be
caused by an acute hypoxic insult. Overshoot has also been described as one component of
a chronic fetal distress pattern which was associated with subsequent cerebral palsy (Shields
& Schifrin, 1988).
A number of mechanisms have been proposed for FHR overshoot. Goodlin and Lowe
(1974) suggested that the FHR overshoot pattern was a marker of fetal hypoxia. It has also
been suggested that overshoot is a marker for fetal acidaemia (Akagi et al., 1988; Saito et al.,
1988) or previous injury (Shields & Schifrin, 1988). In experimental studies in fetal sheep, the
overshoot pattern has been related to development of fetal acidosis and a decrease in cerebral
glucose metabolic rate during recurrent umbilical cord occlusions (Okamura et al., 1989).
Recently, in an ovine model of repeated umbilical cord occlusion, FHR overshoot was
reported to occur under two distinct conditions: long cord occlusions (2 minutes) and during
developing fetal acidosis and hypotension during 1-minute cord occlusions (Westgate et al.,
2001). In my study, overshoot was only seen in 2% of the 0.5-minute occlusions, 40% of the
1-minute cord occlusions, and all of the 1.5-minute cord occlusions in the regular responders.
Two of the fast responders, despite having significant acidosis and hypotension did not
display FHR overshoot but instead had recurrent late decelerations for the duration of the
experimental phase.
The aetiology of FHR overshoot is unknown but potential mechanisms include a
combination of high levels of circulating catecholamines (Goodlin & Lowe, 1974) and
impaired neural control of fetal heart rate as suggested by the progressive impairment of
EEG activity seen with frequent repeated cord occlusions (Westgate et al., 1999). It has been
suggested that it is the interplay between reduced vagal stimulation during the occlusion and
beta-adrenergic myocardial stimulation immediately after the occlusion which results in the
FHR overshoot pattern (Shields & Schifrin, 1988; Westgate et al., 2001). The initial
component of FHR deceleration caused by umbilical cord occlusion is vagal, mediated by the
carotid chemoreflex (Jensen & Hanson, 1995). As the occlusion is continued, the
deceleration is maintained by direct hypoxic myocardial depression. The longer the occlusion
continues, the less vagal tone contributes to bradycardia (Harris et al., 1982; Jensen &
Hanson, 1995). Consistent with a role of reduced myocardial vagal tone in the overshoot
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pattern, atropine produces overshoot tachycardia in humans (Hon & Lee, 1961; Caldeyro-
Barcia et al., 1966) and fetal sheep (Harris et al., 1982; Itskovitz et al., 1982). Catecholamine
stimulation is also required because overshoot induced by atropine can be abolished by
concurrent administration of propanolol (Saito et al., 1988). These experimental findings
suggest that FHR overshoot is caused by beta-adrenergic stimulation that is unopposed
because vagal tone has been inhibited.
In this study, similar to Westgate et al. (2001), FHR overshoot was associated with
hypotension during umbilical cord occlusion. The presence of FHR overshoot in my study
was not associated with systolic or diastolic hypotension between the UCOs, but it had
sensitivity of 84% and 66% for predicting systolic hypotension and diastolic hypotension,
respectively, during the cord occlusion. The mean arterial pH associated with FHR
overshoot was 7.19±0.13 which is similar to 7.17 (Westgate et al., 2001) and 7.15 (Saito et al.,
1988) previously reported; however, overshoot was not a sensitive marker of arterial pH less
than 7.19 in my study (sensitivity 47%, specificity 93%). The findings of my study taken in
conjunction with previous studies investigating FHR overshoot suggest that the association
between the duration of FHR overshoot and the severity of neuronal injury is mediated
through hypotension during UCO and catecholamine release rather than acidosis or hypoxia.
3.4.6.3 Other fetal heart rate patterns and neuropathologic damage
The lack of association between decelerative FHR patterns and the severity of neuronal
injury seen in this study is consistent with decades of clinical research on electronic fetal heart
rate monitoring (EFM). The two studies which followed up cohorts of babies included in
the two randomised controlled trials comparing electronic FHR monitoring with intermittent
auscultation found no significant difference in cerebral palsy rates between the two groups at
the end of the follow-up period (Langendoerfer et al., 1980; Grant et al., 1989). In two of the
larger case-control studies investigating the relationship between EFM and cerebral palsy, a
significant association has been identified between abnormal cardiotocograph (CTG) findings
and cerebral palsy (Gaffney et al., 1994b; Nelson et al., 1996). In the study of Nelson et al.
(1996) the two fetal heart rate patterns found to be associated with an increase risk of
cerebral palsy were multiple late decelerations in the FHR (OR 3.9; 95%CI 1.7-9.3) and
decreased FHR variability (OR 2.7; 95%CI 1.1-5.8); however, only 0.19% of singletons with
these patterns develop cerebral palsy giving a false positive rate of 99.8%. No other FHR
patterns were found to have significant associations with the diagnosis of cerebral palsy.
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3.4.7 Fetal arterial pressure and neuropathologic damage
In my study, the best predictor of the severity of neuronal injury after repeated UCO was the
duration of the arterial pressure changes which occurred during the cord occlusions. The
duration of fetal arterial pressure instability during cord occlusion accounted for 91% of the
variability in grading of neuronal injury 72 hours after repeated UCO. No significant
associations were identified between inter-occlusion arterial pressure and the severity of
subsequent injury.
In animal models of either global asphyxia or partial asphyxia, fetal arterial pressure responses
are associated with the severity of resultant neuronal injury (Myers, 1972; Ting et al., 1983;
Gunn et al., 1992; Mallard et al., 1992; de Haan et al., 1993; de Haan et al., 1997b; Ikeda et al.,
1998a; Fujii et al., 2003). All of these studies have suggested that the key factor precipitating
cerebral damage is hypotension and the consequent compromise of cerebral oxygen delivery.
The pattern of association between hypotension and neuronal injury varies between studies
and experimental models. In each of the aforementioned studies, both metabolic acidosis
and hypotension were required for neuronal injury. In some of the studies it was the severity
of hypotension during asphyxia which correlated with the severity of injury (Ting et al., 1983;
Gunn et al., 1992; Mallard et al., 1992). In other studies the duration of hypotension had
significant correlations with neurologic outcome, with fetuses with the most sustained
hypotension having the most severe injuries (Gunn et al., 1992; de Haan et al., 1997b; Ikeda et
al., 1998a). In one study, the duration of hypotension after the asphyxial insult was
associated with the grade of neuronal injury (Ikeda et al., 1998a).
The lack of association between inter-occlusion arterial pressure and neurologic outcome in
this study may relate to the cord occlusion model used in this study. The majority of
previous studies where a relationship has been found between arterial pressure and
neurologic outcome have used prolonged periods of asphyxia (10-180 minutes) (Myers, 1972;
Ting et al., 1983; Gunn et al., 1992; Mallard et al., 1992; de Haan et al., 1993; de Haan et al.,
1997b; Ikeda et al., 1998a; Fujii et al., 2003). In these studies, the arterial pressure responses
during asphyxia were biphasic. During the asphyxial episode there was an initial increase in
mean arterial pressure which was then followed by hypotension and it was the severity or
duration of this hypotension which correlated with outcome. In my study and in others
using brief repetitive umbilical cord occlusions in near-term fetal sheep (de Haan et al., 1997a;
de Haan et al., 1997b; Westgate et al., 1999), the pattern of arterial pressure responses is
somewhat different to those described in models with prolonged periods of asphyxia. Short
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repetitive cord occlusions of one minute every 2.5 minutes are associated with a sustained
increase in inter-occlusion mean arterial pressure (MAP) (Westgate et al., 1999). A different
pattern is seen in short repetitive cord occlusions of 2 minutes duration every 5 minutes
where a biphasic pattern occurs with the MAP initially increased only to be followed by a
progressive fall in MAP to return to baseline levels after 90 minutes of occlusions (de Haan et
al., 1997a; Westgate et al., 1999). In my study there was a significant increase in the systolic
but not diastolic inter-occlusion pressures in the fetuses that underwent UCO during the 0.5-
minute 1.0-minute cord occlusions. During the 1.5-minute cord occlusions, the systolic
pressures returned to baseline levels. There were no episodes of inter-occlusion hypotension
in my study and this may account for the absence of association between inter-occlusion
arterial pressure and neurologic outcome.
In this study, during the 0.5-, 1.0- and 1.5-minute occlusions there were four distinct patterns
of arterial pressure response. Similar patterns of arterial pressure response during brief
repetitive UCO have been previously reported (de Haan et al., 1997a; de Haan et al., 1997b;
Marumo et al., 2001) but the association of the duration of these patterns with the severity of
neuronal injury has not been previously described. De Haan et al. (1997b) reported an
association between the total duration of arterial pressure below baseline during the entire
experimental protocol and the severity of neuronal injury. Marumo et al. (2001) reported
different patterns of neuronal injury associated with different arterial pressure responses
during UCO. Fetuses with highest arterial pressure both between occlusions and during
occlusions had their dominant neuropathologic lesions in their periventricular white matter.
Fetuses which had both a smaller increase in arterial pressure in response to UCO and those
with systemic hypotension during the final UCO had their dominant lesions in the cerebral
cortex and thalamus (Marumo et al., 2001). In that study there were no differences in the
FHR patterns or acid-base status between the fetuses with lesions in their periventricular
white matter and those with lesions in their cerebral cortex and thalamus. The only
difference between the two groups of fetuses was their arterial pressure response to the brief
repetitive asphyxia.
The mechanism behind the association between the duration of cardiovascular instability
during UCO and the severity of brain injury may relate to changes in cerebral blood flow that
occur during the UCO. Recent published data suggest that the severity of both cerebral
ischaemia during UCO and cerebral reperfusion after UCO correlates with the severity of
fetal neuronal injury (Fujii et al., 2003). The arterial pressure patterns during UCO that have
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been described in my study may be indirect markers of changes in cerebral blood flow that
occur with cord occlusion.
3.4.8 Prediction of rate of response to repeated umbilical cord occlusion
In this study, there were clearly two different patterns of response to repeated UCO which
have arbitrarily been classified as regular or fast. All of the regular responders completed the
UCO and all but one was alive at delivery three days after the occlusion protocol. None of
the fast responders completed the UCO protocol and none was alive at delivery. Fast
responders had more rapid changes in acid-base status, glucose, cortisol, and catecholamines
in response to UCO than regular responders. They also had greater increases in their systolic
and diastolic pressures during the experimental phase of the protocol.
The only variables measured in this study which predicted whether a fetus would have a
regular or fast response to repeated UCO were lactate and glucose levels over the 4 days
prior to the experiment. There were no differences in any other biochemical markers, FHR,
FHR variability, FHR patterns in response to UCO or baseline fetal arterial pressures.
Fetuses with high baseline levels of lactate and glucose may be different from those with low
levels of these analytes in ways which have not been assessed in this study. The small sample
size and the inadvertent creation of this subgroup do not allow detailed or statistical
evaluation of the differences between fast and slow responders. All fetuses in this study were
from singleton pregnancies and there was no apparent difference in maternal condition,
surgical procedure, post-operative recovery, meconium passage or fetal weights between
these two subgroups. I speculate that the fast responder subgroup may have experienced an
insult prior to the experimental phase of this study which increased fetal lactate levels and
mobilized fetal glucose as part of the fetal stress response. Given the small sample size of the
subgroups and the fact that my study was not designed to investigate predictors of rate of
response to repeated UCO, interpretation of these results and extrapolating them to clinical
situations requires much caution. The results of my study do however support the common
clinical practice of careful fetal monitoring of growth restricted fetuses and fetuses of diabetic
mothers in labour.
Growth restricted fetuses, when compared with appropriately grown fetuses, are more
frequently hypoxaemic, hypercapnic and hyperlacticacidaemic (Pardi et al., 1987; Nicolaides et
al., 1988; Nicolaides et al., 1989; Marconi et al., 1990) and human studies have found the
incidence of intrapartum fetal distress approximates 25-50% in growth restricted fetuses
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(Low et al., 1972; Lin et al., 1991; Spinillo et al., 1995). In addition there is evidence that
growth restricted fetuses not only begin labour with lower pH values, but they also have a
faster rate of decrease in pH per hour in labour than normally grown fetuses (Nieto et al.,
1994).
3.4.9 Integration of results into clinical practice
In my study I have found that both lactate and glucose were associated with the pattern of
fetal response to repeated cord occlusion and the severity of neuronal injury associated with
the asphyxial insult whereas no association was identified with traditional markers of fetal
acid-base status. Although one must be careful in interpretation and extrapolation of animal
models of asphyxia with small sample sizes, as used in this study, the consistency of the
results obtained with other animal studies and human data suggests that lactate warrants
further evaluation in the assessment of fetal well-being in labour and its ability to predict
neuronal injury in the newborn.
The lack of association between fetal acid-base status and the severity of neuronal injury after
repeated UCO does not negate the value of umbilical arterial and venous acid-base
assessment at birth. The presence of a significant metabolic acidosis in umbilical arterial
blood at birth confirms that an asphyxial exposure has occurred and provides an indication
of the severity and time of the insult (Belai et al., 1998; MacLennan, 1999). Perhaps more
importantly, the absence of metabolic acidaemia in umbilical arterial blood at birth excludes
the diagnosis. In addition to identifying neonates at risk of birth asphyxia, the presence of
severe metabolic acidosis at birth also identifies a group of neonates at higher risk of
encephalopathy, cardiovascular and renal complications (Nagel et al., 1995; Ingemarsson et al.,
1997).
This study, like the majority of animal studies of asphyxia, suggests that changes in fetal
arterial pressure are the key factors precipitating cerebral damage resulting from asphyxia.
Unfortunately the assessment of the fetal cardiovascular responses as expressed by cardiac
output, systemic arterial pressure and blood flow to the brain and other organ systems are, in
general, not available in labour. Thus in the clinical situation there is little information of
fetal cardiac compensation or decompensation in response to asphyxial exposure. Future
research in this area should include new techniques which can indirectly assess fetal arterial
pressure during asphyxia.
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The ability of lactate to predict which fetuses will undergo rapid physiologic deterioration in
response to UCO and subsequent neuronal injury may aid in the selection of fetuses for early
delivery and for new rescue therapies such as hypothermia (Yager et al., 1993; Edwards et al.,
1995; Laptook et al., 1995a; Laptook et al., 1995b; Thoresen et al., 1995; Thoresen et al., 1996a;
Thoresen et al., 1996b; Gunn et al., 1997; Gunn et al., 1998a; Gunn & Gunn, 1998; Gunn et
al., 1998b; Gunn et al., 1999; Gunn, 2000; Battin et al., 2001; Battin et al., 2003), erythropoietin
(Dame et al., 2001; Chong et al., 2002; Eid & Brines, 2002; Juul, 2002; Kumral et al., 2003;
Solaroglu et al., 2003), and antioxidants (Halks-Miller et al., 1986; Mishra & Delivoria-
Papadopoulos, 1999), especially as early patient selection is important to gain the maximum
benefit from these new experimental techniques.
3.4.10 Future studies
The inadvertent creation of the fast responder subgroup reduced the number of fetuses with
complete biochemical, physiological and histopathological data and it complicated statistical
analysis. If future studies were to utilise the same experimental protocol as that used in this
study, the criteria for cessation of repeated cord occlusion should be modified so that more
fetuses survive 72 hours in utero after the cord occlusion protocol. Catheterisation of blood
vessels to the left side of the fetal brain is a potential confounder in my study. These vessels
were catheterised to enable direct sampling of the blood supply to and from the brain. Even
though no neuronal injury was identified in the right side of the fetal brain of any of the
control fetuses, it would have been better to sample fetal blood and assess arterial pressure
from another vessel, such as the brachial artery or aorta.
The experimental model used in this study involved a repeated cord occlusion protocol on
day four of a seven day study period. There were no significant changes in the fetal pH,
PCO2 or lactate levels in the the control fetuses during the seven day study period suggesting
that the experimental preparation was stable. The “regular responders” who underwent the
cord occlusion protocol continued to have a mild combined respiratory and metabolic
acidosis on day seven of the study period. The etiology of the mild acidosis in these fetuses
three days after the cord occlusion protocol is unknown. The elevated lactate levels may
relate to cellular damage from the brain injury which was known to occur in the “regular
responders”. Alternatively, as discussed in Section 3.4.4, the persistent elevation in lactate
levels may have a role in attenuation of brain injury. The elevation in fetal PCO2 levels in the
regular responders on day seven of the study period suggests that there may have been some
ongoing impairment in fetal gas exchange after the cord occlusion protocol in these fetuses
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despite cessation of umbilical cord occlusion and confirmation of normal umbilical artery
Doppler wave forms at the completion of the experimental phase. The variable biochemical
and physiologic instability over the three days after the uniform cord occlusion protocol has
the potential to confound the histopathologic results of this study; however, as
histopathologic brain damage usually takes three to seven days to become apparent after the
insult (Nakajima et al., 2000) the variation in fetal biochemical status on days 5-7 of the study
period are unlikely to have had a significant impact on the final histopathologic grading. If
future studies were to utilise this experimental protocol more detailed biochemical and
physiologic assessment during the three day recovery phase would be valuable to enable the
integration of data regarding the fetal condition during the recovery phase into the
interpretation of the results.
The model utilised in my study induces neuronal injury in a pattern similar to that which
occurs in humans with intrapartum birth asphyxia (Cowan et al., 2003) and the cord
occlusion protocol mimics events which may occur in labour. Further exploration with this
protocol could be enhanced by the direct assessment of cerebral blood flow, ischaemia and
reperfusion. Cerebral blood flow could be assessed with ultrasonic flow probes around a
carotid artery, flow probes in the superior sagittal sinus or with laser Doppler flow probes on
the superior sagittal sinus. Additional information could also be obtained by assessing the
relationship between oxygen availability and tissue metabolism with near-infrared
spectroscopy in the fetal brain. Near infrared spectroscopy (NIRS) provides continuous,
quantifiable measurements of changes in the concentrations of the oxygen-sensitive
chromophores, oxyhaemoglobin, deoxyhaemoglobin, and the redox state of cytochrome
oxidase from intact tissues such as the fetal brain (Peebles et al., 1992; Marks et al., 1996;
Bennet et al., 1998). Cytochrome oxidase is the terminal enzyme in the oxidative
phosphorylation pathway and changes in the redox state of this enzyme are associated with
changes in oxygen availability as well as with the flow of reducing equivalents down the
phosphorylation pathway and the rate of ATP turnover (Cooper et al., 1997; Cooper &
Springett, 1997). The assessment of FHR and BP after the cord occlusion protocol would
also provide valuable information in future studies.
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3.5 Conclusion
A brief repetitive UCO protocol in near term fetal sheep was used to induce neuropathologic
changes consistent with those identified in human studies in term infants with neonatal
encephalopathy.
Fetal lactate and glucose were associated with the pattern of fetal response to repeated cord
occlusion and with the severity of neuronal injury associated with the asphyxial insult.
Traditional biochemical markers of fetal acid-base status measured prior to cord occlusion
did not predict the rate of cardiovascular or biochemical deterioration in response to repeated
cord occlusion. When these traditional markers of acid-base status were collected after cord
occlusion no significant associations with the severity of neuronal injury were identified.
No significant associations were identified between the severity of neuronal injury and the
duration of decelerative FHR patterns or compound FHR patterns; however, this is the first
study to describe an association between the duration of both increased FHR variability and
FHR overshoot with the severity of subsequent neuronal injury.
In this study fetal arterial pressure between UCO was not associated with the grade of
neuronal injury. This study is the first to describe the strong correlation between the
duration of specific arterial pressure responses during cord occlusions and the grade of
neuronal injury.
Fetal lactate assessment may predict fetal neuronal injury through its association with fetal
arterial pressure responses during UCO. In this study this association was illustrated by fetal
lactate levels having significantly greater area under receiver operator curves predicting
arterial pressure responses during UCO than traditional markers of acid-base status. The
association was also illustrated by the progressive increase in optimum cut off values for
lactate predicting the stages of progression in cardiovascular decompensation during
progressive asphyxia. The traditional markers of acid-base status had stable optimum cut off
scores as the stages of cardiovascular deterioration progressed, suggesting that acid-base
status may function as a threshold for injury, whereas lactate optimum cut off scores
progressively increased parallel to cardiovascular decompensation with asphyxia.
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C h a p t e r 4
THE RELATIONSHIPS BETWEEN FETAL SCALP LACTATE
LEVELS, CENTRAL LACTATE LEVELS AND OXYGEN FREE
RADICAL ACTIVITY IN THE FETAL BRAIN DURING
ASPHYXIA INDUCED BY GRADED CORD OCCLUSION IN THE
OVINE FETUS
The truth is the best you can get with your best endeavour,
the best that the best men accept –
with this you must learn to be satisfied,
retaining at the time with due humility an earnest desire for an ever larger portion.
Sir William Osler (1892)
4.1 Introduction
Fetal scalp blood sampling is the principal technique utilised to collect blood for biochemical
assessment of fetal wellbeing during labour; however, all experimental studies investigating
the ability of biochemical markers to predict fetal brain injury after asphyxia have assessed
blood samples collected from central blood vessels rather than the fetal scalp, due to
experimental design limitations.
In the previous chapter I presented results from my fetal sheep study demonstrating a
significant correlation between fetal carotid arterial lactate levels and brain injury. Similar
associations between central arterial lactate levels and fetal brain injury have been
demonstrated by others in sheep studies of asphyxia (Ikeda et al., 1998a). Prior to the
potential introduction of fetal scalp lactate measurement into clinical practice, two important
issues require investigation. First, what is the relationship between fetal scalp blood and
central blood lactate levels and how does asphyxia alter this relationship? Second, and most
important, do fetal scalp lactate levels, rather than central lactate levels, predict fetal brain
injury? The studies presented in this chapter aim to address these two important questions.
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4.1.1 Development of an experimental model
In both animal and human studies investigating fetal scalp blood samples, a variety of
experimental designs have been utilised. All of these study designs have limitations which
require consideration prior to selection of the experimental model used in this chapter.
Human studies investigating the relationship between central and peripheral biochemical
markers of fetal wellbeing are limited by time delays between scalp blood sample collection
and cord blood collection. In some studies the delay between scalp and cord blood sampling
has been short (1-10 minutes); however, in these studies either the fetal head was on view at
the maternal perineum or the fetus was delivered by forceps or caesarean section under
maternal anaesthesia (Bowe et al., 1970; Yoshioka & Roux, 1970). In other studies the delays
between scalp and cord sampling have been as great as 60 minutes making interpretation of
the results more challenging (Kruger et al., 1998). Human studies investigating the
relationship between fetal scalp blood biochemical markers and brain injury are also limited
by the low incidence of fetal brain injury resulting from intrapartum events.
Animal experimental models can overcome the issue of time delays between sampling from
different fetal sites by allowing simultaneous blood sampling from the fetal scalp and central
blood vessels during asphyxia. Previous studies investigating the relationship between fetal
scalp blood pH and central blood pH have all used acutely instrumented fetuses. These
studies have primarily utilised partially exteriorised fetuses in either monkeys (Adamsons et
al., 1970) or sheep (Gare et al., 1967; Shier & Dilts, 1972; Aarnoudse et al., 1981). There has
been one small study in monkeys where fetuses were instrumented, returned to the uterus
and fetal scalp samples were collected vaginally during induced labour while the mother
remained under anaesthesia. In this study a median of 1 scalp sample could be collected on
each of the 11 monkeys in the study (Adamsons et al., 1970). There have been no
experimental studies sampling both fetal scalp blood and central blood in chronically
instrumented fetuses.
No animal studies have investigated the relationship between brain injury and biochemical
markers measured in fetal scalp blood. The lack of studies in this area probably relates to
experimental design limitations. Although asphyxia can be induced and scalp samples can be
reliably collected in acutely instrumented fetuses, the experimental model is not conducive to
analysis of histopathologic brain damage which can take three to seven days to become
apparent after the insult (Nakajima et al., 2000). It is this significant experimental limitation
which is likely to have limited study in this important area.
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In this study I have chosen to use an acutely catheterised, partially exteriorised, sheep model
to investigate both the relationship between scalp lactate levels and central lactate levels and
also to investigate the relationship between fetal scalp lactate and precursors of brain injury.
This model was chosen because it allowed regular, reliable, repeated collection of adequate
volumes of fetal scalp blood during asphyxia to explore the relationships between fetal scalp
lactate and central lactate levels both at rest and during asphyxia. Concerns have been raised
previously in the interpretation of acutely instrumented models because maternal anaesthesia
is thought to adversely affect autonomic control of the fetal heart (Levinson et al., 1973; Biehl
et al., 1983a; Biehl et al., 1983b; Bachman et al., 1986; Alon et al., 1993) and the responsiveness
of the fetal heart rate to hypoxaemia (Yarnell et al., 1983; Brett et al., 1989). In some studies,
such as those which I have presented in this chapter, the collection of fetal scalp blood
necessitates an acutely instrumented model. In the acutely instrumented model that I have
used for the studies in this chapter the anaesthetic agents were selected to minimise any
adverse affects on the maternal and fetal cardiovascular and autonomic systems. Prior to
exploration of the peripheral-central lactate relationships, I have investigated the merit of the
acute model used in this study by comparing and contrasting it with the chronic model
described in Chapter Three. This evaluation was performed to aid in the interpretation of
the data obtained from the acute model.
4.1.2 The relationship between fetal scalp lactate levels and central lactate levels
Despite the growing evidence that fetal lactate levels may predict fetal brain injury in animal
models of asphyxia (Ikeda et al., 1998a) and observational studies of human labour (Kruger et
al., 1999), little is known about the relationship between scalp blood lactate levels and central
arterial lactate levels. To my knowledge, there have been only two studies that have
investigated the relationship between peripheral and central fetal lactate levels (Yoshioka &
Roux, 1970; Kruger et al., 1998). The first study compared lactate levels from three sampling
sites (fetal scalp, umbilical artery and umbilical vein) in 14 human fetuses, six who were
delivered vaginally with forceps and eight who were delivered by caesarean section (Yoshioka
& Roux, 1970). The scalp blood samples were collected one to three minutes prior to
delivery and collection of umbilical cord samples. There were no significant differences in
the mean lactate concentrations from the three sampling sites in either those fetuses delivered
vaginally or those delivered by caesarean section. On detailed inspection of the results of this
study, lactate levels in scalp blood and umbilical artery blood were identical in babies
delivered by caesarean section whereas scalp lactate levels were lower than umbilical arterial
levels if the fetus was delivered vaginally. Furthermore, if the fetus was considered
159
“distressed” by the authors, scalp lactate levels were less than umbilical artery lactate levels
irrespective of the mode of delivery (Yoshioka & Roux, 1970). These differences, although
potentially clinically significant, did not reach statistical significance; however, only six of the
14 babies studied were delivered vaginally and five of the 14 fetuses studied were considered
“distressed”. The second study addressing the relationship between central and peripheral
lactate levels compared 103 human scalp lactate samples with umbilical artery and vein lactate
samples where the scalp blood was collected within 60 minutes of delivery. Lactate
concentration in fetal scalp blood before delivery showed a modest correlation with umbilical
artery lactate and umbilical vein lactate levels (Kruger et al., 1998). In the study by Yoshioka
et al. (1970) the group means for different sampling sites were compared without considering
the paired nature of the samples and in the study by Kruger et al. (1970) simple univariate
correlation was used to assess the relationship between central and peripheral lactate levels
without considering the wide variation in time between collection of the paired samples. No
published studies have used appropriate paired statistical analysis techniques to evaluate the
differences in lactate levels in blood collected simultaneously from different sites.
Although there have been two studies which have partially investigated the relationship
between fetal scalp lactate levels and umbilical vessel lactate levels, there have been no studies
investigating the relationships between fetal scalp blood lactate, carotid arterial lactate and
jugular venous lactate levels. In addition, there have been no studies investigating the effect
of asphyxia on the relationship between central and peripheral lactate levels. The significance
of investigating the effect of asphyxia on central-peripheral biochemical relationships is
illustrated by studies validating fetal scalp pH measurement, our current biochemical
technique for intrapartum assessment of fetal wellbeing. Although it is generally accepted
that fetal scalp pH levels approximate central fetal pH levels, previous studies have
demonstrated that the relationship between scalp blood pH and fetal carotid and umbilical
arterial pH is influenced by fetal acidaemia, hypoxia, skin oedema and caput succedaneum
(Adamsons et al., 1970; Bowe et al., 1970; Yoshioka & Roux, 1970; Shier & Dilts, 1972;
Greene, 1999).
In the series of studies described in this chapter, I have investigated the relationship between
fetal scalp lactate and carotid arterial and jugular venous lactate levels both at rest and during
asphyxia induced by a cord occlusion protocol designed to mimic events which may occur in
labour.
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4.1.3 The relationship between fetal scalp lactate levels and fetal brain injury
The experiments performed in this chapter were terminal and the passage of several days to
evaluate the evolution of neurological injury was not possible. As an alternative I have
measured oxygen free radical activity in the fetal brain, an indirect marker of potential
neuronal injury. Oxygen free radical activity was assessed both during and after the asphyxial
insult and was related to scalp lactate and other biochemical markers of fetal wellbeing.
4.1.3.1 Oxygen free radical activity and fetal brain injury
Since the first suggestion 25 years ago that oxygen free radicals may be an important
mediator of ischaemic brain injury (Flamm et al., 1978), a vast body of evidence has
developed which suggests that at a molecular level oxygen free radicals are critically involved
in the mechanisms of hypoxic-ischaemic brain injury (Maulik, 1998; Volpe, 2001b; du Plessis
& Volpe, 2002; Gitto et al., 2002; Grow & Barks, 2002; Inder et al., 2002). Substantial
experimental data are available that links oxidative stress with other pathogenic mechanisms
such as excitotoxicity, calcium overload, mitochondrial cytochrome C release, and caspase
activation in the central nervous system (Choi, 1988; Siesjo, 1988; Hockenbery et al., 1993;
Lewen et al., 2000). There is also a large amount of data to suggest that oxygen free radicals
are important in initiating apoptosis (MacManus et al., 1993; Buttke & Sandstrom, 1994;
Fernandez et al., 1995; Polyak et al., 1997; Saikumar et al., 1998), the well known cellular
suicide program which is thought to be an important part of the second wave of neuronal
cell damage that occurs during reperfusion after termination of an hypoxic-ischaemic insult
(Buonocore et al., 2001). Free radical mediated cell death also appears to be the final
common pathway for oligodendroglial cell death after hypoxic-ischaemic insults (Volpe,
2001b).
Oxygen free radicals avidly interact with the essential organic molecules of a cell including
lipids, proteins, and DNA (Figure 4.1). Consequent to these reactions, free radicals affect
cellular and extracellular constituents of neural tissue, profoundly alter its function, and may
lead to immediate necrotic or delayed programmed death of neurons (See Chapter One,
Figure 1.3; Maulik, 1998).
In this study I have focused on lipid peroxidation as a footprint of free radical activity in the
fetal brain. Lipid peroxidation was selected because it has an important role in both
mediating and extending oxidative neuronal injury (Vanucci, 1994). In addition, lipid
peroxidation is one of the earliest signs of oxidative stress, with elevated levels being
detectable within minutes of brain injury (Vagnozzi et al., 1999). Previous ovine studies using
161
ReactiveOxygenSpecies
Lipids Proteins DNA
Lipid PeroxidationLipid Radical Formation
DegredationAggregation
Protein Radical Formation
Strand BreaksBase Alterations
Membrane injury and dysfunctionTransmembrane ionic imbalanceAlteration of membrane fluidity
Alteration of membrane transport proteinsImpaired repair of DNAAltered gene expression
Mitochondrial dysfunction - decoupling of respirationMitochondrial permeability transition formation
Altered nuclear pore permeabilityInitiation of programmed cell death
Figure 4.1 Adverse effects of oxidative stress on neuronal lipids, protein and,
deoxyribonucleic acid. (DNA).
(Adapted from Maulik, 1998)
162
umbilical cord occlusion to induce asphyxia have shown evidence of lipid peroxidation in the
fetal circulation and brain perfusate (Masaoka et al., 1998; Ikeda et al., 1999; Rogers et al.,
2001).
Lipid peroxidation is the process by which unsaturated lipids react with molecular oxygen to
undergo ‘autoxidation’ or ‘peroxidation’ as it is now more widely known (Gutteridge &
Halliwell, 1990). The brain has a large lipid content: 75% of myelin, 55% of white matter,
and 35% of grey matter (Siegel et al., 1989) and the phospholipid bilayer in the neuronal cell
membrane is rich in polyunsaturated fatty acids, thereby increasing the susceptibility of
neurons to peroxidative damage. The presence of unsaturated carbon-to-carbon bonds in
the lipids enhances their roles as electron donors and the lower the saturation of these fatty
acids, the greater the propensity of oxidative reactions with free radicals (Maulik, 1998). The
occurrence of lipid peroxidation in biological membranes has been shown to cause
impairment of membrane function, decreased membrane fluidity, inactivation of membrane-
bound receptors and enzymes, and to increase non-specific permeability to ions such as
calcium (Slater & Sawyer, 1971; Eklow-Lastbom et al., 1986; Comporti, 1987; Gutteridge,
1988; Halliwell & Gutteridge, 1989). In addition, lipid hydroperoxides decompose upon
exposure to transitional metals, metal complexes and some iron proteins including
haemoglobin and myoglobin (Gutteridge, 1988; Halliwell & Gutteridge, 1989) which results
in an ever-expanding chain reaction that leads to the production of more free radicals, lipid
peroxyl and alkoxyl radicals. These attack more lipids and proteins locally, and also diffuse
out of their site of generation and produce distant effects (Halliwell & Chirico, 1993).
There is evidence from both animal and human studies showing that levels of lipid
peroxidation measured in both brain and plasma correlate with both the timing and the
severity of brain injury after hypoxic-ischaemic insults (Russell et al., 1992; Tan et al., 1998;
Ikeda et al., 1999; Candelario-Jalil et al., 2001; Marumo et al., 2001; Yu et al., 2003). When near
term rabbit fetuses were exposed to repetitive ischaemia-reperfusion (RIR) or sustained
uterine ischaemia-reperfusion (IR), the fetuses subjected to RIR were found to have
significantly more brain injury and lower cortical cell viability than IR fetuses and controls
(Tan et al., 1998). In addition the fetuses exposed to RIR had higher levels of lipid
peroxidation, 3-nitrosine, and nitrogen oxides and lower total antioxidant capacity than IR or
control fetuses. It was proposed that the higher free radical production in the fetuses
exposed to repeated ischaemia-reperfusion may be responsible for the greater fetal brain
injury in this experimental group (Tan et al., 1998). In a series of studies of transient cerebral
ischaemia in gerbils, the time course of oxidative damage in different brain regions was
163
assessed. It was found that the timing of maximal disturbance in oxidant-antioxidant balance
in the hippocampus corresponded with the time course of neuronal loss in the hippocampal
CA1 sector (Candelario-Jalil et al., 2001). In preterm human neonates, a fourfold increase in
levels of hypoxanthine (which is broken down with the aid of xanthine oxidase and oxygen
to produce oxygen free radicals) has been noted in neonates who develop cavitating
periventricular leukomalacia compared to age- and weight-matched controls who did not
develop the condition (Russell et al., 1992). Term human infants with asphyxia and hypoxic-
ischaemic encephalopathy (HIE) have been shown to have higher plasma levels of markers
of lipid peroxidation at birth than both asphyxiated neonates without HIE and healthy term
newborn infants (Yu et al., 2003). In addition when the infants were followed up at 5 months
of age, all of the babies with asphyxia without HIE and the healthy term newborn infants
were normal whereas one third of the term neonates with asphyxia, HIE and elevated lipid
peroxidation at birth were neurologically impaired.
In addition to the evidence that elevated levels of markers of lipid peroxidation are associated
with the severity of fetal brain injury, there is also evidence that treatment with novel
inhibitors of lipid peroxidation can protect neurons against damage (Andrus et al., 1997; Tan
et al., 1998, 1999; Wada et al., 1999; Ercan et al., 2001; Chang et al., 2003). When these drugs
are administered after both hypoxic-ischaemic injury (Tan et al., 1998; Chabrier et al., 1999;
Tan et al., 1999) and traumatic brain injury (Inci et al., 1998; Chabrier et al., 1999; Wada et al.,
1999; Ercan et al., 2001; Chang et al., 2003) there is less histopathologic brain damage than in
non-treated animals.
4.1.4 Aim
There are three specific aims for the studies presented in this chapter:
1. to evaluate the acutely instrumented model of repeated cord occlusion to assess
fetal scalp lactate levels during asphyxia,
2. to investigate the relationship between fetal scalp, carotid arterial and jugular
venous blood lactate levels both at rest and during asphyxia, and
3. to investigate the relationship between fetal scalp blood lactate levels and lipid
peroxidation in the fetal brain.
164
4.1.5 Hypothesis
There are two hypothesis for the experiments performed in this chapter:
1. The relationship between fetal scalp blood lactate and central blood lactate levels
are influenced by fetal asphyxia
2. Fetal scalp blood lactate will be a better predictor of lipid peroxidation in the fetal
brain than arterial pH.
165
4.2 Methods
All experimental procedures were approved by the Wentworth Area Health Service Scientific
Advisory Panel and Ethics Committee and the Westmead Hospital Animal Ethics
Committee, New South Wales, Australia. Merino ewes with singleton pregnancies were
transported to the research facility at 125 days of pregnancy to allow for acclimatisation.
4.2.1 Surgical Procedure
Thirty two ewes underwent surgery at 135±4 days (mean±SEM) of pregnancy. Anaesthesia
was induced with Thiopentone 4mg/kg in 15mL saline solution (Abbott Australasia Pty Ltd,
Cronulla, NSW, Australia), intubation performed, and anaesthesia maintained with 0.75-
1.25% Isoflurane in 100% oxygen delivered by positive pressure ventilation with the
ventilator settings selected to maintain maternal blood gases in the normal range. A maternal
femoral arterial catheter was inserted to enable continuous monitoring of maternal blood
pressure and blood gas status. Maternal blood pressure was recorded continuously onto a
computer at 1 KHz after amplification (BP transducer MLT380, bridge amplifier ML110,
PowerLab 8e, ADInstruments, Sydney, Australia). Maternal blood gas status was assessed
every 15 minutes and ventilator settings were selected to maintain maternal blood gases in the
normal range.
The uterus was exposed through a paramedian abdominal incision and a hysterotomy was
performed to deliver the fetal head, neck and upper thorax with care to minimize amniotic
fluid leakage. Vascular catheters were inserted into the left fetal carotid artery and jugular
vein. Fetal electrocardiograph (ECG) leads were sutured to the fetal chest and a vascular
snare was sutured around the umbilical cord to enable intermittent complete cord occlusion.
A circular three crystal ultrasound cuff (10MHz Triton Technology Inc., USA) was loosely
applied around the umbilical vessels to enable confirmation of normal umbilical blood flow
and complete obstruction of umbilical blood flow during cord occlusion. The fetal scalp was
shaved and the fetus was returned to the uterus with its head externalised to facilitate
repeated collection of fetal scalp blood samples. Care was taken to prevent loss of amniotic
fluid and thermal stability was maintained with overhead radiant warmers and bubble wrap
covering the fetal head between scalp blood samples to minimize heat and water loss.
166
After a thirty minute recovery phase, baseline fetal carotid arterial blood samples (0.3mL)
were collected for baseline measurement of arterial gas tensions and pH (Radiometer ABL
300, Radiometer Pty Ltd, Copenhagen, Denmark) and lactate (Accutrend® Lactate, Roche
Diagnostics GmbH, Mannheim, Germany). Repeat fetal arterial samples (0.3mL) were
collected after a further 30 minute rest phase and if the fetal preparation was stable the cord
occlusion protocol was commenced. In three of the 32 fetuses instrumented in this study,
the second arterial pH was higher than the first and a further 30 minute rest phase and
arterial blood sample were required to confirm stability of the fetal preparation.
4.2.2 Cord occlusion protocol
After confirmation that each fetal preparation was stable, each animal entered the
experimental phase of the study. Fetal arterial blood samples (4.0mL) were collected to
measure baseline arterial gas tensions, pH, lactate, glucose, cortisol, organic hydroperoxide
(OHP), malondialdehyde (MDA) and catecholamine concentrations. Fetal blood (0.4mL)
was also collected from the jugular venous catheter to measure lactate levels, OHP and
MDA. Silicone jelly was applied to the fetal scalp and a scalp blood sample was collected in a
heparinised capillary tube (20µL) from a stab wound for lactate measurement.
Twenty-four of the 32 instrumented fetuses were randomly allocated to the same umbilical
cord occlusion (UCO) protocol utilised in the previous chapter and described in detail in
Section 3.2.2. In brief, 27 complete umbilical cord occlusions were performed every three
minutes for a total of 81 minutes; the first nine occlusions were for 0.5-minutes duration
every 3 minutes; the second nine occlusions were 1.0-minute duration every 3 minutes; and
the third nine occlusions were for 1.5-minutes duration every 3 minutes. The remaining eight
fetuses served as controls with blood samples and other measurements taken as scheduled
for the experimental group but without UCO. After each fetal blood sample was withdrawn,
sterile heparinised saline was infused to replace the volume removed for analysis.
Fetal blood samples were collected every nine minutes throughout the experimental phase.
Carotid arterial blood (0.6mL) was sampled to measure arterial gas tensions, pH, lactate,
OHP and MDA. Jugular venous blood (0.5mL) was sampled to measure lactate, OHP and
MDA levels. Fetal scalp blood samples (20µL) were collected to measure lactate levels.
Additional arterial samples (1.0mL) were collected at 27 and 54 minutes to measure fetal
glucose levels. At the end of the experimental phase, a final arterial blood sample (4.0mL)
was collected to measure glucose, cortisol, organic hydroperoxide (OHP), malondialdehyde
167
(MDA) and catecholamine concentrations. The ewes and fetuses were then humanely killed
using a lethal dose of sodium phenobarbitone and the positions of the implanted catheters
were confirmed and fetal weights measured.
4.2.3 Fetal heart rate and arterial pressure assessment
Fetal heart rate and arterial pressure responses to repeated cord occlusion were recorded
throughout the experimental phase to allow validation of the acutely instrumented fetal
preparation. Fetal ECG and blood pressure were recorded continuously onto a computer at
1KHz after amplification (Bioamp, BP transducer MLT380, bridge amplifier ML110,
PowerLab 8e, ADInstruments, Sydney, Australia). FHR and FHR variability were assessed
over the 1 minute prior to each cord occlusion. Mean fetal heart rate, mean minute range
(MMR), short term variation (STV) and long term variability (LTV) were calculated using the
methods described in Section 3.2.3. Fetal arterial pressure was measured for each heart beat
and averaged for the 1 minute preceding each cord occlusion.
4.2.4 Umbilical blood flow
The ultrasound cuff around the umbilical vessels was linked to a vascular spectrum analyser
(Model SP 25 by Vasculab, Meda Sonics, USA) which enabled continuous monitoring of the
umbilical artery Doppler waveforms to confirm normal umbilical blood flow between
occlusions and complete obstruction of umbilical blood flow during cord occlusion.
4.2.5 Biochemical assessment
Plasma samples for glucose, cortisol, OHP, MDA and catecholamine measurement were
recovered after centrifugation at 1500g for 10 minutes at 4°C and were stored at -80°C until
analysed. Plasma cortisol, adrenaline, and noradrenaline were measured using the same
techniques as described in Section 3.2.6. Fetal lipid peroxide measurements were performed
by Dr CC Wang in the Chinese University of Hong Kong. The method used for
determination of organic hydroperoxides was the enzymatic technique described by Heath
and Tapel (1976) with modification as previously published (Wang et al., 1996; Rogers et al.,
2001). Briefly, 50µL plasma and 350µmol/L Tris-ethylenediamine-tetraacetic acid, pH 7.6,
were incubated with 5µmol/L catalase (17,500kU/L) at room temperature for ten minutes.
Then 25µmol/L reduced nicotinamide adenine dinucleotide phosphate (2mmol/L), 5µmol/L
glutathione peroxidase (25,000kU/L) and 50µmol/L glutathione (4.25mmol/L) were added.
168
After further incubation at 33°C for 10 minutes the absorbance of the reaction mixture at
340nm was measured on a response spectrophotometer (Gilford Instruments Laboratory,
Oberlin, Ohio). A 2.5µL aliquot of glutathione reductase (100kU/L) was added immediately,
and the reaction mixture was allowed to proceed for another ten minutes at 33°C. Its
absorbance at 340nm was again measured to determine the net amount of reduced
nicotinamide adenine dinucleotide phosphate oxidised. Values were expressed by
comparison with a standard of butyl hydroperoxide (0 to 600umol/L). The coefficient of
variation (CV) within assays was 5.8% and the between assay CV was 11.4%.
Malondialdehyde levels were estimated as reactive substances by a thiobarbituric acid
adduction method described by Richard et al. (1992) with modification. The primary reagents
were thiobarbituric acid/perchloric acid (2:1, vol/vol) mix, a calibration solution of 1,1,3,3-
tetraethoxypropane (10µmol/L) and butylated hydroxy-toluene. Each analytic run included
the assay of a reagent blank, tetraethoxypropane working standard solutions, plasma
specimens and quality control samples. The continuation of lipid peroxidation during the
reaction was prevented by addition of 10µmol/L butylated hydroxy-toluene (20gm/L). In
each reaction tube 100µL plasma and 750 µmol/L thiobarbituric acid-perchloric acid mix
were combined, vortex mixed, tightly capped and placed in a 95°C water bath. After 60
minutes the samples were chilled on ice to stop the reaction. Reagent blanks, standard
blanks, and assay blanks were left at room temperature. Two millilitres of n-butanol was
added to each tube to extract the thiobarbituric acid-malondialdehyde complex by vigorous
vortex mixing and centrifugation. The thiobarbituric acid-malondialdehyde adduct in the
butanol extract was determined by fluorometry at 532nm excitation and 553nm emission.
The CV was 1.7% within assays and 8.2% between assays.
4.2.6 Study group classification
Recent evidence suggests that spontaneously acidaemic ovine fetuses have altered
cardiovascular, endocrine and metabolic responses to acute hypoxaemia (Gardner et al.,
2002). It has been suggested by these authors that fetuses with mild spontaneous acidaemia
should either be considered as a subgroup or excluded from studies of hypoxia due to their
altered physiology. For this reason I have retrospectively classified the fetuses in this study
into two groups using a classification system previously described in ovine models of
hypoxaemia (Gardner et al., 2002): non-acidaemic (arterial baseline pH>7.25, n=18) and
spontaneously acidaemic (arterial baseline pH<7.25, n=14). As described in Section 4.2.2,
twenty-four fetuses were randomly allocated to the UCO protocol and eight served as
169
controls. Six of the eight controls were non-acidaemic and two were spontaneously
acidaemic. For clarity in presentation of results, the non-acidaemic fetuses will be referred to
as “Normal” (N), the fetuses with a spontaneous baseline metabolic acidosis will be referred
to as “Spontaneously Acidaemic” (SA) and the instrumented fetuses that were not subjected
to UCO will be referred to as “Controls” (C).
4.2.7 Statistical methods
Descriptive data are presented as mean and standard error of the mean (SEM) unless
otherwise stated. Univariate analyses of categorical data were performed with Chi-square test
and continuous data were analysed with t-tests or the non-parametric equivalent as
appropriate. Changes in fetal biochemical variables and cardiovascular parameters were
analysed using multivariate regression modelling with occlusion, time and preparation type
(acute or chronic) modelled as fixed effects and individual animals as random effects repeated
over time. Regression modelling was also used to analyse differences between samples
collected simultaneously from the three sites compared in this study matched by time and
sheep. Variable selection in multivariate modelling was based on stepwise procedures
(forwards and backwards selection) including the assessment of interaction terms to arrive at
the most parsimonious model. Comparisons of effect magnitude were based on evaluation
of changes in each outcome for one standard deviation of each independent variable
considered.
To assess the relationship between fetal scalp lactate and lipid peroxidation in the fetal brain
perfusate two types of analyses were performed. First, fetal arterial and venous OHP levels
were plotted for each individual fetus. The first time point when the venous OHP levels
began to rapidly rise (which exceeded the mean+SEM for venous OHP in controls) was
identified for each fetus. The scalp lactate and carotid artery pH were extracted for these
time points and used to calculate the range in pH and lactate associated with the
commencement of lipid peroxidation in fetal brain perfusate. Comparisons between the
proportions of pH and lactate values which were within this range were performed using
Chi-squared analysis of contingency tables. Second, receiver operator curve (ROC) analysis
was performed to evaluate the ability of biochemical markers to differentiate lipid
peroxidation in the fetal brain perfusate (jugular venous OHP - carotid artery OHP > 0).
(Hanley & McNeil, 1983; Beck & Shultz, 1986; DeLong et al., 1988). Optimum sensitivity
and specificity were chosen from the ROC tables to maximise the total of these two
170
measures of diagnostic accuracy (Hanley & McNeil, 1983). Accuracy is reported for
biochemical prediction of blood pressure responses during UCO and is defined as the
proportion of correctly classified cases (true positives + true negatives) using the biochemical
cut off point selected to maximise the sensitivity and specificity (Zweig & Campbell, 1993).
P-values<0.05 were considered statistically significant and p-values<0.10 were considered
marginally significant. SAS statistical software was used for data analysis (Statistical Analysis
Systems Institute Inc. 1999, Version 8.2., Cary, NC).
171
4.3 Results
4.3.1 Baseline fetal biochemical status prior to the experimental phase
The demographic and baseline fetal biochemical data prior to the experimental phase of this
study are presented in Table 4.1. Fetal body weights and gestational ages were similar
between the two study groups. Meconium-stained amniotic fluid was more common in the
spontaneously acidaemic fetuses (p=0.064). As a result of the retrospective classification,
fetal arterial pH was significantly lower in the spontaneously acidaemic fetuses (7.16±0.02)
when compared to normal fetuses (7.28±0.01; p<0.001). Similarly, arterial base excess was
significantly less in acidaemic fetuses (-10.19±0.84mmol/L) than normal fetuses (-6.51±1.01;
p=0.010). Fetal arterial gas tensions, lactate, glucose, cortisol and catecholamine levels were
not significantly different between the two study groups (Table 4.1).
Table 4.1 Baseline fetal biochemical condition prior to experimental phase.
Normal Acidaemic p-value
Number 18 14
Gestational age (days) 136 ± 1 134 ± 2 0.238
Weight (kg)* 3.40 ± 0.39 3.42 ± 0.37 0.286
Amniotic fluid meconium 1/18 5/14 0.064
Arterial pH 7.28 ± 0.01 7.16 ± 0.02 < 0.001
Arterial PO2 (mmHg) 19.91 ± 1.87 22.13 ± 3.00 0.517
Arterial PCO2 (mmHg) 45.28 ± 3.45 52.93 ± 3.60 0.140
Arterial Base Excess (mmol/L) -6.51 ± 1.01 -10.19 ± 0.84 0.010
Arterial Lactate (mmol/L) 4.01 ± 0.40 4.94 ± 0.80 0.307
Glucose (mmol/L) 1.21 ± 0.15 1.22 ± 0.14 0.970
Cortisol (ng/mL) 12.48 ± 1.98 17.15 ± 3.34 0.263
Adrenaline (ng/L) 0.49 ± 0.28 0.78 ± 0.36 0.532
Noradrenaline (ng/L) 22.58 ± 6.76 20.80 ± 6.38 0.862 * Measured at post-mortem
172
4.3.2 Investigation of the validity of the acutely catheterised model for the
assessment of fetal scalp blood samples
I have compared control and normal fetuses that underwent UCO in this study with fetuses
(regular, fast responders and controls) in the chronically catheterised model described in
Chapter Three. Fetal acid-base status, lactate, glucose, cortisol and catecholamine levels as
well as FHR, measures of FHR variability and inter-occlusion arterial pressure were
compared between the two models.
4.3.2.1 Fetal acid-base status and lactate responses
Fetal arterial PO2, PCO2, and base excess levels in controls were not significantly different
between the acute and chronically instrumented models (p>0.05; Figure 4.2). Acutely
instrumented controls had lower arterial pH (acutely instrumented pH 7.28±0.01 vs.
chronically instrumented pH 7.39±0.02, p=0.002) and higher arterial lactate levels (acutely
instrumented lactate 4.3±0.5mmol/L vs. chronically instrumented lactate 1.4±0.6mmol/L,
p=0.003) than chronically instrumented controls. In fetuses that underwent UCO, the
patterns of change in all measures of fetal acid-base status were similar in both acute and
chronically instrumented fetuses; however, the magnitude of the changes was different for
pH, PO2 and PCO2. Acutely instrumented fetuses that experienced UCO had significantly
greater changes in arterial pH (p=0.002), PO2 (p=0.013) and PCO2 (p<0.001) than chronically
instrumented fetuses exposed to the same occlusion protocol. Lactate and base excess
responses during UCO were not significantly different between experimental models
(p>0.05).
4.3.2.2 Fetal endocrine responses
Fetal arterial glucose, cortisol, and adrenaline levels in controls were not different between
the two experimental models (all p>0.200; Figure 4.3). Fetal noradrenaline levels in acutely
instrumented controls (25.9±6.8ng/L) were higher than chronically instrumented controls
(4.3±1.1ng/L; p=0.002). Cortisol, noradrenaline and adrenaline levels were significantly
increased in fetuses that underwent UCO in both experimental models (all p<0.05; Figure
4.3). Fetal glucose levels in fetuses that underwent UCO were increased compared to
controls in both experimental models; however, p-values associated with these increases were
only marginally significant (p-values 0.05-0.10). The magnitude of the increases in glucose,
cortisol and adrenaline levels in fetuses that underwent UCO were not significantly different
172
Figure 4.2 Comparison of arterial gas tensions, pH, base excess and lactate levels in acute and
chronically instrumented ovine fetuses during repeated umbilical cord occlusion. Data are
presented as mean±SEM. The p-values listed compare the experimental groups between
surgical models (control vs. control; occluded vs. occluded). Non-significant (ns)
comparisons are defined as p-values>0.10. A similar pattern of changes occurred in arterial
gas tensions, pH, base excess and lactate levels in both experimental models. The acidaemia
which developed in acutely instrumented fetuses had a greater respiratory component than
chronically instrumented fetuses, with acutely instrumented fetuses having greater decreases
in pH (p=0.034) and PO2 (p=0.013), and a significantly greater increase in PCO2 (p>0.001)
than chronically instrumented fetuses.
173
0 27 54 816.7
6.9
7.1
7.3
7.5 pH
Time (minutes)
Arte
rial p
H
0 27 54 810
10
20
30
40PO2
Time (minutes)
Arte
rial P
O2
(mm
Hg)
0 27 54 810
50
100
150
200PCO2
Time (minutes)
Arte
rial P
CO
2 (m
mH
g)
0 27 54 816.7
6.9
7.1
7.3
7.5 pH
Time (minutes)
Arte
rial p
H
0 27 54 810
10
20
30
40PO2
Time (minutes)
Arte
rial P
O2
(mm
Hg)
0 27 54 810
50
100
150
200PCO2
Time (minutes)
Arte
rial P
CO
2 (m
mH
g)
0 27 54 81-20
-10
0
10Base Excess
Time (minutes)
Arte
rial B
ase
Exce
ss(m
mol
/L)
0 27 54 810
5
10
15Lactate
Control Occluded
Time (minutes)
Arte
rial L
acta
te(m
mol
/L)
0 27 54 81-20
-10
0
10Base Excess
Time (minutes)
Arte
rial B
ase
Exce
ss(m
mol
/L)
Chronically InstrumentedModel
0 27 54 810
5
10
15Lactate
Control Occluded
Time (minutes)
Arte
rial L
acta
te(m
mol
/L)
Acutely InstrumentedModel
Control(p=0.002)
Occluded(p=0.002)
Control(ns)
Occluded(p=0.013)
Occluded(p<0.001)
Control(ns)
Control(ns)
Occluded(ns)
Occluded(ns)
Control(p=0.003)
173
Figure 4.3. Comparison of endocrine responses in acute and chronically instrumented ovine
fetuses during repeated umbilical cord occlusion. The p-values compare similar experimental
groups between models and non-significant (ns) comparisons are defined as p-values>0.10.
Glucose, cortisol and adrenaline levels in both controls and fetuses that underwent UCO
were not significantly different between the two experimental models. The acutely
instrumented controls had higher baseline noradrenaline levels than chronically instrumented
controls (p=0.002) and the magnitude of the increase in noradrenaline in acutely
instrumented fetuses that underwent UCO was less than the increase seen in chronically
instrumented fetuses that underwent UCO (p=0.024).
174
0 27 54 810
10
20
30
40Cortisol
Time (minutes)C
ortis
ol (n
g/m
L)
0 27 54 810
50
100
150Noradrenaline
Time (minutes)
Nor
adre
nalin
e (n
g/L)
0 27 54 810
50
100
150Noradrenaline
Time (minutes)
Nor
adre
nalin
e (n
g/L)
0 27 54 810
10
20
30
40Cortisol
Time (minutes)
Cor
tisol
(ng/
mL)
0 27 54 810
20
40
60
80Adrenaline
Control Occluded
Time (minutes)
Adre
nalin
e (n
g/L)
0 27 54 810
1
2
3
4
5Glucose
Time (minutes)
Glu
cose
(mm
ol/L
)
0 27 54 810
1
2
3
4
5Glucose
Time (minutes)
Glu
cose
(mm
ol/L
)
0 27 54 810
20
40
60
80Adrenaline
Control Occluded
Time (minutes)
Adre
nalin
e (n
g/L)
Control(ns)
Occluded(ns)
Control(ns)
Occluded(ns)
Occluded(p=0.024)
Control(p=0.002)
Control(ns)
Occluded(ns)
Chronically InstrumentedModel
Acutely InstrumentedModel
175
between the two models (p>0.05) whereas the magnitude of the increase in noradrenaline
levels in acutely instrumented fetuses (22.5±4.9ng/L to 45.4±6.4ng/L, p=0.009) was
significantly less than in chronically instrumented fetuses (5.2±1.3ng/L to 114.2±31.4ng/L,
p<0.001; group difference p=0.024).
4.3.2.3 Fetal heart rate responses
The pattern of FHR responses to the experimental phase of the study was similar in both the
acute and chronically instrumented fetuses (Figure 4.4). FHR in acutely instrumented
controls (141.3±4.3bpm) was significantly lower than chronically instrumented controls
(173.0±4.4bpm; p<0.001). Similarly, the three measures of FHR variability in acutely
instrumented controls were lower than in chronically instrumented controls (acute MMR
27.5±2.3msec vs. chronic MMR 36.7±2.4msec, p<0.001; acute STV 2.00±0.33msec vs.
chronic STV 2.87±0.37msec, p<0.001; acute LTV 12.4±1.0bpm vs. chronic LTV 16.4±1.0
bpm, P=0.006).
In both acutely instrumented fetuses that underwent UCO (p<0.001) and chronically
instrumented fetuses that underwent UCO (p=0.003) there was a significant increase in inter-
occlusion FHR over the duration of the study. The magnitude of the increase in inter-
occlusion FHR in acutely instrumented fetuses was significantly greater than chronically
instrumented fetuses (p=0.007). MMR, STV and LTV were significantly increased in fetuses
that experienced UCO compared to controls in both experimental models (p>0.05; Figure
4.4) and the magnitude of these increases were not significantly different between models
(MMR p=0.267, STV p=0.241, LTV p<0.967).
4.3.2.4 Fetal arterial pressure responses
The inter-occlusion arterial pressure for the acutely instrumented fetuses in this study and the
chronically instrumented fetuses described in the previous chapter are presented in Figure
4.5. Fetal arterial systolic and diastolic pressures in both controls and fetuses that underwent
UCO were not significantly different between the two experimental models (p>0.10).
175
Figure 4.4 Comparison of FHR and FHR variability responses in acute and chronically
instrumented fetuses during repeated umbilical cord occlusion. The p-values shown compare
similar experimental groups between models. The magnitude of increase in inter-occlusion
FHR in acutely instrumented fetuses in response to UCO was significantly greater than
chronically instrumented fetuses (p=0.007). The MMR, STV and LTV responses to UCO
were not significant different (ns) between the two experimental models (p>0.241).
176
0 9 18 27 36 45 54 63 72 810
40
80
120
160
Time (minutes)
FHR
MM
R (m
sec)
0 9 18 27 36 45 54 63 72 810
3
6
9
12
15
Time (minutes)
FHR
STV
(mse
c)
0 9 18 27 36 45 54 63 72 810
10203040506070
OccludedControl
Time (Minutes)
FHR
LTV
(bpm
)
0 9 18 27 36 45 54 63 72 81100
150
200
250
Time (min)
FHR
(bpm
)
0 9 18 27 36 45 54 63 72 81100
150
200
250
Time (minutes)
FHR
(bpm
)
0 9 18 27 36 45 54 63 72 810
40
80
120
160
Time (minutes)
FHR
MM
R (m
sec)
0 9 18 27 36 45 54 63 72 810
3
6
9
12
15
Time (minutes)
FHR
STV
(mse
c)
0 9 18 27 36 45 54 63 72 810
10203040506070
Control Occluded
Time (Minutes)
FHR
LTV
(bpm
)
Acutely InstrumentedModel
Chronically InstrumentedModel
Control(p<0.001)
Occluded(p=0.007)
Control(p<0.001)
Occluded(ns)
Occluded(ns)
Control(p<0.001)
Control(p=0.006)
Occluded(ns)
177
70
80
90
100
110
Syst
olic
Pre
ssur
e(m
mH
g)
70
80
90
100
110
Syst
olic
Pre
ssur
e(m
mH
g)
0 9 18 27 36 45 54 63 72 8120
30
40
50
60
Control Occluded
Time (minutes)
Dia
stol
ic P
ress
ure
(mm
Hg)
0 9 18 27 36 45 54 63 72 8120
30
40
50
60
Control Occluded
Time (minutes)
Dia
stol
ic P
ress
ure
(mm
Hg)
Acutely Instrumented Model Chronically Instrumented Model
Control(ns)
Occluded(ns)
Control(ns)
Occluded(ns)
Figure 4.5 Comparison of inter-occlusion fetal arterial pressure responses in acute and
chronically instrumented fetuses during repeated cord occlusion. Fetal arterial pressure
responses in acutely instrumented fetuses were not significantly different from those of
chronically instrumented fetuses (p>0.10).
178
4.3.3 Biochemical responses of normal (non-acidaemic) and spontaneously
acidaemic fetuses to repeated umbilical cord occlusion
4.3.3.1 Controls
Fetal arterial pH, gas tensions, base excess and lactate levels in controls did not significantly
change over the duration of the experimental phase (p>0.913 for all; Figure 4.2). Similarly,
fetal arterial glucose, cortisol, noradrenaline and adrenaline levels in controls did not change
over the duration of the experimental phase (p>0.324; Figure 4.3).
4.3.3.2 Fetal acid-base status and lactate responses
Fetal arterial pH (p=0.002) and PO2 (p<0.001) levels in normal fetuses decreased during the
experimental protocol whilst arterial PCO2 (p=0.046) and lactate (p<0.001) increased
compared to controls (Figure 4.2). Arterial base excess in normal fetuses decreased in
response to repeated UCO compared to controls but the difference did not reach statistical
significance (p=0.451).
The changes in pH, PO2 and base excess levels in spontaneously acidaemic fetuses in
response to the experimental protocol were smaller in magnitude than similar changes in
normal fetuses exposed to the identical experimental protocol (pH, p=0.045; PO2, p=0.013;
base excess, p=0.065; Figure 4.6). Similarly, arterial PCO2 in spontaneously acidaemic fetuses
did not significantly increase over the duration of the experimental protocol (p=0.394)
whereas in normal fetuses PCO2 levels significantly increased during the UCO protocol
(p=0.007). Arterial lactate levels increased significantly in spontaneously acidaemic fetuses
that experienced UCO (p=0.046) and there was no difference in the lactate response to UCO
between normal and spontaneously acidaemic fetuses (p=0.929).
4.3.3.3 Fetal endocrine responses
Arterial glucose, cortisol, noradrenaline and adrenaline levels increased in normal fetuses
exposed to UCO compared to controls (glucose, p=0.07; cortisol, p=0.050; noradrenaline,
p=0.009; adrenaline, p=0.001; Figure 4.7). Similar increases occurred in noradrenaline
(p<0.001) and adrenaline (p<0.001) levels in spontaneously acidaemic fetuses exposed to
UCO and there was no difference in the magnitude of these responses between normal and
spontaneously acidaemic fetuses (noradrenaline, p=0.414; adrenaline, p=0.269; Figure 4.7).
The fetal arterial glucose and cortisol levels in spontaneously acidaemic fetuses that
experienced UCO were not significantly different from controls (glucose, p=0.583; cortisol,
p=0.370).
179
-30
6.8
7.0
7.2
7.4
SA(p=0.021)
0 27 54 81
N(p<0.001)
pH
N vs. SA p=0.045
Time (minutes)
pH
-30
-16
-12
-8
-4
0
SA(p=0.855)
0 27 54 81
N(p=0.579)
Base Excess
N vs. SA p=0.065
Time (minutes)
Base
Exc
ess
(mm
ol/L
)
-300
50
100
150
200
SA(p=0.394)
0 27 54 81
N(p=0.007)
PCO2
Time (minutes)P
CO
2(m
mH
g)
N vs. SA p=0.839
-300.02.55.07.5
10.012.515.0
SA(p=0.046)
0 27 54 81
N(p=0.012)
Lactate
N vs. SA p=0.929
Time (minutes)
Lact
ate
(mm
ol/L
)
-300
10
20
30
SA(p=0.141)
0 27 54 81
N(p<0.001)
PO2
N vs. SA p=0.013
Time (minutes)
PO
2(m
mH
g)
Figure 4.6 Fetal arterial gas tensions, pH, base excess and lactate in normal (N) and
spontaneously acidaemic (SA) fetuses during experimental phase. The p-values in the legend
for each graph relate to the change in the specific variable in that subgroup over time. The p-
values in boxes compare the normal and spontaneously acidaemic fetuses over the duration
of the experimental phase considering group and time as fixed effects and individual animals
as random effects repeated over time. Despite being exposed to the same UCO protocol,
the magnitude of the reduction in arterial pH, PO2 and base excess in response to UCO in
spontaneously acidaemic fetuses were smaller than in normal fetuses (p=0.045, p=0.013 and
p=0.065 respectively). The increase in arterial lactate levels in response to repeated UCO was
not significantly different between groups (p=0.929).
180
0 810
10203040506070
Noradrenalinep=0.414
C
N(p=0.001)
SA(p<0.001)
Time (minutes)
Nor
adre
nalin
e (n
g/L)
0 810
10
20
30
40Cortisol p=0.526
C
N(p=0.050)
SA(p=0.370)
Time (minutes)
Cor
tisol
(ng/
mL)
0 810
10
20
30
40Adrenaline
p=0.269
C
N(p=0.001)
SA(p<0.001)
Time (minutes)
Adre
nalin
e (n
g/L)
0 27 54 810
1
2
3
4Glucose
p=0.09
C
N(p=0.07)
SA(p=0.583)
Time (minutes)
Glu
cose
(mm
ol/L
)
Figure 4.7 Fetal endocrine responses to repeated cord occlusion in normal (N), control (C)
and spontaneously acidaemic (SA) fetuses. P-values attached to the legends compare the
experimental group to controls over the duration of the protocol and were calculated using
regression modelling considering group and time as fixed effects and individual animals as
random effects repeated over time. In both normal and spontaneously acidaemic fetuses,
catecholamine levels were significantly increased after repeated UCO compared to controls
(p≤0.001). Arterial glucose and cortisol levels in normal fetuses exposed to UCO were
increased compared to controls (glucose, p=0.07; cortisol p=0.05); however glucose and
cortisol levels in spontaneously acidaemic fetuses that underwent UCO were not significantly
different from controls.
181
4.3.4 The relationship between fetal scalp blood lactate levels and central lactate
levels
Fetal scalp, carotid arterial and jugular venous blood lactate levels for controls, normal and
spontaneously acidaemic fetuses during the experimental phase are presented in Figures 4.8
to 4.10. These figures also present graphically the differences between fetal scalp and jugular
venous lactate levels compared to carotid arterial lactate levels (∆lactate) based on paired
analysis within the experimental group. Comparisons between the three sampling sites in the
three experimental groups are presented in Figure 4.11.
4.3.4.1 Fetal scalp blood lactate levels and carotid arterial lactate levels
The relationship between fetal scalp blood and carotid arterial lactate levels was modified by
experimental group (p=0.015). In controls (scalp-artery, -0.37± 0.46mmol/L, p=0.496;
Figure 4.8A) and spontaneously acidaemic fetuses during UCO (0.05±0.25mmol/L,
p=0.852; Figure 4.10A) fetal scalp blood lactate levels were not significantly different from
carotid arterial lactate levels. In contrast, in normal fetuses prior to UCO (time -30 minutes to
0 minutes), scalp blood lactate levels were not significantly different from carotid arterial
levels (0.10±0.44mmol/L, p=0.748; Figure 4.9A); however, during UCO fetal scalp blood
levels underestimated carotid arterial lactate levels by 0.62±0.28mmol/L in normal fetuses
(p=0.034).
The relationship between fetal scalp blood lactate levels and carotid arterial lactate levels was
not influenced by fetal pH (p=0.687), PO2 (p=0.260), PCO2 (p=0.975), base excess (p=0.957),
glucose (p=0.261), noradrenaline level (p=0.410) or time during the experimental phase
(p=0.920); however, the scalp-carotid arterial relationship was modified by adrenaline levels
(p=0.048) in all experimental groups and carotid arterial lactate levels in normal fetuses
(p=0.002). As fetal adrenaline levels increased, the difference between scalp and carotid
arterial blood lactate levels became smaller. Conversely, as arterial lactate levels increased
during cord occlusion the difference between scalp and carotid arterial lactate levels became
larger with scalp blood lactate levels tending to underestimate carotid arterial lactate levels.
The analysis of interactions suggested that the effect of arterial lactate levels on scalp lactate
levels was greater in normal than spontaneously acidaemic fetuses (p=0.002). The net result
of these combined influences on the relationship between the two sampling sites was such
that in normal fetuses scalp blood lactate levels underestimated carotid arterial lactate levels
during cord occlusion whereas in the spontaneously acidaemic fetuses scalp lactate levels
were not significantly different from carotid arterial lactate levels.
181
Figure 4.8 The relationships between fetal scalp blood, carotid arterial blood and jugular
venous blood lactate levels in controls. Fetal scalp blood lactate was not significantly
different from either carotid arterial lactate (Figure A, p=0.496) or jugular vein lactate (Figure
B, p=0.472). ∆lactate in Figure C compares fetal scalp blood lactate and jugular venous
lactate to carotid arterial lactate levels; scalp-artery and vein-artery respectively.
182
Scalp Vein-1.0
-0.5
0.0
0.5
1.0
p=0.496 p=0.893
∆La
ctat
e (m
mol
/L)
Control Fetuses
-300
3
6
9
12
15
0 27 54 81
CarotidArtery
FetalScalp
Time (min)
Lact
ate
(mm
ol/L
)
-300
3
6
9
12
15
0 27 54 81
JugularVein
FetalScalp
Time (min)
Lact
ate
(mm
ol/L
)
A
B
C
182
Figure 4.9 The relationships between fetal scalp blood, carotid arterial blood and jugular
venous blood lactate levels in normal fetuses during repeated cord occlusion. Fetal scalp
blood lactate was significantly lower than carotid arterial lactate by 0.62±0.28mmol/L (Figure
A, p=0.034) but not significantly different from jugular venous lactate (Figure B, p=0.529).
∆lactate in Figure C compares fetal scalp blood lactate and jugular venous lactate to carotid
arterial lactate levels; scalp-artery and vein-artery respectively. This figure illustrates that
during repeated cord occlusion in normal fetuses scalp blood lactate levels were less than
jugular venous lactate levels which were less than carotid arterial lactate levels.
183
Scalp Vein-1.0
-0.5
0.0
0.5
1.0
p=0.034 p=0.061
∆La
ctat
e (m
mol
/L)
Normal Fetuses
-300
3
6
9
12
15
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
-300
3
6
9
12
15
0 27 54 81
FetalScalp
JugularVein
Time (min)
Lact
ate
(mm
ol/L
)
A
B
C
183
Figure 4.10 The relationships between fetal scalp blood, carotid arterial blood and jugular
venous blood lactate levels in spontaneously acidaemic fetuses during repeated cord
occlusion. Fetal scalp blood lactate was not significantly different from either carotid arterial
lactate (Figure A, p=0.852) or jugular venous lactate levels (Figure B, p=0.615). ∆lactate in
Figure C compares fetal scalp blood lactate and jugular venous lactate to carotid arterial
lactate levels; scalp-artery and vein-artery respectively.
184
Spontaneously AcidaemicFetuses
-300
3
6
9
12
15
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
-300
3
6
9
12
15
0 27 54 81
FetalScalp
JugularVein
Time (min)
Lact
ate
(mm
ol/L
)
Scalp Vein-1.0
-0.5
0.0
0.5
1.0
∆La
ctat
e (m
mol
/L)
p=0.852 p=0.240
A
B
C
184
Figure 4.11 Comparison of three lactate sampling sites in control (C), normal (N) and
spontaneously acidaemic (SA) fetuses. Figure A illustrates the relationship between fetal
scalp blood lactate and carotid arterial lactate in the three experimental groups with normal
fetuses having a greater difference between fetal scalp lactate and carotid arterial lactate levels
than spontaneously acidaemic fetuses (p=0.082). Figure B illustrates the relationship
between fetal scalp lactate and jugular venous lactate levels which was constant and
independent of experimental group. Figure C illustrates the relationship between jugular
venous and carotid arterial lactate levels in the three experimental groups with divergent
relationships present between normal and spontaneously acidaemic fetuses (p=0.026).
185
∆ Lactate (Scalp - Artery)
-1.0
-0.5
0.0
0.5
1.0
p=0.082
C N SA
∆La
ctat
e (m
mol
/L)
∆ Lactate (Scalp-Vein)
-1.0
-0.5
0.0
0.5
1.0
C N SA
∆La
ctat
e (m
mol
/L)
∆ Lactate (Vein - Artery)
-1.0
-0.5
0.0
0.5
1.0
p=0.026
C N SA
∆La
ctat
e (m
mol
/L)
A
B
C
186
4.3.4.2 Fetal scalp blood lactate levels and jugular venous lactate levels
Fetal scalp lactate levels were not significantly different from jugular venous lactate levels in
controls (scalp-vein, -0.25±0.34mmol/L, p=0.472), normal fetuses that experienced UCO
(-0.18± 0.28 mmol/L, p=0.529) and spontaneously acidaemic fetuses that experienced UCO
(0.12±0.2, p=0.615; Figure 4.11).
4.3.4.3 Fetal carotid artery and jugular vein blood lactate levels
Fetal jugular venous lactate levels were not significantly different from carotid arterial levels
in either controls (vein-artery, -0.03±0.21mmol/L, p=0.893) or spontaneously acidaemic
fetuses (0.18±0.16mmol/L, p=0.240). In normal fetuses that experienced UCO fetal jugular
venous lactate levels were less than carotid arterial lactate levels (-0.36±0.18mmol/L,
p=0.061).
In all groups of fetuses in this study, the relationship between venous and arterial lactate
levels was not influenced by fetal arterial pH (p=0.200), PCO2 (p=0.369), base excess
(p=0.849), noradrenaline (p=0.829), adrenaline (p=0.297), glucose levels (p=0.827) or time
during the experimental phase (p=0.218). However, the relationship between venous and
arterial lactate levels was modified by arterial lactate (p=0.021) and PO2 (p=0.047). As arterial
lactate levels increased the venous-arterial lactate difference became significantly less
(p=0.021). The relationship between the venous-arterial lactate difference and PO2 is more
complex. In normal fetuses, as PO2 levels fell during cord occlusion, the venous-arterial
lactate difference tended to decrease in size (i.e. venous and arterial levels become more
similar). In contradistinction, when PO2 levels fell during cord occlusion in spontaneously
acidaemic fetuses, the venous-arterial lactate difference tended to increase in size with jugular
venous lactate levels tending to be higher than carotid arterial lactate levels.
187
4.3.5 The relationship between fetal scalp blood lactate and lipid peroxidation in
the fetal brain
4.3.5.1 Controls
Neither fetal carotid arterial organic hydroperoxide (OHP) levels nor jugular venous OHP
levels changed significantly over the duration of the experimental phase in controls (arterial
OHP 0.060±0.004µmol/L, p=0.128; venous OHP 0.058±0.004µmol/L, p=0.871; Figure
4.12). Fetal carotid arterial OHP was 3.4% higher than jugular venous OHP levels in
controls (artery-vein 0.002±0.0005, p<0.001). An identical pattern was observed for
malondialdehyde (MDA) levels where both carotid arterial MDA and jugular venous MDA
levels did not change significantly over time (arterial MDA 1.317±0.038µmol/L, p=0.680;
venous OHP 1.297±0.047µmol/L, p=0.848; Figure 4.12). Arterial MDA levels were
significantly greater (1.8%) than venous MDA levels in controls (artery-vein
0.023±0.002µmol/L, p<0.001).
4.3.5.2 Normal and spontaneously acidaemic fetuses
Carotid artery and jugular vein OHP and MDA levels in normal and spontaneously
acidaemic fetuses during UCO are presented in Figure 4.13. Carotid artery OHP levels were
not significantly different between normal and spontaneously acidaemic fetuses experiencing
UCO (p=0.836). Similarly jugular venous OHP levels were not significantly different
between the two groups (p=0.670). An identical response was present for MDA levels;
carotid arterial levels were not significantly different between the two groups (p=0.294) and
jugular venous levels were also not significantly different between the two groups (p=0.262).
Therefore, for all further discussions relating to lipid peroxidation in the fetal brain, the two
groups of fetuses that underwent UCO will be considered as one group.
In fetuses that underwent UCO carotid arterial OHP levels were not significantly different
from arterial OHP levels in controls (p=0.152); however, carotid arterial OHP varied over
the duration of the experimental phase of the study (p<0.001). There was an initial increase
in arterial OHP levels between nine and 27 minutes of UCO compared to baseline OHP
levels (p<0.008). This increase was followed by a decrease toward baseline levels between 36
and 54 minutes of UCO and a secondary increase from 63-81 minutes (p<0.027) compared
to OHP levels collected prior to UCO.
188
Control OHP
-300.00
0.05
0.10
0.15
JugularVein(p=0.871)
0 27 54 81
CarotidArtery(p=0.128)
Time (min)
OH
P (u
mol
/L)
Control MDA
-301.00
1.25
1.50
1.75
2.00
JugularVein(p=0.848)
0 27 54 81
CarotidArtery(p=0.680)
Time (min)
MD
A (u
mol
/L)
Figure 4.12 Lipid peroxidation in carotid arterial and jugular venous blood of control fetuses.
Data are presented as mean±SEM for carotid arterial and jugular venous blood OHP and
MDA levels in control fetuses. P-values represent change in each variable over the duration
of the experimental protocol. Both measures of lipid peroxidation did not change
significantly over the duration of this study (P>0.128).
189
Normal and SpontaneouslyAcidaemic OHP
-300.00
0.05
0.10
0.15
0 27 54 81
N ArterySA Artery
N VeinSA Vein
Time (min)
OH
P (u
mol
/L)
Normal and SpontaneouslyAcidaemic MDA
-301.00
1.25
1.50
1.75
2.00
0 27 54 81
N ArterySA Artery
N VeinSA Vein
Time (min)
MD
A (u
mol
/L)
Figure 4.13 Lipid peroxidation in carotid arterial and jugular venous blood of fetuses that
experienced umbilical cord occlusion. Carotid artery OHP (p=0.836) and MDA (p=0.294)
levels were not significantly different between the normal (N) and spontaneously acidaemic
(SA) fetuses. Similarly jugular vein OHP (p=0.670) and MDA (p=0.262) were not
significantly different between groups. Umbilical cord occlusion produced a 260% increase
in venous OHP levels (p<0.001) and a 125% increase in venous MDA levels (p<0.001)
compared to controls.
190
Jugular venous OHP levels in fetuses that underwent UCO were significantly different from
controls (p<0.001). In the first 27 minutes of the experimental phase jugular venous OHP
levels were not significantly different from controls (p>0.232). This was followed by a
massive increase in venous OHP levels during both the 1.0-minute occlusions (36-54
minutes, p<0.01) and the 1.5-minute occlusions (61-81minutes, p<0.001) such that levels
during the 1.5-minute occlusions were 260% higher than controls.
Carotid arterial MDA levels in fetuses that underwent UCO were not significantly different
from controls (p=0.595). Repeated UCO was associated with a 125% increase in venous
MDA levels compared to controls (p<0.001); no difference between the UCO and control
groups was identified until after 54 minutes of UCO (P<0.015).
4.3.5.3 Prediction of the lipid peroxidation in fetal brain perfusate
The time point at which the rapid increase in jugular venous OHP levels commenced was
determined for each fetus as described in Section 4.2.7. The rapid increase in venous OHP
levels occurred over a range of scalp lactate values from 6.2 to 8.0mmol/L and a range of
carotid arterial pH values from 7.04 to 7.19. These ranges are plotted graphically in Figure
4.14 against the arterial pH and scalp blood lactate changes seen in the two groups of fetuses
that experienced UCO. This figure illustrates the observation that the rapid increase in
venous OHP levels occurred over a wider range of arterial pH levels than scalp lactate levels.
If one were to use either arterial pH levels or scalp lactate levels as a technique of predicting
when the rapid increase in venous OHP levels began, a greater proportion of arterial pH
values (117/307; 38%) than fetal scalp blood lactate levels (52/274; 25) were within the
window where OHP production commenced (OR 2.63, 95%CI 1.80-3.94; p<0.001).
Therefore if one were to predict the rapid increase in venous OHP levels from scalp lactate
values, it could occur anywhere between 18 and 54 minutes of the experimental phase
(Figure 4.14).
In order to reflect oxidative stress in an organ, significantly higher levels of lipid peroxidation
must be detected in the venous drainage than found in the blood perfusing the organ (i.e. a
positive venous-arterial difference must exist; Rogers et al., 2001). Receiver operator curves
(ROC) were calculated for biochemical markers of fetal well-being to predict OHP
production (jugular OHP-carotid artery OHP > 0) and these are presented in Figure 4.15.
191
-30
6.8
7.0
7.2
7.4
SA
0 27 54 81
N
pH
StartOHP
Production
Time (minutes)
Car
otid
Arte
rial p
H
-300
5
10
15
SA
0 27 54 81
NLactate
StartOHP
Production
Time (minutes)
Scal
p La
ctat
e(m
mol
/L)
Figure 4.14 Range of carotid arterial pH and scalp blood lactate associated with lipid
peroxidation in jugular venous blood. The rapid increase in jugular venous OHP levels
occurred over a larger range of pH values than lactate levels during repeated UCO (OR 2.63,
95%CI 1.80-3.94; p<0.001).
192
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity (false positives)
Sen
sitiv
ity (t
rue
posi
tives
)
No discriminationScalp LactateArterial LactateArterial pH
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity (false positives)
Sen
sitiv
ity (t
rue
posi
tives
)
No discriminationScalp LactateArterial PO2Arterial PCO2
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity (false positives)
Sen
sitiv
ity (t
rue
posi
tives
)
No discrimination
Scalp Lactate
Arterial BaseExcess
Figure 4.15 Receiver operator curves for the prediction of OHP production (jugular venous
OHP–carotid arterial OHP >0) in jugular venous blood.
Scalp lactate vs.
Arterial pH p=0.056
Scalp lactate vs.
Arterial PO2 p<0.001
Scalp lactate vs. Arterial Base Excess
p<0.001
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Three hundred and forty nine data points were used for the generation of these curves. Fetal
scalp blood lactate (area under ROC 0.782) had significantly greater area under the ROC than
carotid arterial pH (area 0.717, p=0.056), base excess (area 0.640, p<0.001), and PO2 (0.647,
p<0.001). The area under the carotid arterial lactate ROC was not significantly different
from the fetal scalp blood lactate ROC (p>0.05). The sensitivity and specificity for optimum
cut off points and accuracy associated with these points are presented in Table 4.2. Even
though scalp blood lactate levels had significantly greater area under the ROC than routine
blood gas measures, the optimum sensitivity was still only 62.7% and specificity was 83.3%
for the prediction of OHP production in the fetal jugular venous blood.
Table 4.2 Accuracy of biochemical prediction of OHP production in brain perfusate
Area Under ROC Optimum Cut Off Sensitivity Specificity Accuracy§
Scalp Lactate (mmol/L)
0.782 (0.726-0.839) 7.0 62% 83% 72%
Arterial Lactate** (mmol/L)
0.759 (0.699-0.819) 8.7 63% 81% 72%
Arterial pH** 0.717 (0.653-0.781) 7.125 41% 92% 66%
Arterial PCO2** (mmHg)
0.724 (0.663-0.786) 65.3 59% 77% 62%
Arterial PO2 ** (mmHg)
0.647 (0.580-0.714) 14.0 62% 62% 62%
Arterial Base Excess**
0.640 (0.571-0.708) -7.3 68% 55% 61%
* OHP production ** All arterial samples collected from carotid artery catheter § Accuracy is the proportion of correctly classified cases using the biochemical cut off point selected to maximise the sensitivity and specificity
Receiver operator curves were also calculated for biochemical markers of fetal well-being to
predict MDA production (jugular MDA-carotid artery MDA > 0). Similar to OHP
production, scalp lactate had the largest area under the curve (0.725) for predicting MDA
production in the fetal brain perfusate, followed by arterial PCO2 (0.682), pH (0.614), base
excess (0.580), and PO2 (0.543). The area under the scalp lactate curve was significantly
greater than the area under the oxygen partial pressure curve (p=0.041). No significant
differences were identified between the areas under the ROC for the other biochemical
markers assessed.
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4.4 Discussion
In labour the fetal scalp is the window for biochemical assessment of fetal wellbeing.
Despite the many technological advances in obstetrics over the last 40 years, the technique
for acquiring fetal blood during labour has not changed since its original description in 1962
(Saling, 1962). Intrapartum fetal blood sampling is limited to the skin covering the fetal
presenting part. It requires ruptured membranes and the cervix to be at least two to three
centimetres dilated. Sample collection is further complicated by the fact that the presenting
part can vary from one to ten centimetres away from the perineum. Although other
techniques such as ultrasound guided cordocentesis can provide access to fetal blood, these
techniques are invasive and are generally considered not suitable to perform on women in
labour.
The purpose of this chapter was to investigate the relationships between fetal scalp lactate
levels, central lactate levels, and oxygen free radical activity in the fetal brain during asphyxia.
Prior to exploration of these relationships I have compared and contrasted the acutely
instrumented experimental model used in this study which allowed regular, reliable access to
fetal scalp blood during asphyxia with responses from chronically instrumented fetuses.
4.4.1 Comparison of the acute and chronic instrumented models
As detailed in Section 4.1.1, all study designs to investigate fetal scalp blood in humans and
animals have important limitations. Human experimental models are limited by the
infrequent nature of birth asphyxia in human labour and the delays between the collection of
fetal scalp blood and the collection of umbilical cord blood which can vary from 1 to 60
minutes (Bowe et al., 1970; Yoshioka & Roux, 1970; Kruger et al., 1998). Animal
experimental models, whilst allowing both experimental exposure to asphyxia and
simultaneous collection of fetal scalp blood and central blood, have all been performed in
acutely instrumented fetuses with mothers under anaesthesia (Gare et al., 1967; Adamsons et
al., 1970; Shier & Dilts, 1972; Aarnoudse et al., 1981).
It is almost universally accepted that chronically instrumented fetal preparations rather than
acutely instrumented preparations should be used when investigating fetal responses to
asphyxia. However, although chronic preparations are considered optimal, there are some
circumstances, such as the studies presented in this chapter, where acute preparations are
necessary. One area of concern is the effect of maternal anaesthesia on the fetus (Rudolph &
Heymann, 1967; Levinson et al., 1973; Palahniuk & Shnider, 1974; Palahniuk & Cumming,
195
1977; Biehl et al., 1983a; Alon et al., 1993) and fetal physiologic responses to asphyxia (Smith
et al., 1975; Yarnell et al., 1983; Cheek et al., 1987; Swartz et al., 1987; Morishima et al., 1989).
The pattern and magnitude of the effects of maternal anaesthesia on the fetus and the fetal
response to asphyxia vary widely in previously published literature depending on both the
anaesthetic agents used and their doses.
4.4.1.1 Selection of maternal anaesthesia
To optimise the experimental preparation used in this study, careful consideration was given
to maternal anaesthesia. Anaesthetic agents were selected specifically to minimise the fetal
biochemical, endocrine, cardiovascular and autonomic responses to maternal anaesthesia. In
addition, the specific agents were selected to produce minimal or no change to the fetal
responses to asphyxia. A single low dose of thiopentone (4mg/kg) was chosen as the
induction agent and isoflurane at an inspired concentration of 0.75-1.25% in oxygen was
chosen as the volatile agent to maintain anaesthesia. It can be seen from Table 4.3 that the
maternal anaesthesia used in this study is significantly different from that used in the original
studies using acutely instrumented fetuses to validate fetal scalp pH assessment.
Table 4.3 Comparison of maternal anaesthesia techniques between experimental models
Author Induction of Anaesthesia Maintenance of Anaesthesia
Gare et al., 1967 2% Lidocaine local anaesthesia
2% Lidocaine local anaesthesia
Adamsons et al., 1970 Pentobarbital 10mg/kg IM* Nitrous oxide Halothane Further pentobarbital if required
Shier & Dilts, 1972 Pentobarbital 15.2mg/kg IV† Supplemental pentobarbital if required
Aarnoudse et al., 1981 Pentobarbital 30mg/kg IV† Nitrous oxide Piritramide 7.5mg SC‡ 2 hourly Pancuronium 0.08mg/kg IV† hourly
Chapter 4, Pennell, 2003 Thiopentone§ 4mg/kg IV† Isoflurane 0.75-1.25% *IM intramuscular injection, IV† intravenous injection, SC‡ subcutaneously § Conversion of pentobarbital:thiopentone is 3:1(Fragen & Avram, 1999)
196
A single low dose of thiopentone was chosen as the anaesthetic induction agent for the
studies in this chapter. In pregnant ewes (125 days gestational age), low dose thiopentone
has been shown to have transient effects on both the mother and fetus (Alon et al., 1993). A
single intravenous bolus of 5mg/kg produced a transient decrease in maternal mean arterial
pressure (MAP) and uterine blood flow. This was associated with a transient increase in
FHR, MAP and PCO2 and a decrease in fetal pH. The duration of these effects was
approximately five minutes after which all the physiological variables returned to their pre-
anaesthetic levels (Alon et al., 1993). In humans, when considering Apgar scores and the
need for neonatal resuscitation, low doses of thiopentone have been shown to not depress
the fetus whereas larger doses, such as 6mg/kg and 8mg/kg, are associated with increased
risk of neonatal depression at birth (Kosaka et al., 1969). In women given 8mg/kg
thiopentone for induction of anaesthesia prior to caesarean section, 43% of the babies had
depressed Apgar scores at birth (Kosaka et al., 1969). The single low dose of thiopentone
was chosen because both repeated and high doses of this drug, although not altering the
maternal or fetal peak concentrations, have been shown to significantly increase the time
taken to clear the drug from both the maternal and fetal circulation (Kosaka et al., 1969).
There is a swift decline in thiopentone concentration in maternal blood after a single
intravenous administration with levels falling to 20% of the initial peak level within three
minutes of administration. Although the drug is highly lipid soluble and maximum fetal
levels are reached within three minutes of administration, the maximum fetal blood levels are
only 15% of the initial maternal peak level and within ten minutes of maternal administration,
the fetal arterial levels have fallen to 5% of the peak maternal concentration (Kosaka et al.,
1969).
Induction of anaesthesia can be induced with other agents such as high dose volatile agents
or ketamine; these were avoided in this study due to their adverse fetal effects. High dose
volatile agents have been shown to induce greater depression of myocardial contractility than
low dose barbiturates (Frankl & Poole-Wilson, 1981) by altering calcium uptake by cardiac
sarcoplasmic reticulum (Blanck & Stevenson, 1988). In ovine fetuses, ketamine abolished the
fetal hypertension and bradycardia produced by partial cord occlusion (Swartz et al., 1987).
In this study, isoflurane was chosen as the volatile agent to maintain anaesthesia because it
has significantly less effect on baroreceptor reflexes than other inhaled anaesthetics (Kotrly et
al., 1984). In general, all volatile anaesthetics depress baroreceptor reflexes, but not equally
(Fragen & Avram, 1999). When comparing volatile anaesthetic agents it is typical to compare
effects at specific minimum alveolar concentrations (MAC). The MAC of an anaesthetic is
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the concentration in the alveolus which is just capable of blocking response to a noxious
stimulus in 50% of a population. As the alveolar concentration is proportional to blood and
brain concentration at equilibrium, MAC’s can be used to compare therapeutic ratios of
various drugs. Halothane depresses baroreceptor reflexes such that by 2.5 MAC there is
virtually no tachycardic response to changes in blood pressure (Duke et al., 1977). Nitrous
oxide combined with halothane produced less depression in baroreceptor reflexes than
halothane alone at the same MAC levels (Duke & Trosky, 1980). Nitrous oxide combined
with enflurane produced virtually the same degree of depression as nitrous oxide and
halothane (Morton et al., 1980). At one MAC, isoflurane has been shown to reduce the
sensitivity of the baroreceptors controlling heart rate by 10-15% whereas halothane
combined with nitrous oxide or enflurane alone reduced baroreceptor sensitivity by 70% and
halothane alone reduced it by 85% (Kotrly et al., 1984).
A variety of agents to maintain anaesthesia have been used previously to investigate the
effects of maternal anaesthesia on the ovine fetus. Isoflurane at an inspired concentration of
2% in oxygen has been shown to not significantly alter fetal arterial pressure or FHR over 90
minutes of anaesthesia; however, a 25% decrease in fetal cardiac index and small decreases
in pH and base excess were noted (Biehl et al., 1983b). When isoflurane was used at lower
concentrations it did not result in fetal acidosis or significant falls in cardiac output (Bachman
et al., 1986) Halothane, whilst not altering uterine artery blood flow (Cheek et al., 1987), has
been shown to be associated with a 9-33% fall in fetal mean arterial pressure. Despite the fall
in fetal MAP, no change was demonstrated in cardiac output, placental blood flow or
regional blood flow to vital organs (Biehl et al., 1983a; Yarnell et al., 1983; Cheek et al., 1987).
Medroxyflurane at one MAC has been shown to be associated with moderate falls in
maternal arterial pressures, cardiac output and uterine blood flow whilst no significant effects
on fetal heart rate, MAP or acid-base status were noted after 90 minutes of anaesthesia
(Smith et al., 1975). It was only when the dose of medroxyflurane was increased to two MAC
that marked changes in maternal arterial pressures, cardiac output and uterine blood flow
became apparent and resulted in a mixed respiratory and metabolic acidosis in the fetus
(Smith et al., 1975).
A variety of anaesthetic agents have also been used in studies investigating the effects of
anaesthesia on the fetal responses to asphyxia. When the ewe was anaesthetised with
halothane during fetal asphyxia it was noted that the increase in arterial pressure in response
to asphyxia was slightly smaller (9%) and the increase in FHR was greater than with asphyxia
alone (Yarnell et al., 1983; Cheek et al., 1987). Despite these differences, the addition of
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halothane did not alter the ability of the fetus to increase cardiac output or cerebral blood
flow in response to asphyxia, nor were vital organ blood flows altered by (Yarnell et al., 1983;
Cheek et al., 1987). Both ketamine and lidocaine adversely affected the fetal response to
asphyxia. Ketamine has been shown to abolish fetal hypertension and bradycardia produced
by partial cord occlusion (Swartz et al., 1987). Lidocaine has been shown to be associated with
loss of fetal cardiovascular adaptations to asphyxia with significant increase in fetal PCO2, and
decreases in pH, MAP, and blood flow to the brain, heart and adrenals (Morishima et al.,
1989).
The anaesthetic agents used in this study have previously been used in studies involving fetal
sheep at similar doses (Baker et al., 1990; Alon et al., 1993). I chose to use 0.75% to 1.25%
inspired concentration of isoflurane in oxygen, as the MAC for isoflurane previously
determined for both pregnant ewes and fetuses were 0.86% and 0.34% respectively
(Bachman et al., 1986). In pregnant ewes, isoflurane at 1% inspired concentration has been
shown to be associated with a 28% increase in uterine blood flow, a 25% increase in maternal
heart rate and a 20% decrease in maternal blood pressure (Baker et al., 1990; Alon et al.,
1993). Similar responses are seen in pregnant humans under isoflurane anaesthesia (Jouppila
et al., 1979). Despite these cardiovascular changes in pregnant ewes, fetal arterial pressures,
heart rate and acid-base status were not altered by isoflurane anaesthesia compared with non-
anaesthetised controls (Alon et al., 1993). Fetal heart rate variability (long term variability)
was however reduced in the presence of 1% isoflurane (Alon et al., 1993).
The anaesthetic agents used in this study have also been used previously in an ovine model of
asphyxia and were shown to not alter fetal responses during asphyxia (Baker et al., 1990).
The same pattern of changes occurred in fetal acid-base status, fetal arterial pressure, cerebral
oxygen consumption and oxygen delivery in the asphyxia-alone and the isoflurane-asphyxia
groups. In addition, during asphyxia similar responses were seen in the two groups with total
brain, heart and adrenal blood flows increasing while flow to the spleen and carcass
decreased. The only difference between the asphyxia-alone and the isoflurane-asphyxia
groups was that the magnitude of the changes in pH and PCO2 were greater in the isoflurane-
asphyxia group (Baker et al., 1990). Similar results have also been shown in young lambs
exposed to asphyxia with and without isoflurane (Brett et al., 1989).
In summary, based on previous studies, the agents used to anaesthetise pregnant ewes in this
study have been chosen to have minimal adverse effects on fetal responses to asphyxia and
were unlikely to have any significant effects on fetal scalp blood collection or interpretation.
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4.4.1.2 Similarities and differences between the acute and chronically instrumented
models
4.4.1.2.1 Control fetuses
The acutely instrumented model used in this chapter has been compared and contrasted with
the chronically instrumented model described in the previous chapter. In control fetuses, no
significant differences were found in fetal PO2, PCO2, base excess, glucose, cortisol or
adrenaline levels between the two models. However, the acutely instrumented fetuses had
lower pH levels, and higher lactate and noradrenaline levels. In addition, compared to
chronically instrumented fetuses, the acutely instrumented fetuses in this study had lower
baseline FHR and a 25% reduction in FHR variability. Similar reductions in FHR variability
have previously been reported in fetuses under the influence of maternal isoflurane
anaesthesia (Alon et al., 1993). Differences between the acute and chronically instrumented
control are likely to have resulted from the combination of fetal manipulation,
instrumentation, and the effect of maternal anaesthesia (Biehl et al., 1983b; Taylor et al., 1992).
The higher baseline FHR in the chronically instrumented fetuses may suggest subclinical
inflammation or infection; however, I have provided no direct evidence for this in my study.
In addition, previous studies that have measured white blood cell counts in chronically
catheterised fetuses (Nitsos et al., 2002) found levels that were not different from levels
reported by others in non-catheterised fetuses (Kallapur et al., 2001) suggesting that chronic
catheterisation of fetal sheep did not elicit a systemic inflammatory response.
4.4.1.2.2 Fetuses subjected to repeated cord occlusion
In this study a similar pattern of changes in fetal acid-base status, fetal arterial pressures and
fetal heart rate occurred in both the chronically instrumented fetuses exposed to asphyxia and
acutely instrumented fetuses exposed to the same insult whilst under anaesthesia. These
observations have previously been reported by other authors (Baker et al., 1990). In this
study I have demonstrated for the first time that there were no differences in the pattern of
endocrine responses seen in acute and chronically instrumented fetuses exposed to the same
asphyxial challenge. In fetuses subjected to UCO there were no significant differences in
glucose, cortisol or adrenaline levels between the two models. Despite the 25% reduction in
FHR variability in acutely instrumented controls, when the acutely instrumented fetuses were
exposed to an asphyxial insult, all three measures of FHR variability increased significantly
and were no longer significantly different from values obtained in chronically instrumented
fetuses exposed to the identical asphyxial insult. This similarity between the acute and
chronically instrumented fetal preparations has not previously been reported.
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The acutely instrumented fetuses subjected to UCO had the same lactate and base excess
responses as chronically instrumented fetuses exposed to the same asphyxial insult. Despite
the same degree of metabolic acidosis, the acutely instrumented fetuses exposed to asphyxia
developed a more severe respiratory acidosis than those in the chronic preparation. This was
demonstrated in the acutely instrumented fetuses by a more rapid fall in arterial pH and more
dramatic changes in PO2 and PCO2 than seen in the chronically instrumented fetuses exposed
to the identical asphyxial insult. This response has previously been reported in ovine fetuses
exposed to asphyxia under the influence of isoflurane anaesthesia (Baker et al., 1990) and may
be related to impaired placental function under anaesthesia (Taylor et al., 1992). Fetal PO2
and PCO2 equilibrate rapidly across the placenta (Daniels, 1966), unlike lactate, which is slow
to equilibrate across the placenta as it is reliant on facilitated diffusion coupled with proton
transfer (Piquard et al., 1990). Fetal respiratory gas levels are reliant on both umbilical blood
flow and uterine blood flow and the additional respiratory acidosis which developed in the
acute fetal preparation during asphyxia may have resulted from the maternal left lateral
position, the mechanical ventilation used in the acute preparation, or from alterations in the
fetus’ ability to direct blood toward the placenta during asphyxia. It is less likely that changes
in fetal cardiac output or uterine blood flow during asphyxia were responsible for the
additional acidosis as both have been shown to be preserved during isoflurane anaesthesia
and asphyxia (Baker et al., 1990; Alon et al., 1993).
Another difference between the two models was the magnitude of increase in FHR during
asphyxia. The inter-occlusion FHR in the acutely instrumented controls was less than the
chronically instrumented controls; however, when exposed to asphyxia the FHR increased to
a higher level in the acute than the chronic fetal preparation. This does not suggest blunting
of the hypoxaemia-induced tachycardia as previously suggested in lambs exposed to
isoflurane (Brett et al., 1989), but rather it suggests enhanced responsiveness of the FHR to
hypoxia. This may be secondary to the elevated noradrenaline levels seen in acutely
instrumented fetuses prior to asphyxia rather than enhanced sensitivity of the fetal heart to
catecholamines as both thiopentone (Skovsted et al., 1970) and isoflurane (Joas & Stevens,
1971) have previously been shown to not sensitise the heart to catecholamines. The elevated
levels of noradrenaline in controls in the acute compared to chronic preparation may relate to
the maternal and fetal stresses associated with the surgery (Palahniuk & Cumming, 1977). An
alternative more likely hypothesis is that the elevated levels of noradrenaline are secondary to
the isoflurane anaesthesia because there were no differences between the adrenaline and
cortisol levels between models and isoflurane anaesthesia has previously been shown to
201
increase noradrenaline levels in animals (Hellyer et al., 1989) and humans (Tanaka et al., 1996;
Segawa et al., 1998).
4.4.1.3 What is the optimum model for assessing fetal scalp blood during asphyxia?
There is little doubt that when exploring fetal responses to asphyxia it is ideal for the
experimental design to replicate the natural setting in as many ways as practically possible.
Acutely instrumented fetal preparations are at risk of maternal and fetal effects from
anaesthesia; however, as described both in this chapter and by others (Brett et al., 1989; Baker
et al., 1990) this can be minimised by appropriate selection of anaesthetic agents and their
doses. Chronic fetal preparations, although not influenced by maternal anaesthesia, are
influenced by other potential confounders which include surgical recovery, confinement
during experiments, multiple fetal catheters, and potential infection. There is also evidence
that placental function is temporarily disturbed after fetal surgery as illustrated by impaired
fetal growth after surgery to implant fetal catheters (Newnham et al., 1986) and the 15-30
days taken for amniotic fluid volume to return to normal after simple fetal surgery to implant
two catheters into the amniotic sac (Pennell et al., 2003).
After considering the fetal biochemical, endocrine and cardiovascular responses to asphyxia,
I have found the acute preparation used in this study does not appear to impair the ability of
the fetal lamb to coordinate neural, endocrine and local tissue responses to asphyxia.
Specifically, during asphyxia there was no evidence identified in this study that maternal
anaesthesia adversely affected autonomic control of the fetal heart or the responsiveness of
the FHR to hypoxemia. Although there were some differences between the acutely
instrumented model used in this chapter and the chronically instrumented model used in the
previous chapter, the differences identified are unlikely to be detrimental to the investigation
of relationships between fetal scalp blood lactate levels, central blood lactate levels and fetal
brain injury.
I propose that the model that I have chosen to use in this study is currently the best that is
available to assess fetal scalp blood in a regular, reliable, repeated manner during asphyxia,
allowing for collection of adequate volumes of fetal scalp blood for biochemical assessment.
However, in the absence of the requirement to collect fetal scalp blood, chronic fetal
catheterisation remains the model of choice for other experiments.
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4.4.2 The relationship between fetal scalp blood, carotid arterial and jugular venous
blood lactate levels
Despite growing evidence that fetal lactate levels may predict fetal brain injury, both in
animal models of asphyxia (Ikeda et al., 1998a) and observational studies of human labour
(Kruger et al., 1999), little is known about the relationship between scalp blood lactate levels
and central arterial lactate levels. My study is the first to explore the relationships between
fetal scalp lactate levels and carotid arterial and jugular venous lactate levels. Two previous
studies have partially investigated the relationship between fetal scalp blood lactate and
umbilical artery and vein lactate levels (Yoshioka & Roux, 1970; Kruger et al., 1998); however
the statistical analyses performed in these studies were limited. Unlike previous studies which
have compared group mean lactate levels between different sampling sites (Yoshioka &
Roux, 1970) or used simple univariate correlation to compare blood from different sampling
sites at different times (Kruger et al., 1998), I have used statistical techniques which evaluate
differences between matched samples collected simultaneously whilst taking into account the
repeated nature of the sampling on each of the fetuses.
4.4.2.1 Scalp lactate and central lactate levels in control and normal fetuses during
asphyxia
In this chapter I have shown that in the absence of asphyxia, scalp lactate levels are not
significantly different from carotid arterial and jugular venous lactate levels; however, when
fetuses are exposed to asphyxia, the relationship between fetal scalp blood lactate and carotid
arterial lactate is altered. During asphyxia fetal scalp blood lactate levels were found to
underestimate carotid arterial lactate levels by a mean of 0.6mmol/L (lactates<lactatea).
These observations are similar to those which were suggested by Yoshioka et al. (1970) where
mean scalp blood lactate levels and mean umbilical artery lactate levels were not significantly
different when collected from “non-distressed” fetuses at elective caesarean section;
however, when samples were collected from “distressed” fetuses, scalp blood lactate levels
were less than umbilical artery lactate levels. The differences identified by Yoshioka et al.
(1970) were clinically significant but failed to reach statistical significance which probably
relates to their small sample size (five “distressed” and nine “non-distressed” fetuses) and
their statistical analysis.
The observation in my study that during asphyxia fetal scalp lactate tends to underestimate
carotid arterial lactate levels is the opposite of the relationship previously described for pH
assessment from the same two sampling sites. During induced labour in monkeys, fetal scalp
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pH overestimated carotid arterial pH by an average of 0.047 pH units (pHs<pHa) (Adamsons
et al., 1970). Similarly, in human fetuses scalp blood pH can be lower than umbilical arterial
pH by as much as 0.06 pH units (Bretscher & Saling, 1967; Bowe et al., 1970). These
observations considered together suggest that the additional acidosis that develops in the
fetal scalp during asphyxia is respiratory rather than metabolic in origin. This is supported by
evidence that during hypoxia fetal scalp PO2 is not significantly different from arterial PO2
(difference 0.2mmHg) whereas fetal scalp PCO2 is significantly higher than arterial PCO2
(difference 8mmHg; Shier & Dilts, 1972). The additional carbon dioxide can combine with
water to form carbonic acid and result in a respiratory acidosis and an additional fall in fetal
scalp blood pH.
In my study fetal scalp blood lactate levels were similar to jugular vein lactate levels and this
relationship was not influenced by asphyxia or fetal condition as demonstrated by the
preservation of this relationship in spontaneously acidaemic fetuses during asphyxia. A
similar response has previously been described in fetal monkeys and fetal sheep where pH,
PO2 and PCO2 in scalp capillary blood closely resembled jugular venous blood levels even
when pH levels were as low as 6.75 (Adamsons et al., 1970; Shier & Dilts, 1972). The
observation that fetal scalp blood more closely resembles jugular venous blood than carotid
arterial blood may be advantageous. The composition of jugular venous blood is determined
not only by the composition of arterial blood but also by the relationship between flow rate
and metabolic needs of the brain (Figure 4.16). Therefore, jugular venous blood and fetal
scalp blood lactate levels may be more informative than arterial lactate levels regarding the
operational conditions of the fetal brain.
In this study I have used multivariate regression techniques to explore factors which
influence the relationship between fetal scalp blood lactate levels and carotid arterial lactate
levels in an attempt to elucidate potential mechanisms for why fetal scalp blood
underestimates carotid arterial lactate levels during asphyxia. Through this exploratory
analysis I have found that the scalp-arterial lactate difference was not influenced by fetal
arterial pH, PO2, PCO2, base excess, glucose levels or noradrenaline levels; however, it was
influenced by arterial lactate levels and adrenaline levels. In all fetuses in this study, as arterial
lactate levels increased the scalp-arterial lactate difference became greater. Conversely, as
fetal adrenaline levels increased, the scalp-arterial lactate difference became smaller with the
magnitude of the adrenaline effect being approximately half the size of the lactate effect. The
association between arterial lactate levels, adrenaline levels and the difference between scalp
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Figure 4.16 Control of jugular venous and scalp capillary blood content. The composition of jugular venous blood and scalp capillary blood is determined not
only by the composition of carotid arterial blood, but also by the relationship between flow rate and metabolic needs of the fetal brain and scalp tissues
respectively. This figure illustrates the multiple factors which directly influence carotid arterial blood content, blood flow and tissue metabolism and therefore
indirectly influence the content of jugular venous or scalp capillary blood.
BLOOD
FLOW
CAROTID
ARTERIAL BLOOD
TISSUE
METABOLISM
JUGULAR VENOUS BLOOD OR
SCALP CAPILLARY BLOOD
Placental Function
Cardiovascular and Neuroendocrine
Reflexes
Nutrient Supply
Previous Stresses on Fetus
Metabolic Processes to Facilitate Energy
Supply to Fetal Brain
205
and arterial lactate levels suggests that the difference between these sites during asphyxia may
be secondary to changes in fetal scalp perfusion.
During asphyxia fetal scalp blood flow rates are altered as part of the fetal cardiovascular
responses to redistribute cardiac output to vital organs (Cohn et al., 1974; Itskovitz et al.,
1987; Jensen et al., 1987; Jensen & Berger, 1991; Jensen et al., 1991; Reid et al., 1991). The
changes in skin blood flow which occur during asphyxia appear to be influenced by the
mechanism producing the asphyxia (Jensen et al., 1991; Bobby & Santos, 2000) and also may
vary with the severity of the resulting acidaemia (Cohn et al., 1974). During asphyxia induced
by a reduction in uterine artery blood flow in fetal lambs, fetal skin, carcass and lung blood
flow were decreased whilst blood flow to the myocardium, cerebral cortex and adrenals were
increased (Jensen et al., 1987; Jensen et al., 1991). In contrast, when asphyxia was induced by
partial umbilical cord occlusion in fetal lambs, blood flow to the skin overlying the head and
hindquarters increased three fold and there also was an increase in blood flow to the fetal
carcass which did not occur at the expense of other cardiovascular adaptations to asphyxia
(Itskovitz et al., 1987; Bobby & Santos, 2000). It has been postulated that this seemingly
paradoxical increase in blood flow to the fetal carcass during hypoxaemia induced by reduced
umbilical perfusion may be because the rapid blood flow through the carcass may act as a
fast time-constant vascular bed thus maintaining venous return under conditions where
placental blood flow is restricted, such as may occur with umbilical cord occlusion (Itskovitz
et al., 1987).
In my study fetal scalp blood flow was not measured; however, it can be postulated that the
widening of the scalp-arterial lactate level difference during asphyxia may be secondary to an
increase in scalp blood flow similar to the three-fold increase in scalp blood flow previously
described with 90 minutes of partial cord occlusion (Bobby & Santos, 2000). To understand
how improved scalp blood flow reduces scalp lactate levels during asphyxia it is important to
remember that the fetal scalp blood collected in this study (and also in human fetuses in
labour) is actually capillary blood. Therefore, similar to jugular venous lactate levels which
are influenced by carotid arterial lactate levels, cerebral blood flow and brain metabolism,
fetal scalp capillary lactate levels are influenced by carotid arterial lactate levels, scalp blood
flow rate and scalp metabolism (Figure 4.16). As local lactate levels are determined by the
balance between tissue lactate production and the overall capacity to clear lactate from the
tissue (Perret & Enrico, 1978) it becomes apparent that increased scalp blood flow during
asphyxia may be responsible for the lower levels obtained in scalp capillary blood than arterial
blood.
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The widening of the scalp-arterial lactate difference with increasing arterial lactate levels may
be because arterial lactate levels are an indirect marker of the severity of asphyxia. As the
severity of the asphyxial insult increases, the magnitude of the cardiovascular responses to
maintain cardiac output and redirect blood flow to vital organs have been shown to increase
(Jensen et al., 1991). Therefore, in an experimental model using repeated UCO to induce
asphyxia, as the severity of the asphyxia increases the fetal scalp blood flow may continue to
increase which potentially reduces fetal scalp capillary blood lactate levels.
The narrowing of the scalp-arterial lactate level difference in response to increasing
adrenaline levels is not surprising if one considers the effect of adrenaline on fetal circulation.
Adrenaline is released from the adrenal medulla, partly as a result of increased sympathetic
outflow, but also because the fetal adrenal gland is directly responsive to falls in PO2 (Hanson
& Kisured, 2001; Wood, 2001) Adrenaline is a potent vasoconstrictor which has been shown
to decrease scalp blood flow in sheep during asphyxia (Jensen et al., 1987) and therefore may
reduce the clearance of lactate from fetal scalp tissues resulting in higher capillary lactate
levels.
4.4.2.2 Information gained from spontaneously acidaemic fetuses
Recent evidence suggests that ovine fetuses which are spontaneously acidaemic have altered
cardiovascular, endocrine and metabolic responses to acute hypoxaemia (Gardner et al.,
2002). In the studies performed in this chapter I have confirmed that spontaneously
acidaemic fetuses have altered pH, base excess, PO2 and glucose responses to asphyxia whilst
their lactate, cortisol and catecholamine responses were not different from non-acidaemic
fetuses exposed to an acute asphyxial challenge. Rather than exclude these fetuses from
analysis, I have considered them as a subgroup because they have the ability to provide
potentially useful information about the fetal response to asphyxia.
The spontaneously acidaemic fetuses provided evidence to suggest that fetal scalp lactate
levels in previously compromised fetuses accurately reflect carotid arterial lactate levels rather
than underestimating lactate levels as demonstrated in the healthy fetuses exposed to
asphyxia. The spontaneously acidaemic fetuses also provided evidence to suggest that scalp
blood lactate levels reliably represents jugular venous lactate levels at rest and during asphyxia
in fetuses and previously compromised fetuses.
It has been suggested that fetal metabolism of glucose and lactate during acute hypoxaemia is
altered in fetuses exposed to an adverse intrauterine environment prior to the acute challenge
(Gardner et al., 2003). My study provided further evidence in support of this hypothesis
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because the spontaneously acidaemic fetuses displayed different cranial metabolism of lactate
and different glucose responses than non-acidaemic fetuses exposed to the same asphyxial
challenge. Considering arterio-venous lactate differences across the fetal cranial structures,
the normal fetus exposed to an asphyxial insult appeared to metabolise lactate as a source of
fuel during asphyxia whereas the spontaneously acidaemic fetus exposed to the same
challenge produced lactate. Both human and sheep fetuses are capable of using lactate as a
source of metabolic fuel (Fowden, 1994). In fact, in vitro studies have demonstrated that
neurones can carry out synaptic function with lactate as the only available carbon source
(Schurr et al., 1997). The altered lactate metabolism in the cranial structures of the
spontaneously acidaemic fetuses exposed to asphyxia may reflect attempts by the fetus to
mobilise an additional source of energy for the fetal brain during asphyxia because these
fetuses did not mount a hyperglycaemic response to the asphyxial insult which would have
provided energy to the fetal brain during asphyxia. There is evidence from rat models of
hypoxic-ischaemic brain injury that an increase in lactate output by brain tissue during
hypoxia-ischaemia serves to meet the energy needs of glutamate-activated neurons and may
be important to neuronal survival after asphyxia (Schurr & Rigor, 1998; Schurr et al., 1999).
Care is required in interpreting data derived from spontaneously acidaemic fetuses because
the cause of the specific reduction in fetal arterial pH and base excess despite appropriate
lactate concentrations is not known. No differences between the spontaneously acidaemic
fetuses and normal fetuses were observed in maternal condition, maternal cardiovascular
stability during anaesthesia, surgical duration or complications, or fetal biochemical status
prior to the experimental phase. Although the spontaneously acidaemic subgroup may have
developed during the instrumentation phase of the fetal preparation used in this study, the
significant alterations observed in fetal biochemical, endocrine and metabolic responses
during asphyxia seen in this subgroup suggest that these fetuses may have been inherently
different prior to the commencement of the fetal instrumentation. Arterial pH has been
shown to change independently of the concentration of circulating metabolic acids (Hooper
et al., 1990; Gardner et al., 2001a, b), and it has been postulated that this response may be
related to changes in specific metabolically-active hormone concentrations, in particular
catecholamines and prostaglandin E2 (PGE2; Hooper et al, 1990). Although my study
suggests that there were no catecholamine differences between the two groups, a role for
changes in PGE2 concentration or the placental and/or renal handling of acid cannot be
ruled out.
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4.4.3 Fetal scalp blood lactate and lipid peroxidation in the fetal brain
Although understanding the relationship between fetal scalp blood and central blood lactate
levels is important prior to the potential introduction of fetal scalp lactate measurement into
widespread clinical practice, the most important relationship that requires investigation is that
between fetal scalp blood lactate levels and fetal brain injury. No previous studies have
investigated the relationship between fetal scalp blood biochemical measurements and fetal
brain injury. This is probably because experimental models which afford regular, reliable
collection of adequate volumes of fetal scalp blood are not conducive to waiting three to
seven days after the insult for histopathologic brain injury to develop.
This study is the first to attempt to investigate the relationship between fetal scalp blood
biochemical measurements and potential brain injury through investigating the relationship
between fetal scalp blood lactate levels and oxygen free radical activity in the fetal brain. I
have shown during repeated umbilical cord occlusion there is evidence of increased lipid
peroxidation in the brain perfusate and that lactate levels are better predictors of the presence
of lipid peroxidation in the brain perfusate than routine acid-base analysis. Of particular
note, scalp lactate predicts oxygen free radical activity in the fetal brain at least as well as
carotid arterial lactate levels; perhaps slightly better.
4.4.3.1 Markers of lipid peroxidation
The two markers of lipid peroxidation measured in this study were organic hydroperoxides
(OHP) and malondialdehyde (MDA). Organic hydroperoxides are relatively stable (half-life
approximately three hours) products of free radical attack on lipids contained in cell
membranes (Ward & Peters, 1995; Wang et al., 1996) which appear to have limited transfer
across the human placenta (Rogers et al., 1999). Malondialdehyde is a by-product of lipid
peroxidation due to di-ene conjugation, which is itself responsible for some of the damaging
effects of free radicals on DNA and cell membranes (Ward & Peters, 1995). Unlike OHP,
MDA is thought to be actively transported across the placenta from the fetus to the mother
(Rogers et al., 1999) which would provide one mechanism to protect the fetus from the
known toxic effects of MDA on cellular metabolism (Gutteridge & Halliwell, 1990).
Malondialdehyde, as measured in this study by thiobarbituric acid adduction, includes not
only MDA produced by metabolism of OHP in vivo, but also that produced from
breakdown of OHP and endoperoxides in vitro (Gutteridge & Halliwell, 1990). Therefore,
of the two techniques to measure lipid peroxidation, OHP is the more robust measurement
for assessing fetal response to asphyxia (Rogers et al., 1997; Rogers et al., 1999).
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4.4.3.2 Patterns of lipid peroxidation during asphyxia
In this study I have shown a 260% increase in jugular venous levels of OHP and a 125%
increase in jugular venous levels of MDA in the fetuses exposed to repeated umbilical cord
occlusion. These results are similar to previous studies using similar cord occlusion protocols
in instrumented ovine fetuses (Rogers et al., 2001). It is likely that the increased lipid
peroxidation that appears evident in my study is secondary to the repeated hypoxia-
ischaemia-reperfusion that occurs with repeated umbilical cord occlusions. Similar dramatic
elevations of markers of lipid peroxide have previously been described in repeated ischaemia-
reperfusion models in rabbits and rats (Tan et al., 1998; Al Nita et al., 2001). During umbilical
cord occlusion, fetal hypoxia leads to depletion of the adenosine triphosphate (ATP) and an
increase in its degradation products (Harkness et al., 1982). Fetal hypoxia also results in the
accumulation of lactate and the conversion of xanthine dehydrogenase to xanthine oxidase
(Figure 4.17). During reoxygenation and reperfusion between umbilical cord occlusions,
xanthine oxidase catalysed by calcium causes the breakdown of hypoxanthine and its
derivatives generating oxygen free radicals. It is thought that with re-oxygenation,
metabolism of polyunsaturated fatty acids via the cyclooxygenase pathway and adenine
nucleotides via the xanthine oxidase pathway (Figure 4.17), are the most likely sources of
oxygen radicals responsible for subsequent lipid peroxidation (Fridovich, 1978).
It is likely that the OHP measured in my experiments is derived from vascular endothelial
cells in the brain and some from lipids in the plasma rather than directly from neuronal cells
themselves. However, neuronal and vascular endothelial cells are almost certainly subject to
similar degrees of oxidative stress to the blood perfusing the organ. In controls and during
the 0.5-minute cord occlusions, jugular venous OHP levels were consistently lower than the
matched carotid arterial samples, suggesting somatic rather than neural production of OHP
with scavenging and metabolism of peroxides by the brain, possibly by copper-zinc-
superoxide dismutase and anti-apoptotic protein Bcl-2 (Chan, 1996). The absence of an
increase in venous OHP levels during the 0.5-minute cord occlusions may be because the
occlusions were of insufficient duration to produce significant changes in oxygenation to the
fetal brain because of cardiovascular shunting of oxygenated blood to the fetal brain in
response to hypoxia. This hypothesis is supported by the receiver operator curve analysis of
lipid peroxidation in the brain perfusate which suggested that lipid peroxidation does not
reliably start in the fetal brain until oxygen tensions fall below 14mmHg. It has previously
been shown that arterial PO2 levels fall approximately 30% during 1.0-minute umbilical cord
occlusions to levels in the order of 14mmHg (Richardson et al., 1996). Therefore, it is
probable that during 0.5-minute cord occlusions, small reductions in carotid arterial PO2
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Oxygen FreeRadicals
Oxygen FreeRadicals
CordOcclusion
ONHypoxia
XDH XO LactateAccumulationATP Hydrolysis
Hypoxanthine
Xanthine
CordOcclusion
OFF
Urate
O2
XO
Figure 4.17 Mechanism of oxygen free radical production with umbilical cord occlusion.
Cord occlusion leads to fetal hypoxia which results in the accumulation of lactate, the
conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and accumulation
of hypoxanthine due to depletion of adenosine triphosphate (ATP). When oxygen is re-
introduced during intervals between cord occlusions, XO catalysed by calcium causes the
breakdown of hypoxanthine in two steps, each generating oxygen free radicals.
211
occur, but oxygen levels in the fetal brain are not low enough to stimulate lipid peroxidation.
Following from this, during 1.0-minute cord occlusions one would expect that oxygen levels
in the fetal brain would reach levels that would stimulate lipid peroxidation at least during
cord occlusion. This in fact is what occurred in this study. The absence of an increase in
venous OHP during the 0.5-minute cord occlusions may also have occurred because the
antioxidant defences in the fetal brain were not overwhelmed by the free radical production
in the early phase of the experimental protocol. Somatic OHP production during the 0.5-
minute cord occlusions may be secondary to hypoperfusion in peripheral, renal and
gastrointestinal circulations that occurs in response to cardiovascular adaptation to asphyxia
induced by umbilical cord occlusion (Itskovitz et al., 1987; Bobby & Santos, 2000).On
increasing the duration of cord occlusion to 1.0-minute and decreasing the inter-occlusion
recovery periods, the jugular venous OHP levels increased 260%. It is reasonable to assume
that by this stage of the experiment plasma low density lipoproteins were fully oxidised and
the predominate source of OHP release would be from vascular endothelial cells. During the
1.0-minute cord occlusions fetal carotid arterial OHP concentrations started to decrease. This
may have been due to severe restriction of oxygen supply to somatic tissues causing either a
decrease in peroxidation or a shutdown of these somatic vascular beds, or both. On further
increasing the duration of cord occlusions to 1.5-minutes, OHP production started to
plateau. The decrease in peroxidation may have been due to severe restriction of oxygen
supply in the fetal neural circulation by this stage of the experiment. Alternatively, there may
be a maximum rate of release of peroxides from endothelial cell membranes into the
circulation which was reached during the 1.0-minute occlusions.
A similar pattern of changes occurred in the MDA levels during this experiment; however,
the increase in jugular venous MDA levels did not occur until during the 1.5-minute cord
occlusions. The delayed response in MDA compared to OHP is not surprising as OHPs are
the primary molecular products of oxidative stress while MDA is one of the end products of
peroxidative cleavage of fatty acids. Malondialdehyde production involves the formation of
hydroperoxides, cleavage to yield hydro-peroxy-aldehydes, and finally, di-ene conjugation via
a second scission and dismutation of lipid peroxyl radicals (Gutteridge, 1988).
Malondialdehyde is also less stable than OHP, and given its particularly toxic effects on
cellular metabolism, the fetal brain may have better MDA scavenging mechanisms than exists
for OHP.
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4.4.3.3 The relationship between fetal scalp lactate and lipid peroxidation in the
fetal brain
In order to reflect oxidative stress in an organ, significantly higher levels of lipid peroxidation
must be detected in the venous drainage than found in the blood perfusing the organ (i.e. a
positive venous-arterial difference must exist). In this study I have shown that both fetal
scalp lactate levels and carotid arterial lactate levels are significantly better predictors of lipid
peroxidation in the fetal brain than acid-base assessment. This observation has not
previously been reported; however, it is consistent with the experimental findings presented
in chapter three and also with the current understanding of the mechanisms of fetal hypoxic-
ischaemic brain injury.
The fetal biochemical responses observed in the normal and spontaneously acidaemic fetuses
in response to asphyxia in this study may aid in unravelling the explanation of the potential
mechanisms responsible for the ability of fetal lactate levels to predict lipid peroxidation in
the fetal brain. The normal and spontaneously acidaemic fetuses in this study had the same
changes in lipid peroxidation in response to repeated UCO despite their different responses
in arterial pH, PO2, base excess and glucose. The two groups of fetuses also had the same
responses in arterial lactate levels, catecholamines and cortisol. These observations suggest
that changes in lactate, catecholamine and lipid peroxidation relate more to the insult than to
fetal status prior to acute asphyxia. In addition, similar findings in the two study groups
suggest that the common link may be perfusion and control of fetal blood pressure.
I propose that the ability of fetal lactate levels to predict lipid peroxidation in the fetal brain
during asphyxia is secondary to the ability of lactate to predict blood pressure instability
during umbilical cord occlusion and cerebral hypo-perfusion during asphyxia. In Chapter
Three evidence was presented that fetal lactate levels were better predictors of fetal blood
pressure instability during cord occlusion than other markers of fetal acid-base status. This is
particularly relevant because the duration of fetal blood pressure instability during umbilical
cord occlusion accounted for 91% of the variability in grading of histopathologic brain injury
72 hours after the same cord occlusion protocol as used in this study. In addition, there is
substantial evidence from adult critical care medicine that lactate levels are better predictors
of tissue hypoperfusion than pH and base excess (Perret & Enrico, 1978; Bakker et al., 1996;
Husain et al., 2003). As our understanding of the mechanisms responsible for hypoxic-
ischaemic brain injury has improved over the last 30 years, it has become increasingly
apparent that it is the severity of the changes in fetal blood pressure and cerebral blood flow
during asphyxia which predict fetal brain injury rather than acid-base and blood gas
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alterations (Myers, 1972; Ting et al., 1983; Gunn et al., 1992; Mallard et al., 1992; de Haan et al.,
1993; de Haan et al., 1997b; Ikeda et al., 1998a; Fujii et al., 2003).
The limited ability of routine acid-base assessment to predict lipid peroxidation in the fetal
brain is consistent with previous studies showing that the process of raised lipid peroxidation
products derived from reactive oxygen species may occur independently of changes in acid-
base status (Mongelli et al., 1997; Rogers et al., 1999). It is also consistent with animal (Myers,
1972; Ting et al., 1983; Gunn et al., 1992; Mallard et al., 1992; Ikeda et al., 1998a) and human
studies (Winkler et al., 1991; Low et al., 1994; Kruger et al., 1999) which suggest that fetal acid-
base status is a poor predictor of the severity of neuronal injury after asphyxia.
In human labour, the fetal scalp is the window for biochemical assessment of fetal wellbeing.
It was reassuring to find in this study that fetal scalp lactate was at least as good at predicting
lipid peroxidation in the fetal brain perfusate as carotid arterial lactate levels; perhaps even
better.
4.4.4 Future studies
The studies presented in this chapter I have significantly increased the understanding of the
relationship between fetal scalp blood lactate levels and central blood lactate levels during
asphyxia. I have not addressed the issue of the effect of fetal scalp oedema or caput
succedaneum on the reliability of fetal scalp lactate assessment, nor have I assessed the effect
of fetal head compression or moulding. The effect of caput on fetal scalp blood pH
assessment varies between studies. Some studies suggest that scalp oedema and caput can
result in scalp blood pH levels being up to 0.06 pH units lower than carotid arterial or
umbilical arterial blood levels in as many as 10-20% of samples (Bowe et al., 1970; Parer &
Livingston, 1990; Greene, 1999). Others authors have found that caput does not alter the
accuracy of fetal scalp blood pH assessment with no difference in the haematocrit between
scalp blood samples collected vaginally and those collected from the carotid artery and
jugular vein via fetal catheters (Adamsons et al., 1970). Fetal head compression and moulding
have not been shown to alter the accuracy of fetal scalp blood pH assessment (Adamsons et
al., 1970). Although it is important to understand the effect of caput succedaneum, scalp
oedema and moulding on the accuracy of fetal lactate assessment, these factors did not
appear to adversely affect the ability of fetal scalp blood lactate levels to predict low Apgar
scores at five minutes or moderate-severe hypoxic-ischaemic encephalopathy in humans
(Kruger et al., 1999).
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Fetal scalp pH measurements were not collected in this study to compare directly with fetal
scalp blood lactate levels because the blood gas machine in the research laboratory at the time
these studies were performed required 80µL of blood to perform a pH assessment in
contrast to the 15µL required to analyse lactate on the Accutrend® handheld lactate meter.
Although it is possible to collect large volumes of blood from the fetal scalp, I chose not to
attempt this to optimise the quality of the results obtained in this study. The collection of
large volumes of blood from the fetal scalp takes time and it has previously been shown that
the longer scalp blood is exposed to air, the less accurate the result (Zernickow, 1966). In
addition, the repeated nature of the sampling required in this study protocol would have
compromised the stability of the preparation as a substantial amount of time would have
been devoted to attempting to collect adequate volumes of blood rather than maintaining
thermal stability of the preparation and minimising fetal stimulation and manipulation. These
issues are particularly important given that one of the primary aims of the studies in this
chapter was to validate the model utilised for collecting fetal scalp samples.
This study raised the issue that lactate metabolism may be altered in the brains of fetuses in
an adverse uterine environment. This is potentially important given that many complications
of human pregnancy are associated with an adverse uterine environment. Limited
conclusions regarding cerebral metabolism can be made using only carotid artery and jugular
venous blood. More detailed evaluation of this area would require blood sampling from the
superior sagittal sinus and assessment of cerebral blood flow.
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4.5 Conclusion
In this chapter, three important issues relating to the collection of fetal scalp blood lactate
levels rather than central lactate levels during intrapartum asphyxia have been addressed.
The first aim was to compare the experimental model used to collect fetal scalp blood for
lactate assessment during asphyxia with the chronically instrumented model described in
Chapter Three. After considering the fetal biochemical, endocrine and cardiovascular
responses to asphyxia, the acutely instrumented fetal preparation used in this study did not
appear to impair the ability of the fetal lamb to coordinate neural, endocrine and local tissue
responses to asphyxia. Specifically, there was no evidence identified that the selected
maternal anaesthesia adversely affected autonomic control of the fetal heart during asphyxia
or the responsiveness of the FHR to hypoxaemia. I propose that the experimental model
chosen in this study is currently the best that is available to assess fetal scalp blood in a
regular, reliable, repeated manner during asphyxia, allowing collection of adequate volumes of
fetal scalp blood for biochemical assessment.
The second aim of this study was to evaluate the relationship between fetal scalp blood
lactate and central blood lactate levels during asphyxia. I have shown that in the absence of
asphyxia, scalp blood lactate levels are not significantly different from carotid arterial lactate
levels; however, when the fetus is exposed to asphyxia, fetal scalp blood lactate levels were
found to underestimate the carotid arterial lactate levels by a mean of 0.6mmol/L. Although
the mechanism responsible for scalp lactate underestimating central lactate during asphyxia is
unknown, I have provided evidence to suggest the underestimate during asphyxia may be due
to the cardiovascular changes which occur in response to umbilical cord occlusion and the
influence of adrenaline on the fetal scalp. In this study fetal scalp blood lactate levels were
found to be not significantly different from jugular venous lactate levels and this relationship
was not influenced by fetal asphyxia or fetal condition prior to asphyxia. This observation
may be advantageous because the composition of jugular venous blood is determined not
only by the composition of arterial blood, but also by the relationship between flow rate and
the metabolic needs of the fetal brain.
The final aim of this chapter was to investigate the relationship between fetal scalp lactate
levels and an indirect marker of fetal brain injury, lipid peroxidation. I have shown that fetal
scalp lactate levels are significantly better than carotid arterial pH, PO2, and base excess at
predicting lipid peroxidation in the fetal brain during asphyxia. The potential mechanism
responsible for the improved ability of lactate to predict lipid peroxidation may be secondary
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to the ability of lactate levels to predict fetal blood pressure instability during umbilical cord
occlusion and tissue hypoperfusion during asphyxia.
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C h a p t e r 5
FETAL LACTATE RESPONSES IN THE GROWTH RESTRICTED
OVINE FETUS DURING ASPHYXIA INDUCED BY GRADED
CORD OCCLUSION
After all, concentration is the price the modern student pays for success.
Thoroughness is the most difficult habit to acquire,
but it is the pearl of great price,
worth all the worry and trouble of the search.
Sir William Osler (1892)
5.1 Introduction
The motivation to perform the studies presented in this chapter arose from the observation
in both Chapters Three and Four that an adverse intrauterine environment, as demonstrated
by high fetal lactate levels or a spontaneous mild acidaemia prior to the experimental
protocol, altered cardiovascular, endocrine and metabolic responses to acute hypoxaemia in
the ovine fetus. The study subgroups in Chapters Three and Four occurred spontaneously
and their aetiology is uncertain. This limits the interpretation of the results relating to these
subgroups; however, it does suggest that formal investigation of lactate responses during
asphyxia in fetuses with an adverse intrauterine environment should be considered as part of
the evaluation of the role of lactate in intrapartum assessment of fetal wellbeing.
There are many potential causes of an adverse intrauterine environment in human pregnancy.
These include uteroplacental insufficiency, diabetes mellitus, preterm premature rupture of
the fetal membranes, intrauterine infection, maternal medical conditions, and exposure to
maternal medications. In the studies presented in this chapter, I have specifically chosen to
investigate the effect of the adverse intrauterine environment associated with growth
restriction due to uteroplacental insufficiency.
The intrauterine environment of the growth restricted fetus is significantly different from that
of appropriately grown fetuses. Growth restricted human fetuses have been shown to be
218
hypoxaemic, hypercapnic, hyperlactaemic, and acidaemic when compared to appropriately
grown gestational age-matched controls (Nicolaides et al., 1989; Marconi et al., 1990). It has
also been shown that growth restricted fetuses have altered lactate metabolism (Marconi et al.,
1990), glucose metabolism (Jansson et al., 1993; Marconi et al., 1996; Pardi et al., 2002), amino
acid metabolism (Cetin et al., 1996; Marconi et al., 1999) and fatty acid metabolism (Cetin et
al., 2002) when compared to appropriately grown controls.
Fetal growth restriction occurs in between three and five percent of pregnancies. These
fetuses are over-represented in morbidity and mortality data, with infants born between 38
and 42 weeks gestation, whose weights are between 1500g and 2500g, having perinatal
morbidity and mortality rates 3-30 times those of infants whose weights are between the 10th
and 90th centile (Williams et al., 1982; Dashe et al., 2000).
There is a growing body of evidence to suggest that growth restricted fetuses are at increased
risk of adverse events during labour (Low et al., 1976; Low et al., 1981; Nieto et al., 1994;
Dashe et al., 2000) and, more importantly, they are at increased risk of adverse neurologic
outcomes related to acute intrapartum hypoxic events than appropriately grown fetuses
(Bukowski et al., 2003). Observational studies in humans have shown a significant inverse
relationship between birthweight percentile and frequency of abnormal fetal heart rate
patterns such as total and late decelerations (Low et al., 1976; Dashe et al., 2000). Similarly,
growth restricted fetuses have been shown to commence labour with slightly lower scalp pH
levels and also to have more rapid decreases in scalp pH levels during labour than normally
grown fetuses (Nieto et al., 1994). As the birthweight centile falls, the incidence of metabolic
acidaemia in umbilical arterial blood samples increases. Infants with birthweight less than the
3rd centile are three times more likely to have a metabolic acidaemia than those born with
weights in the 50th centile (Low et al., 1981).
Growth restricted human fetuses are at increased risk of neonatal encephalopathy and
cerebral palsy when compared with fetuses demonstrating normal growth (Blair & Stanley,
1990, 1992; Badawi et al., 1998a; Bukowski et al., 2003). Although there are data to suggest
that a proportion of cases of neonatal encephalopathy have an antenatal origin (Badawi et al.,
1998a) recent evidence suggests that fetuses with reduced growth potential are 20 times more
likely to develop neonatal encephalopathy secondary to an acute intrapartum hypoxic event
than normally grown fetuses (Bukowski et al., 2003). A consistent relationship has been
shown between the risk of cerebral palsy and birth weight centile (Stanley et al., 2000) with
22% of cases of spastic cerebral palsy occurring in babies born below the tenth centile (Blair
& Stanley, 1990). Fifty percent of cases of cerebral palsy in infants born between 34 and 37
219
weeks occur in infants with birth weights below the 10th centile (Rantakallio, 1985). Although
the majority of cases of cerebral palsy are thought to have an antenatal origin, between 5 and
28% of cases of cerebral palsy can be attributed to intrapartum events (Nelson, 1988;
MacLennan, 1999; Hagberg et al., 2001). It is not known whether fetuses with reduced
growth potential are more likely to develop cerebral palsy secondary to acute intrapartum
hypoxic events than fetuses with normal growth potential; however, the relationship between
growth restriction, acute intrapartum hypoxic events, and neonatal encephalopathy would
suggest that growth restricted fetuses may also be at an increased risk of cerebral palsy
attributable to intrapartum events. Therefore, considering the evidence that growth restricted
fetuses are more likely to experience adverse events in labour than normally grown fetuses, I
believe that it is important to explore any new technique proposed to assess fetal wellbeing in
labour in this important group of fetuses prior to its potential introduction into clinical
practice.
Despite the known importance of fetal growth disorders, there have been no published
experimental studies using animal models in which fetal heart rate patterns, acid-base, lactate
and endocrine responses to asphyxia have been investigated using a standardised intervention
in fetuses in which growth has previously been restricted. The studies presented in this
chapter were designed to investigate the lactate responses during asphyxia in growth
restricted fetuses. The experimental model was specifically designed to investigate central
and scalp lactate levels during asphyxia. In addition to characterising the biochemical and
physiologic responses of the growth restricted fetus during repeated UCO, I have also
investigated the ability of FHR patterns, pH and lactate levels to predict fetal arterial pressure
responses during cord occlusion, the factor previously identified (Section 3.3.5.3.3) as the
single best predictor of severity of fetal brain injury after repeated umbilical cord occlusion.
In the studies presented in this chapter, I used repeated umbilicoplacental embolisation to
induce fetal growth restriction because it reproduces many features seen in human growth
restricted fetuses (Nicolaides et al., 1989) and it has been widely used to study fetal endocrine,
renal, metabolic and cardiovascular responses to chronic placental insufficiency (Clapp et al.,
1981; Trudinger et al., 1987; Gagnon et al., 1995; Gagnon et al., 1996; Cock & Harding, 1997).
Umbilicoplacental embolisation has been shown to induce asymmetric growth restriction
and it reproduces the fetal acid-base and lactate changes seen in growth restricted human
fetuses (Nicolaides et al., 1989). In addition, this model of growth restriction has been shown
to result in high systolic-diastolic ratios in umbilical artery Doppler flow velocity waveform
analysis (Trudinger et al., 1987; Morrow et al., 1989; Gagnon et al., 1994) similar to those
220
which occur in growth restricted human fetuses (Giles et al., 1985; Trudinger et al., 1985;
McCowan et al., 1987). Umbilicoplacental embolisation has also been shown to result in fetal
cerebral white matter injury (Mallard et al., 1998; Duncan et al., 2000) similar to that which can
occur in severely growth restricted human fetuses (Leviton & Paneth, 1990; Gaffney et al.,
1994c). After asymmetric growth restriction had been induced, fetuses were acutely
instrumented using the protocol described in Chapter Four to enable the assessment of both
carotid arterial and scalp blood lactate levels during asphyxia induced by repeated umbilical
cord occlusion.
5.1.1 Aim
There are four specific aims for the studies presented in this chapter:
1. to characterise the physiologic responses of growth restricted fetuses to repeated
UCO using a pattern designed to mimic events which may occur in labour,
2. to investigate the fetal lactate responses during UCO in the growth restricted
fetus,
3. to investigate the relationship between fetal scalp, carotid arterial and jugular
venous blood lactate levels both at rest and during asphyxia in the growth
restricted fetus, and
4. to investigate the ability of fetal scalp blood lactate levels to predict fetal arterial
pressure changes during asphyxia in the growth restricted fetus.
5.1.2 Hypothesis
I hypothesise that:
1. growth restricted ovine fetuses exposed to UCO will have more rapid
deterioration in their cardiovascular, biochemical and endocrine responses during
asphyxia than normally grown fetuses,
2. lactate responses in growth restricted fetuses will be similar to those in normally
grown fetuses exposed to the same asphyxial challenge,
3. fetal scalp lactate levels will reliably predict jugular venous lactate levels in growth
restricted fetuses and growth restriction will alter the relationship between fetal
scalp blood lactate levels and carotid arterial lactate levels, and
4. the ability of fetal lactate levels to predict fetal arterial pressure responses during
UCO will not be altered in growth restricted fetuses.
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5.2 Methods
All experimental procedures were approved by the Animal Experimentation Ethics
Committee of The University of Western Australia. Merino ewes with singleton pregnancies
were transported to the research facility at 96 days gestational age to allow for acclimatisation
to the facility. The experimental protocol utilised in this study had three consecutive stages
which are outlined in Figure 5.1.
5.2.1 First surgical preparation
Twenty four ewes underwent surgery at 101±1 days (mean±SEM) of pregnancy.
Anaesthesia was induced with Thiopentone 4mg/kg in 15mL saline solution (Abbott
Australasia Pty Ltd, Cronulla, NSW, Australia), ewes were intubated, and anaesthesia was
maintained with 0.75-1.25% Isoflurane (Abbott Australasia Pty Ltd, Cronulla, NSW,
Australia) in 100% oxygen delivered by positive pressure ventilation with ventilator settings
selected to maintain maternal blood gases in the normal range.
The uterus was exposed through a paramedian incision in the anterior abdominal wall and a
fetal hind limb was manipulated to an avascular region of a uterine horn. A small
hysterotomy was performed with care taken to avoid loss of amniotic fluid. Using an
established procedure (Trudinger et al., 1987; Gagnon et al., 1995; Cock & Harding, 1997;
Murotsuki et al., 1997a; Mallard et al., 1998; Duncan et al., 2000), a 1.5mm polyvinylchloride
catheter was implanted into the fetal descending abdominal aorta via a femoral artery, such
that the tip lay 1-2cm below the renal arteries and above the common umbilical artery
(approximately 7cm). This catheter subsequently enabled fetal blood sampling and injection
of microspheres into the umbilicoplacental circulation. After replacement of the hind limb, a
1-mL injection of Benacillin (procaine penicillin, 150mg/mL; benzathine penicillin,
150mg/mL; procaine hydrochloride, 20mg/mL; Troy Laboratories, Smithfield, Australia)
was injected into the amniotic fluid. The uterus was closed and the catheter was tunneled
subcutaneously to the ewe’s flank. The maternal abdomen was closed and 3mL
intramuscular Benacillin was given to the ewe at completion of the surgery. After surgery,
sheep were held in individual pens with access to food and water and catheter patency was
maintained by daily flushing with heparinised saline (2IU/1mL 0.9% NaCl).
222
Figure 5.1 Outline of experimental protocol. Twenty four ovine fetuses were instrumented
at 101 days to allow umbilicoplacental embolisation for 23 days. At 129 days the fetuses were
acutely instrumented to allow the collection of both central and peripheral blood samples
during asphyxia induced by repeated umbilical cord occlusion. Emb = embolised, Non-Emb
= non-embolised, Occl = cord occlusion protocol, Non-Occl = instrumented but no cord
occlusion.
Instrumented101 days
n = 24
UmbilicoplacentalEmbolisation
23 daysn = 12
Non-Embolised23 daysn = 12
CordOcclusion129 days
n = 7
Control129 days
n = 5
CordOcclusion129 days
n = 7
Control129 days
n = 5
Emb / Occl Emb / Non-Occl
Non-Emb /Occl
Non-Emb /Non-Occl
5 dayRecovery
Em
bolis
atio
nP
hase
Exp
erim
enta
lP
hase
223
5.2.2 Embolisation protocol
After a five day recovery period (106±1days gestational age), each ewe was allocated at
random to one of two treatment groups: embolised (n=12) or non-embolised (n=12; Figure
5.1). In both groups, fetal blood was sampled daily (0.3mL) for measurement of arterial gas
tensions and pH (Rapidlab 228, Bayer Diagnostics, Tarrytown NY) and lactate (Accutrend®
Lactate, Roche Diagnostics GmbH, Mannheim, Germany). For 23±1 days, the fetuses
allocated to the embolisation group were given daily aortic injections of nonsoluble,
nonradiolabeled, mucosaccharide microspheres (Sephadex Superfine G-25, 40-70µm,
Pharmacia, Sweden) suspended in a 1%wt/vol solution of sterile isotonic saline with 0.02%
Tween 80 (≈1 million spheres/mL). Once per day repeated injections of microspheres (0.05-
0.20 million) were made every 20 minutes until the fetal arterial PO2 levels were ≈8mmHg or
more below pretreatment levels (daily total dose 0.05 to 2.00X106 spheres). The catheters of
fetuses in the non-embolised group were sampled and flushed daily, but no microspheres
were injected. Arterial blood samples (4.0mL) were collected from both groups at the
beginning and end of the embolisation phase for assessment of fetal catecholamine,
adrenocorticotropic hormone (ACTH), cortisol and glucose concentrations. Fetal blood was
centrifuged at 1500g for 10 minutes at 4°C. Plasma was removed and stored at -80°C until
analysed.
5.2.3 Second surgical preparation
At 129±1 days gestational age, all ewes were acutely instrumented using the experimental
protocol described in Chapter Four. Maternal anaesthesia, arterial pressure monitoring and
biochemical monitoring were the same as described in Section 4.1. As described previously,
great care was taken to minimise the use of anaesthetic agents and to maintain stability of
maternal arterial pressure and acid-base status during the procedure.
The fetuses were instrumented as described in Section 4.2.1 to allow collection of carotid
arterial blood samples, jugular venous blood samples, fetal electrocardiogram (ECG) and
fetal arterial pressure. To assess lactate metabolism in the fetal brain an additional catheter
was inserted into the superior sagittal sinus to allow collection of blood directly flowing from
the fetal brain in three fetuses of each of the four experimental groups. A vascular snare and
a circular three-crystal ultrasound cuff (10MHz Triton Technology Inc., USA) were applied
around the umbilical vessels (Section 4.2.1) to enable confirmation of normal umbilical blood
flow and complete obstruction of umbilical blood flow during cord occlusion. The fetal
scalp was shaved and the fetus was returned to the uterus with its head externalised to
224
facilitate repeated collection of fetal scalp blood samples. Care was taken to prevent loss of
amniotic fluid and thermal stability was maintained with overhead radiant warmers and
bubble wrap covering the fetal head between scalp blood samples to minimise heat and water
loss.
After a thirty minute recovery phase, baseline fetal carotid blood samples (0.3mL) were
collected for baseline measurement of arterial gas tensions and pH. Repeated fetal arterial
samples (0.3mL) were collected after a further 30 minute rest phase and if the fetal
preparation was stable the cord occlusion protocol was commenced.
5.2.4 Cord occlusion protocol
After confirmation that each fetal preparation was stable, the fetuses entered the
experimental phase of the study. Fetal arterial blood samples (4.0mL) were collected to
measure baseline arterial gas tensions, pH, lactate, glucose, ACTH, cortisol and
catecholamine concentrations. Fetal blood (0.2mL) was collected from the jugular vein to
measure venous pH, gas tensions and lactate levels. Silicone jelly was applied to the fetal
scalp and a scalp blood sample was collected in a heparinised capillary tube (40µL) from a
stab wound for lactate, pH and gas tension measurements. In the subgroup of fetuses
instrumented with a superior sagittal sinus catheter, an additional blood sample (0.2mL) was
collected to measure pH, gas tensions and lactate levels.
Seven fetuses from the embolised group and a further seven fetuses from the non-embolised
group were randomly allocated to the cord occlusion protocol. The remaining five fetuses in
each group acted as controls (Figure 5.1). The umbilical cord occlusion (UCO) protocol
used in this study is identical to that used in Chapters Three and Four (see Section 3.2.2).
Control fetuses underwent identical physiological monitoring and blood sampling as the
fetuses exposed to UCO. After each blood sample was withdrawn, sterile saline was infused
to replace the volume of blood removed for analysis.
Throughout the experimental phase, fetal blood samples were collected every nine minutes.
Carotid arterial blood (0.2mL), jugular venous blood (0.2mL) and fetal scalp blood (40µL)
were collected to measure gas tensions, pH, and lactate levels. In fetuses with superior
sagittal sinus catheters, an additional blood sample (0.2mL) was collected to measure gas
tensions, pH, and lactate levels. Additional arterial samples (1.0mL) were collected at 27 and
54 minutes to measure fetal glucose levels. At the end of the experimental phase, a final
arterial blood sample (4.0mL) was collected to measure glucose, ACTH, cortisol, and
225
catecholamine concentrations. The ewes and fetuses were then humanely killed using a lethal
dose of sodium phenobarbitone and the positions of the implanted catheters were confirmed
and fetal weights measured.
5.2.5 Fetal heart rate, arterial pressure and umbilical blood flow assessment
Fetal ECG and arterial pressure were recorded continuously onto computer at 1 KHz after
amplification (Bio amp, BP transducer MLT380, bridge amplifier ML110, PowerLab 8e,
ADInstruments, Sydney, Australia). Over the 1 minute prior to each cord occlusion, FHR
and FHR variability were assessed to calculate mean fetal heart rate, mean minute range
(MMR), short term variation (STV) and long term variability (LTV) as previously described in
Section 3.2.3. FHR traces were visually assessed to classify the fetal heart rate pattern during
each of the 27 cord occlusions using the classification system described in Section 3.2.3.
Fetal arterial pressure was measured for each heart beat and averaged for the 1 minute
preceding each cord occlusion. The fetal arterial pressure responses during UCO were also
assessed and classified using the arbitrary classification system described in Section 3.3.3.2
and illustrated in Figure 3.14. The ultrasound cuff around the umbilical vessels was linked to
a vascular spectrum analyser (Model SP 25 by Vasculab, Meda Sonics, USA) which enabled
continuous monitoring of the umbilical artery Doppler waveforms to confirm normal
umbilical blood flow between occlusions and complete obstruction of umbilical blood flow
during cord occlusion.
5.2.6 Biochemical methods
Plasma glucose, cortisol, and catecholamine concentrations were measured using the same
techniques as described in Section 3.2.6. Plasma immunoreactive ACTH concentrations
were measured using a commercial radioimmunoassay (Incstar, Stillwater, MN, USA)
validated previously for use in fetal sheep (Norman et al., 1985). The intra-assay coefficient
of variation was 4.5% and the inter assay 4%. The mean assay sensitivity was 15pg/mL.
5.2.7 Statistical methods
Descriptive data are presented as mean and standard error of the mean (SEM) unless
otherwise stated. Univariate analyses of categorical data were performed with Chi-square test
and continuous data were analysed with t-tests or the non-parametric equivalent as
appropriate. Levine’s test of equality of variance was used to compare group differences in
variation about the mean. Time to development of abnormal fetal heart rate patterns was
226
assessed using t-tests and Wilcoxon log rank tests and exact inference due to small sample
sizes.
Changes in fetal biochemical variables and cardiovascular parameters were analysed using
multivariate regression modeling with occlusion, time and experimental group modeled as
fixed effects and individual animals as random effects repeated over time. Regression
modeling was also used to analyse differences between samples collected simultaneously
from the four sites compared in this study matched by time and sheep. Variable selection in
multivariate modeling was based on stepwise procedures (forwards and backwards selection)
including the assessment of interaction terms to arrive at the most parsimonious model.
Comparisons of effect magnitude were based on evaluation of changes in each outcome for
one standard deviation of each independent variable considered.
When investigating the different physiological associations with FHR patterns in both the
embolised and non-embolised fetuses, analyses of FHR patterns consisted either of all 27
occlusion periods (648 observations in total) or were limited to occlusions 1, 2, 26 and 27 in
order to incorporate catecholamine concentrations as candidate covariates (96 observations
in total). Final multivariate models were obtained via backward selection methods following
a careful examination of all two-way interaction effects. Covariate effects for each FHR
pattern considered were presented as odds ratios and 95% confidence intervals (CI). Odds
ratios for continuous measurements were reported for a change of 0.5 standard deviation for
each covariate, relative to no change in this covariate. A positive effect was reported when
the odds ratio for a particular FHR pattern increased as the value of the considered variable
increased. An inverse effect was reported when the odds ratio for a FHR pattern increased
as the value of the considered variable decreased. In cases of wide confidence intervals,
conservative comparisons of effect magnitudes were based on the confidence interval limit
closest to one. Receiver operator curve (ROC) analysis was used to identify optimum cut off
values that maximised sensitivity and specificity for variables found to be associated with
abnormal FHR patterns in both groups of fetuses (Hanley & McNeil, 1983; Beck & Shultz,
1986; DeLong et al., 1988).
Sensitivity and specificity were calculated for the ability of specific FHR patterns to predict
each pattern of fetal arterial pressure response during UCO. ROC analysis was also used to
assess the ability of biochemical measures to predict fetal arterial pressure responses during
UCO and also to identify optimum cut off values that maximised sensitivity and specificity.
Accuracy is reported for the prediction of arterial pressure responses during UCO and is
defined as the proportion of correctly classified cases (true positives + true negatives) using
227
either the specific abnormal FHR pattern or the biochemical cut off point selected to
maximise the sensitivity and specificity (Zweig & Campbell, 1993).
P-values <0.05 were considered statistically significant and p-values <0.10 were considered
marginally significant. SAS statistical software was used for data analysis (Statistical Analysis
Systems Institute Inc. 1999, Version 8.2., Cary, NC).
228
5.3 Results
Results are presented in three sections, each addressing one of the following specific aims:
1. assessment of physiologic responses of the growth restricted fetus during
umbilical cord occlusion,
2. assessment of the relationship between fetal scalp blood lactate levels and central
lactate levels, and
3. the ability of FHR patterns and biochemical markers to predict arterial pressure
responses during UCO.
To aid with comparison between this and previous chapters, similar analyses and graphic
presentations are used. For all graphical representations fetuses exposed to umbilicoplacental
embolisation are represented as solid circles or bars and the non-embolised fetuses are
represented as open circles or bars.
5.3.1 Physiologic responses of the growth restricted fetus during umbilical cord
occlusion
5.3.1.1 Umbilicoplacental embolisation
Umbilicoplacental embolisation produced a 30 percent reduction in fetal arterial PO2 (Figure
5.2). The PO2 levels in the embolised fetuses were significantly lower than in the non-
embolised fetuses by day 5 of the 23±1 day embolisation protocol and remained significantly
lower thereafter (embolised 12.29±0.15mmHg; vs. non-embolised 17.31±0.15mmHg,
p<0.001). Fetal pH was also slightly lower in the embolised fetuses than in the non-
embolised fetuses during this phase (embolised 7.371±0.002; vs. non-embolised
7.392±0.002, p=0.003). Fetal arterial lactate (p=0.370), PCO2 (p=0.487), and base excess
levels (p=0.361) were not significantly different between groups.
At completion of the embolisation phase, the fetuses subjected to repeated umbilicoplacental
embolisation had lower PO2 levels than non-embolised fetuses (12.09±0.64mmHg vs.
17.64±0.73mmHg in non-embolised; p<0.001) and a 2-3 fold increase in adrenaline and
noradrenaline levels (Table 5.1). All other blood gas variables were not significantly different
between groups. Fetal glucose levels were lower in the embolised fetuses
(0.90±0.09mmol/L) than non-embolised fetuses (1.11±0.07mmol/L; p=0.084) but the
differences did not reach statistical significance. ACTH and cortisol levels were not
significantly different between groups. Umbilicoplacental embolisation produced a 29%
229
Figure 5.2 Acid-base changes during 23 days of umbilicoplacental embolisation from 106 to
129 days gestational age. Arterial PO2, pH and lactate concentration (mean ± SEM) for
embolised (E, solid circles, n=12) and non-embolised fetuses (NE, open circles, n=12). Fetal
PO2 was significantly lower in the embolised fetuses by day 5 of the experimental protocol
(p<0.001) and fetal pH was also marginally lower in the embolised fetuses during the
embolisation phase (p=0.003). Fetal lactate levels were not significantly different between
groups (p=0.370).
5
10
15
20
25
p<0.001
NE
E
PO2
PO
2 (m
mH
g)
7.30
7.35
7.40
7.45
NE
E
pH
pH
0 2 4 6 8 10 12 14 16 18 20 220
1
2
3
NE
E
Lactate
Days
Lact
ate
(mm
ol/L
)
230
reduction in body weight (embolised, 2.30±0.15kg vs. non-embolised, 3.22±0.07kg; p<0.001)
at the time of UCO phase.
Table 5.1 Effect of 23 days of umbilicoplacental embolisation on fetal status
Non-Embolised Embolised P-value
pH 7.33 ± 0.01 7.34 ± 0.01 0.650
PO2 (mmHg) 17.64 ± 0.73 12.09 ± 0.64 <0.001
Lactate (mmol/L) 2.77 ± 0.23 2.92 ± 0.40 0.748
Glucose (mmol/L) 1.11 ± 0.07 0.90 ± 0.09 0.084
ACTH (pg/mL) 23.44±2.66 20.80±1.81 0.529
Cortisol (ng/mL) 4.57 ± 0.64 6.20 ± 0.84 0.165
Adrenaline (ng/L) 0.23 ± 0.05 0.60 ± 0.16 0.025
Noradrenaline (ng/L) 4.48 ± 1.08 9.16 ± 1.57 0.021
Weight* (kg) 3.22 ± 0.07 2.30 ± 0.07 <0.001 *measured at completion of experimental phase
5.3.1.2 Fetal acid-base responses to repeated cord occlusion
Fetal arterial pH, PO2, PCO2, and base excess levels were not significantly different between
non-embolised controls and embolised controls. In addition, there were no changes in these
variables over the duration of the experimental phase in both control groups (Figure 5.3).
Arterial pH levels fell significantly during repeated UCO in both non-embolised fetuses
(p<0.001) and embolised fetuses (p<0.001) when compared to controls. The rate of fall of
arterial pH during UCO was significantly greater in embolised fetuses (p=0.002), and
considering the entire experimental phase, the embolised fetuses were significantly more
acidaemic than the non-embolised fetuses (mean group difference: -0.08 pH units [95% C.I.:-
0.14, -0.02], p=0.013).
Fetal arterial PO2 fell significantly during repeated UCO in both non-embolised fetuses
(p=0.003) and embolised fetuses (p<0.001) when compared to controls; however, the
decrease in PO2 levels occurred earlier and was more severe in the embolised fetuses
(p=0.027). The embolised fetuses had a significantly lower arterial PO2 than non-embolised
fetuses over the duration of the cord occlusion protocol (mean group difference: 5.17mmHg
[95% C.I. 3.67, 6.66], p<0.002).
230
Figure 5.3 Fetal acid-base responses during repeated umbilical cord occlusion. P-values on
each graph compare the fetuses that experienced UCO (occluded fetuses) with controls
whereas p-values between boxes compare the two groups of controls (non-embolised and
embolised) or the two groups of fetuses that experienced UCO. All measures of fetal acid-
base balance presented in this figure were not significantly different between the two groups
of controls. All fetuses exposed to repeated UCO developed hypoxia and respiratory
acidaemia. Although base excess appeared to fall in both groups of fetuses experiencing
UCO, these differences did not reach statistical significance. The magnitude of the hypoxia,
hypercapnia and acidaemia were significantly greater in the embolised fetuses than the non-
embolised fetuses.
231
Occluded(p=0.027)
0 27 54 81
7.0
7.1
7.2
7.3
7.4 pH
Time (minutes)
pH
p<0.001
0 27 54 81
7.0
7.1
7.2
7.3
7.4 pH
p<0.001
Time (minutes)
pH0 27 54 81
10
15
20
25
30
35 PO2
Time (minutes)
PO
2(m
mH
g)
p=0.003
0 27 54 81
10
15
20
25
30
35 PO2
p<0.001
Time (minutes)P
O2
(mm
Hg)
0 27 54 81
30
50
70
90PCO2
p=0.003
Time (minutes)
PC
O2
(mm
Hg)
0 27 54 81
30
50
70
90PCO2
p<0.001
Time (minutes)
PC
O2
(mm
Hg)
0 27 54 81
-10
-6
-2
2
Control Occluded
Base Excess
Time (minutes)
Base
Exc
ess
p=393
0 27 54 81
-10
-6
-2
2Base Excess
Control Occluded
Time (minutes)
Base
Exc
ess p=0.226
NON-EMBOLISED EMBOLISED
Control(ns)
Occluded(p=0.002)
Control(ns)
Occluded(p=0.131)
Control(ns)
Occluded(p=0.027)
Control(ns)
231
Figure 5.4 Fetal arterial lactate responses during repeated umbilical cord occlusion. P-values
on each graph compare fetuses experiencing UCO (occluded fetuses) with controls whereas
the statistical comparisons between the boxes compare the two groups of controls (non-
embolised and embolised) or the two groups of fetuses experiencing UCO. Arterial lactate
levels in controls did not vary during the experimental phase and were not significantly
different between groups. Repeated UCO was associated with an increase in arterial lactate
levels in non-embolised and embolised fetuses (p<0.001). The lactate response in the
embolised fetuses exposed to UCO was not significantly different from the lactate response
in the non-embolised fetuses experiencing UCO (p=0.300).
232
0 27 54 81
0
2
4
6
8
10 Lactate
Control
Occluded(p<0.001)
Time (minutes)
Lact
ate
(mm
ol/L
)
0 27 54 81
0
2
4
6
8
10 Lactate
Control
Occluded(p=0.002)
Time (minutes)
Lact
ate
(mm
ol/L
)
NON-EMBOLISED
EMBOLISED
Control(ns)
Occluded(ns)
233
Fetal arterial PCO2 levels increased significantly during repeated UCO in both non-embolised
fetuses (p=0.003) and embolised fetuses (p<0.001) when compared to controls. Similar to
arterial pH and PO2 responses, the magnitude of the change in arterial PCO2 levels in the
embolised fetuses was significantly greater than non-embolised fetuses (Figure 5.3).
5.3.1.3 Fetal arterial lactate response to repeated cord occlusion
Fetal arterial lactate levels were not significantly different between non-embolised controls
and embolised controls and there were no significant changes in these levels during the
experimental phase (Figure 5.4). Repeated UCO significantly increases arterial lactate levels
in both non-embolised fetuses (p<0.001) and embolised fetuses (p=0.002) when compared
to controls and there were no differences in lactate responses between groups (p=0.300).
5.3.1.4 Fetal endocrine responses to repeated cord occlusion
Fetal arterial glucose, cortisol, noradrenaline and adrenaline levels in both non-embolised and
embolised controls did not change significantly over the duration of the experimental phase
(Figure 5.5). Glucose levels were significantly lower in embolised controls (0.87±
0.08mmol/L) than non-embolised controls (1.13±0.08mmol/L; p=0.024); however cortisol,
noradrenaline and adrenaline levels were not significantly different between the two control
groups.
Fetal arterial glucose levels were not significantly different between non-embolised controls
and non-embolised fetuses that underwent UCO (p=0.101) In embolised fetuses UCO was
associated with a significant increase in fetal glucose levels (p=0.041). The opposite pattern
of responses occurred in fetal cortisol levels; non-embolised fetuses subjected to UCO
significantly increased their cortisol levels compared to controls (p=0.048) whilst embolised
fetuses exposed to the same UCO protocol did not significantly increase their cortisol levels
(p=0.156) when compared to appropriate controls. The different cortisol responses between
the two groups exposed to UCO occurred despite fetal ACTH levels increasing by the same
amount in both groups in response to asphyxia (Figure 5.6).
In both non-embolised and embolised fetuses, repeated UCO was associated with significant
increases in both noradrenaline (non-embolised p=0.022, embolised p=0.006) and adrenaline
levels (non-embolised p=0.008, embolised p=0.001) when compared to controls (Figure 5.5).
The magnitude of the increase in catecholamines was significantly greater in the embolised
fetuses (noradrenaline p=0.024, adrenaline p<0.001).
233
Figure 5.5 Fetal endocrine responses during umbilical cord occlusion. P-values on each
graph compare fetuses experiencing UCO (occluded fetuses) with controls whereas p-values
between the boxes compare the two groups of controls (non-embolised and embolised) or
the two groups of fetuses experiencing UCO. Repeated UCO was associated with an
increase in blood glucose levels in embolised occluded fetuses (p=0.041) which did not occur
in the non-embolised fetuses exposed to the same asphyxial insults (p=0.078). Conversely,
an increase in arterial cortisol levels in response to asphyxia only occurred in the non-
embolised fetuses (p=0.048) and not in the embolised fetuses during UCO. Both groups of
fetuses that experienced UCO increased their catecholamine levels in response to asphyxia;
however, the magnitude of this response was greater in the embolised fetuses (p<0.024).
234
0
1
2
3
0 27 54 81
Glucose
Time (minutes)
Glu
cose
(mm
ol/L
) p=0.041
0
10
20
30
40
50
0 81
Cortisol p=0.048
Time (minutes)
Cor
tisol
(ng/
mL)
0
20
40
60
80
0 81
Noradrenaline p=0.022
Time (minutes)
Nor
adre
nalin
e (n
g/L)
0
20
40
60
80
0 81
Noradrenaline
Time (minutes)
Nor
adre
nalin
e (n
g/L) p=0.006
0
20
40
60
80
0 81
Adrenaline
Control Occluded
p=0.018
Time (minutes)
Adre
nalin
e (n
g/L)
0
20
40
60
80
0 81
Adrenaline
Control Occluded
Time (minutes)
Adre
nalin
e (n
g/L)
p=0.001
NON-EMBOLISED EMBOLISED
Occluded(p=0.078)
Control(p=0.024)
Occluded(p<0.001)
Control(ns)
Occluded(p=0.018)
Control(ns)
Occluded(0.024)
Control(ns)
0
1
2
3
0 27 54 81
Glucose p=0.101
Time (minutes)
Glu
cose
(mm
ol/L
)
0
10
20
30
40
50
0 81
Cortisol p=0.156
Time (minutes)C
ortis
ol (n
g/m
L)
235
Non-em
bolis
ed 0
min
Contro
l 81 m
in
Occlud
ed 81
min
0
200
400
600
800
ACTHp=0.008
AC
TH (p
g/m
L)
Embolis
ed 0
min
Contro
l 81 m
in
Occlud
ed 81
min
0
200
400
600
800
ACTHp=0.001
AC
TH (p
g/m
L)
Non-em
bolis
ed 0
min
Contro
l 81 m
in
Occlud
ed 81
min
0
10
20
30
40
50
Cortisol
p=0.038
Cor
tisol
(ng/
mL)
Embolis
ed 0
min
Contro
l 81 m
in
Occlud
ed 81
min
0
10
20
30
40
50
Cortisol
p=0.160
Cor
tisol
(ng/
mL)
NON-EMBOLISED EMBOLISED
Occluded(ns)
Control(p=0.074)
Occluded(p=0.018)
Control(ns)
Figure 5.6 Fetal ACTH and cortisol responses during umbilical cord occlusion. P-values on
each graph compare fetuses experiencing UCO with baseline levels collected at the beginning
of the experimental phase. P-values between the boxes compare the two groups of controls
(non-embolised and embolised) or the two groups of fetuses experiencing UCO. Fetal
ACTH levels were significantly increased in both non-embolised fetuses (p=0.008) and
embolised fetuses (p=0.001) experiencing UCO when compared to baseline levels and post-
occlusion ACTH levels were not significantly different between groups of fetuses exposed to
UCO (p=0.768). Despite similar ACTH responses, the cortisol responses were different in
the two groups of occluded fetuses with a significant increase in cortisol occurring only in the
non-embolised fetuses subjected to UCO (p=0.038)
236
5.3.1.5 Fetal heart rate responses to repeated cord occlusion
5.3.1.5.1 Baseline fetal heart rate response to repeated cord occlusion
Baseline FHR, assessed during the one minute prior to each UCO, was not significantly
different between non-embolised controls (137.6±5.1bpm) and embolised controls
(138.0±5.9bpm; p=0.955; Figure 5.7) and there was no significant change in baseline FHR
over the duration of the experimental phase in both control groups (p>0.893). Fetal heart
rate increased significantly in both non-embolised fetuses exposed to UCO (p=0.008) and
embolised fetuses exposed to UCO (p=0.044) when compared to appropriate controls.
Visually the two occluded groups had different patterns of response in baseline FHR during
the experimental phase (Figure 5.8); however, these differences were not statistically
significant (p=0.125). Fetal heart rate in the embolised fetuses increased earlier in response
to UCO than occurred in non-embolised fetuses (Figure 5.7). In addition, the maximum
increase in FHR in embolised fetuses experiencing UCO was less than non-embolised fetuses
during UCO (p=0.042).
5.3.1.5.2 Fetal heart rate variability response to repeated cord occlusion
Fetal heart rate variability (MMR, STV and LTV) was lower in embolised controls than in
non-embolised controls; however, these differences did not reach statistical significance
(non-embolised vs. embolised; MMR 22.49±3.39msec vs. 20.72±3.11msec, p=0.695; STV
1.60±0.20msec vs. 1.53±0.19msec, p=0.804; LTV 6.15±1.54bpm vs. 3.69±1.42bpm,
p=0.383). All measures of FHR variability increased in both non-embolised and embolised
fetuses subjected to UCO (Figure 5.7). The magnitude of the increase in STV and LTV was
significantly greater in non-embolised than embolised fetuses experiencing UCO (STV,
p=0.001; LTV, p=0.012). Fetal heart rate variability responses of the two groups of fetuses
subjected to UCO are presented graphically in Figure 5.8.
5.3.1.5.3 Fetal heart rate patterns during repeated cord occlusion
The ultrasound cuff around the umbilical vessels confirmed normal umbilical blood flow in
controls and between cord occlusions in the occluded fetuses. Doppler assessment also
confirmed complete obstruction of umbilical blood flow during each cord occlusion during
the experimental phase.
In controls, no accelerations or decelerations were present during the 81 minute experimental
phase. In five of the seven non-embolised fetuses subjected to UCO, FHR accelerations
were present primarily during the 0.5-minute cord occlusions; in three fetuses these were
236
Figure 5.7 Fetal heart rate responses during umbilical cord occlusion. P-values on each graph
compare fetuses experiencing UCO (occluded fetuses) with controls whereas p-values
between the boxes compare the two groups of controls (non-embolised and embolised) or
the two groups of fetuses experiencing UCO. All fetuses subjected to UCO increased their
inter-occlusion FHR and FHR variability; however, the magnitude of these increases were
less in the embolised fetuses compared to the non-embolised fetuses. The maximum
increase in FHR in embolised fetuses was less than in non-embolised fetuses during UCO
(p=0.042). Similarly, the magnitude of the increase in STV and LTV in embolised fetuses
was less than in non-embolised fetuses during UCO (p=0.001 and p=0.012 respectively).
237
-27 0 27 54 81100
150
200
250
p=0.008
Time (minutes)
FHR
(bpm
)
-27 0 27 54 81100
150
200
250
p=0.044
Time (minutes)
FHR
(bpm
)
-27 0 27 54 810
20
40
60
80
100
Time (minutes)
FHR
MM
R (m
sec)
p<0.001
-27 0 27 54 810
20
40
60
80
100
Time (minutes)
FHR
MM
R (m
sec)
p=0.057
-27 0 27 54 810
3
6
9
Time (minutes)
FHR
STV
(mse
c)
p<0.001
-27 0 27 54 810
3
6
9
Time (minutes)
FHR
STV
(mse
c)
p=0.717
-27 0 27 54 810
20
40
60
p<0.001
Control Occluded
Time (minutes)
FHR
LTV
(bpm
)
-27 0 27 54 810
20
40
60
p=0.018
Control Occluded
Time (minutes)
FHR
LTV
(bpm
)
NON-EMBOLISED EMBOLISED
Occluded(ns)
Control(ns)
Occluded(0.012)
Control(ns)
Occluded(ns)
Control(ns)
Occluded(p=0.001)
Control(ns)
237
5.8 Fetal heart rate responses in the two groups of fetuses exposed to repeated cord
occlusion. P-values on each graph compare the non-embolised fetuses subjected to UCO
with the embolised fetuses subjected to UCO. Although both groups of fetuses experiencing
UCO increased their inter-occlusion FHR and FHR variability, the pattern of these responses
was altered by umbilicoplacental embolisation.
238
-27 0 27 54 81100
150
200
250
Embolised
Non-embolised
Time (minutes)
FHR
(bpm
)
p=0.125
-27 0 27 54 810
20
40
60
80
100
Non-embolised
Embolised
Time (minutes)
FHR
MM
R (m
sec) p=0.129
-27 0 27 54 810
3
6
9
Non-embolised
Embolised
Time (minutes)
FHR
STV
(mse
c) p=0.549
-27 0 27 54 810
20
40
60
Non-embolised
Embolised
Time (minutes)
FHR
LTV
(bpm
) p=0.447
239
repeated events and in two they were isolated events. A similar pattern was observed in the
embolised fetuses subjected to UCO with FHR accelerations present in four of the seven
fetuses; in two fetuses they were repeated during the 0.5-minute occlusions and in two they
were isolated events. Variable FHR decelerations were seen in all fetuses subjected to UCO.
Severe variable decelerations occurred in five of the seven non-embolised fetuses and six of
the seven embolised fetuses during UCO. Similarly late components to decelerations
occurred in five of 7 and six of 7 fetuses in these groups, respectively. Overshoot instability
was present in all of the non-embolised fetuses at some time during the experimental phase
but was only present in four of the seven embolised fetuses subjected to UCO. Both severe
variable decelerations (Figure 5.9A and 5.9B) and late components to decelerations (Figure
5.9C and 5.9D) occurred significantly earlier during repeated cord occlusions in embolised
occluded fetuses when compared to non-embolised occluded fetuses (severe variable
decelerations, p=0.030; late component, p=0.042). Embolised fetuses also had more
variability in the time of onset of both of these decelerative patterns than non-embolised
fetuses (severe variable deceleration, p=0.025; late component, p=0.013). Conversely,
overshoot instability after severe variable decelerations (Figure 5.9E and 5.9F) occurred later
in embolised occluded fetuses than in non-embolised occluded fetuses (p=0.031).
5.3.1.6 Fetal arterial pressure responses to repeated cord occlusion
5.3.1.6.1 Fetal arterial pressure between cord occlusions
Fetal arterial pressures did not change significantly in either control group over the duration
of the experimental phase (p=0.576; Figure 5.10 and 5.11). Both systolic and diastolic
pressures in embolised controls were significantly higher than non-embolised controls
(systolic pressure 62.6±2.4mmHg vs. 55.6±2.4mmHg, p<0.001; diastolic pressure
46.7±2.6mmHg vs. 36.8±2.6mmHg, p<0.001).
In non-embolised fetuses both systolic and diastolic pressures increased significantly in
response to UCO when compared with controls (Figures 5.10 and 5.11; both p<0.001). In
contrast, fetal systolic (p=0.236) and diastolic pressures (p=0.753) in embolised fetuses that
underwent UCO were not significantly different to controls. Over the duration of the
experimental phase, the systolic and diastolic pressure responses in the non-embolised
fetuses subjected to UCO were significantly different from embolised fetuses subjected to
UCO (systolic pressures, p=0.002; diastolic pressures, p=0.012).
240
Severe VariableDecelerations
0
27
54
81
Embolised Occluded
Non-embolised Occluded
p = 0.030
Tim
e of
Ons
et (m
in)
Severe VariableDecelerations
0 9 18 27 36 45 54 63 72 810
25
50
75
100
p = 0.138
Time (min)
Patte
rn P
rese
nt (%
)
Late Components toDecelerations
0 9 18 27 36 45 54 63 72 810
25
50
75
100
p = 0.042
Time (min)
Patte
rn P
rese
nt (%
)
Late Components toDecelerations
0
27
54
81
Embolised Occluded
Non-embolised Occluded
p = 0.029
Tim
e of
Ons
et (m
in)
Overshoot
0 9 18 27 36 45 54 63 72 810
25
50
75
100
p = 0.031
Non-embolised / OccludedEmbolised / Occluded
Time (min)
Patte
rn P
rese
nt (%
)
Overshoot
0
27
54
81
Embolised Occluded
Non-embolised Occluded
p = 0.083
Tim
e of
Ons
et (m
in)
A B
C D
E F
Figure 5.9 Fetal heart rate decelerative patterns during umbilical cord occlusion. Figures A, C
and E show the percentage of fetuses at each time point with particular FHR patterns.
Figures B, D and F show the median time of onset of each FHR pattern. Both severe
variable decelerations and late components to decelerations occurred earlier and with more
variability in time of onset in embolised fetuses that underwent repeated UCO than non-
embolised fetuses exposed to the same asphyxial insult. In contrast, overshoot instability
occurred later, and only in 57% of embolised fetuses experiencing UCO.
241
-27 -18 -9 0 9 18 27 36 45 54 63 72 8140
50
60
70
80
90
Control
Occluded
p<0.001
Time (min)
Feta
l Sys
tolic
Pres
sure
(mm
Hg)
-27 -18 -9 0 9 18 27 36 45 54 63 72 8140
50
60
70
80
90
Control
Occluded
Time (min)
Feta
l Sys
tolic
Pres
sure
(mm
Hg) p=0.236
NON-EMBOLISED
EMBOLISED
Control(p<0.001)
Occluded(p=0.002)
Figure 5.10 Fetal systolic pressure responses between umbilical cord occlusions. P-values on
each graph compare fetuses experiencing UCO with controls whereas p-values between the
boxes compare the two groups of controls (non-embolised and embolised) or the two groups
of fetuses that experienced UCO during the experimental phase. The systolic pressures in
the embolised controls were significantly higher than in the non-embolised controls
(p<0.001) and, when subjected to UCO the embolised fetuses did not significantly increase
their inter-occlusion systolic pressures (p=0.236). The non-embolised fetuses increased their
systolic pressures between the 1.0-minute and 1.5-minute cord occlusions (p<0.001).
242
-27 -18 -9 0 9 18 27 36 45 54 63 72 8120
30
40
50
60
70
Control
Occluded
Time (min)
Fet
al D
iast
olic
Pres
sure
(mm
Hg) p<0.001
-27 -18 -9 0 9 18 27 36 45 54 63 72 8120
30
40
50
60
70
Occluded
Control
Time (min)
Fet
al D
iast
olic
Pres
sure
(mm
Hg) p=0.753
NON-EMBOLISED
EMBOLISED
Control(p<0.001)
Occluded(p=0.002)
Figure 5.11 Fetal diastolic pressure responses between umbilical cord occlusions. P-values
on each graph compare fetuses experiencing UCO with controls whereas p-values between
the boxes compare the two groups of controls or the two groups of fetuses that experienced
UCO during the experimental phase. The diastolic pressures in embolised controls were
significantly higher than non-embolised controls (p<0.001). In response to UCO the non-
embolised fetuses significantly increased their diastolic pressures compared to non-embolised
controls (p<0.001) whereas the diastolic pressures in the embolised fetuses experiencing
UCO were not significantly different from embolised controls (p=0.753).
243
5.3.1.6.2 Fetal arterial pressure during cord occlusions
Both non-embolised and embolised fetuses subjected to UCO displayed the four distinct
patterns of fetal arterial pressure responses during cord occlusion (hypertensive, decreasing
hypertensive, biphasic systolic and biphasic diastolic) that were described in detail in Section
3.3.3.2 and illustrated in Figure 3.14. The rate of progression through the four distinct
patterns was similar in embolised and non-embolised fetuses with no significant differences
between groups in the timing of decreasing hypertensive response and biphasic systolic
response (Figure 5.12). The embolised fetuses that experienced repeated UCO developed
the biphasic diastolic response earlier (p=0.056) and in a more variable manner (p=0.046)
than the non-embolised fetuses exposed to the same UCO protocol.
5.3.1.7 Physiologic associations with fetal heart rate patterns in embolised and non-
embolised fetuses
To investigate the potential mechanisms responsible in embolised fetuses for the earlier
decelerative FHR patterns when compared to non-embolised fetuses, univariate and
multivariate regression modeling was performed.
Summaries of the univariate analyses of risk factors associated with particular heart rate
patterns, including fetal blood gas variables, fetal arterial pressure between cord occlusions
and fetal catecholamine concentrations, are presented in Table 5.2. A number of significant
associations were identified with each FHR pattern except overshoot after variable
decelerations which did not appear to be associated with any of the variables considered in
the analysis.
Summaries of the multivariate analyses considering all of the acid-base measures, lactate
levels and arterial pressures simultaneously as risk factors for particular FHR patterns are
presented in Table 5.3. A subset analysis of occlusions 1, 2, 26 and 27 was also performed to
assess fetal catecholamine concentrations in the multivariate analysis together with fetal blood
gases and fetal arterial pressures. Significant associations with FHR patterns are presented in
Table 5.4. Optimal cut off values for significant predictors of FHR patterns were estimated
using ROC analysis and are summarised in Table 5.5.
243
Figure 5.12 Fetal arterial pressure responses during umbilical cord occlusions. The rate of
progression through the four distinct patterns was similar in embolised and non-embolised
fetuses with no significant differences between groups in the timing of decreasing
hypertensive response and biphasic systolic response (Figure 5.12). The embolised fetuses
that experienced repeated UCO developed the biphasic diastolic response earlier (p=0.056)
and in a more variable manner (p=0.046) than the non-embolised fetuses exposed to the
same UCO protocol
244
0 27 54 810
25
50
75
100
p=0.173
Time (min)
Patte
rn P
rese
nt (%
)
0
27
54
81p=0.171
EmbolisedOccluded
Non-embolisedOccluded
Tim
e (m
in)
0 27 54 810
25
50
75
100
Time (min)
Patte
rn P
rese
nt (%
)
p=0.219
0
25
50
75
100p=0.175
EmbolisedOccluded
Non-embolisedOccluded
Tim
e (m
in)
0 27 54 810
25
50
75
100
p=0.086
Non-embolised / Occluded
Embolised / Occluded
Time (min)
Patte
rn P
rese
nt (%
)
0
25
50
75
100p=0.056
EmbolisedOccluded
Non-embolisedOccluded
Tim
e (m
in)
Decreasing Hypertensive Response
Biphasic Systolic Response
Biphasic Diastolic Response
245
Table 5.2 Univariate associations of FHR patterns with blood gas, arterial pressure and catecholamine values
Univariate Associations FHR Pattern Association a
Blood Gases Arterial Pressure Catecholamines
Positive Base excess‡ pH*
Acceleration
Inverse Lactate‡ PCO2
* Diastolic pressure† Adrenaline* Noradrenaline*
Positive Lactate‡ PCO2
‡ Diastolic pressure‡ Systolic pressure†
Adrenaline* Noradrenaline†
Late Deceleration
Inverse pH‡ Base excess‡ PO2
†
Positive Lactate‡ PCO2
* Adrenaline*
Noradrenaline* Severe Variable Deceleration Inverse
Base excess‡ pH† PO2
†
Positive Lactate† PCO2
* Diastolic pressure* Adrenaline† Increased Variability
Inverse Base excess‡ pH† PO2
†
Positive Overshoot
Inverse
a A positive association indicates that the odds of the FHR pattern occurring increased as the value of the variable increased. An inverse association indicates the odds of the FHR pattern occurring increased as the value of the variable decreased. *p≤0.05, † p<0.01, ‡ p<0.001
246
Table 5.3 Multivariate associations of FHR patterns considering all blood gas variables and fetal arterial pressure variables simultaneously
Group
Embolised Non-embolised
FHR Pattern Associationa Variable Variable P-value
Positive pH Base excess
0.027 0.022
Acceleration
Inverse PO2
Systolic pressure -
PO2 Systolic pressure
PCO2
<0.001 0.020 0.041
Positive Lactate Systolic pressure*
Lactate Systolic pressure
<0.001 0.025 Late
Deceleration Inverse Base excess
pH Base excess
- 0.017
<0.001
Positive
Systolic pressure Lactate†
- PCO2
Systolic pressure Lactate
Base excess -
<0.001 0.003 0.001 0.026 Severe
Variable Deceleration
Inverse - Base excess
pH -
0.011 0.001
Positive -
PCO2 Systolic pressure
Diastolic pressure - -
<0.001 <0.001 <0.001
Increased Variability
Inverse
- pH
Base excess Lactate
Diastolic pressure
Systolic pressure - - - -
<0.001 <0.001 <0.001 <0.001 <0.001
Positive Overshoot
Inverse
a A positive association indicates that the odds of the FHR pattern occurring increased as the value of the variable increased. An inverse association indicates the odds of the FHR pattern occurring increased as the value of the variable decreased. *Effect significantly greater in embolised fetuses, p=0.003 † Effect significantly greater in non-embolised fetuses, p=0.031
247
Table 5.4 Multivariate associations of FHR patterns considering all blood gas, fetal arterial pressure and catecholamine variables simultaneously
FHR Pattern Associationa Multivariate Associations
(blood gas, arterial pressure, catecholamines)
P-value
Positive
Acceleration Inverse PO2
Noradrenaline 0.016 0.006
Positive Adrenaline 0.007 Late Deceleration
Inverse Base excess <0.001*
Positive Systolic pressure 0.006 Severe Variable Deceleration
Inverse pH 0.043
Positive Diastolic PCO2
0.009 0.011 Increased Variability
Inverse Systolic pressure Base excess
0.016 <0.001
Positive Overshoot
Inverse
a A positive association indicates that the odds of the FHR pattern occurring increased as the value of the variable increased. An inverse association indicates that the odds of the FHR pattern occurring increased as the value of the variable decreased. *Greater effect in embolised fetuses (p=0.021)
248
Table 5.5. Receiver operator curve analysis for the prediction of FHR patterns from fetal physiologic parameters
FHR Pattern Physiologic Variable
Umbilicoplacental Circulation
Optimum Cut Off Sensitivity Specificity
Non-embolised 22.1 88.9 61.2 Arterial PO2 (mmHg) Embolised 21.1 100.0 50.0
Embolised 65 100.0 43.9 Acceleration
Systolic pressure (mmHg) Non-embolised 50 26.7 95.4
Non-embolised 3.1 85.7 67.3 Lactate (mmol/L) Embolised 3.1 97.0 77.7
Non-embolised -1.8 80.0 60.5 Base Excess
Embolised -4.1 69.7 79.8
Non-embolised 61 74.3 64.1
Late Deceleration
Systolic pressure (mmHg) Embolised 55 95.5 55.0
Non-embolised 61 97.2 67.0 Systolic pressure (mmHg) Embolised 63 87.1 68.7
Non-embolised 4.5 77.8 93.5
Severe Variable Deceleration Lactate
(mmol/L) Embolised 3.5 92.9 78.5
Non-embolised 53 92.7 29.1 Systolic pressure (mmHg) Embolised 67 89.5 72.1
Non-embolised 35 97.6 33.3 Increased Variability Diastolic
pressure (mmHg) Embolised 41 100.0 47.8
5.3.1.7.1 Fetal heart rate accelerations
Multivariate analyses indicated that the only variables associated with FHR accelerations in
both groups of fetuses were fetal arterial PO2 (p<0.001) and fetal systolic pressure (p=0.020).
The magnitudes of these effects were similar with the odds ratio for accelerations increasing
by 2.73 (95%CI 2.06-3.71) for each fall in PO2 of 5 mmHg and decreasing by 1.40 (95%CI
1.05-1.86) for each increase in systolic pressure of 5mmHg. After inclusion of catecholamine
data into the multivariate analysis, a significant inverse association was also found between
noradrenaline (p=0.006). ROC analysis indicated the very close optimum cut offs for PO2 in
both groups with high sensitivity and modest specificity (Table 5.5). The optimum cut off
values for systolic pressure was different in the embolised and non-embolised fetuses with
249
very poor sensitivity in the non-embolised fetuses. In the non-embolised fetuses, additional
associations were found between pH, PCO2, and base excess and the presence of FHR
accelerations. The magnitudes of these effects were at least five times greater than the effect
of PO2 or systolic pressure. Arterial pH, PO2 and base excess were not associated with FHR
accelerations in fetuses that underwent embolisation.
5.3.1.7.2 Late component to decelerations
Multivariate analyses indicated that the only variables associated with a late component to
decelerations in both groups of fetuses were arterial lactate concentrations (p<0.001), systolic
pressure (p=0.025) and base excess (p=0.017; Table 5.3). In both groups of fetuses the
magnitude of effect of lactate and base excess were similar with the odds ratio of late
decelerations increasing by 1.62 (95%CI 1.09-2.41) for each fall of 2 base excess units and
increasing by 2.90 (95%CI 1.60-5.24) for each 2mmol/L increase in lactate. The effect of
systolic pressure on late decelerations was significantly greater in the embolised fetuses
(p=0.003) with each 5 mmHg increase in fetal systolic pressure increasing the odds ratio of
late decelerations by 6.44 (95%CI 2.36-17.55) in the embolised fetuses, in contrast to 1.33
(95%CI 1.01-1.76) in the non-embolised fetuses. Fetal arterial pH was only associated with
late decelerations in the embolised group, with each fall in pH of 0.05 pH units increasing the
odds ratio of late decelerations by 4.63 (95%CI 2.37-9.06, p<0.001). Inclusion of
catecholamine data into the model identified an additional significant positive association
with adrenaline (p=0.007; Table 5.4).
ROC analysis for the presence of late decelerations found the same lactate cut off
(3.1mmol/L) for both groups of fetuses (Table 5.5). Embolised fetuses developed late
decelerations at lower systolic pressure (embolised 55mmHg vs. non-embolised 61mmHg)
and lower base excess values than the non-embolised fetuses (embolised base excess -4.1 vs.
non-embolised -1.8).
5.3.1.7.3 Severe variable decelerations.
Multivariate analysis identified different associations between severe variable decelerations
and fetal physiological variables in embolised and non-embolised fetuses (Table 5.3). Fetal
lactate (p=0.003) and systolic pressure (p<0.001) were found to be significantly associated
with severe variable decelerations in both groups of fetuses; however, changes in fetal lactate
concentration had a significantly greater effect on the odds ratio of severe variable
decelerations in the non-embolised fetuses (p=0.031). For each increase of 1mmol/L of
lactate, the odds ratio of severe variable decelerations was increased by 2.30 (95%CI 1.33-
250
3.97) in the non-embolised fetuses compared to 1.22 (95%CI 1.02-1.45) in the embolised
fetuses. For each increase in systolic pressure of 5mmHg, the odds ratio of severe variable
decelerations increased by 2.14 (95%CI 1.45-3.17).
With multivariate analysis, arterial pH was associated only with severe variable decelerations
in the non-embolised group of fetuses (Table 5.3). The magnitude of this inverse association
was greater than that with systolic pressure or lactate, with each decrease in pH of 0.05 units
increasing the odds ratio of severe variable decelerations by 8.67 (95%CI 2.41-31.1, p=0.011).
Conversely, arterial PCO2 was associated with severe variable decelerations only in the
embolised group of fetuses and the magnitude of this positive association was at least twice
that of systolic pressure and lactate. Each increase in PCO2 of 5mmHg increased the odds
ratio of severe variable decelerations by 4.64 (95%CI 1.04-20.82, p=0.026).
The covariate adjusted association between base excess and severe variable decelerations was
different between embolised and non-embolised fetuses. In non-embolised fetuses, a
decrease in base excess by 2 units resulted in a reduction of the odds ratio for severe variable
decelerations of 1.83 (95%CI 1.45-2.31, p=0.001), while in the embolised fetuses the same
change resulted in a large increase in the odds ratio for severe variable decelerations of 10.5
(95%CI 1.79-61.45, p=0.001).
No association was found between severe variable decelerations and concentrations of
catecholamines. ROC analysis identified the same optimum cut offs in systolic pressure for
the prediction of severe variable decelerations in both groups of fetuses, however they
occurred at lower lactate concentrations in the embolised fetuses (embolised 3.5mmol/L,
non-embolised 4.5mmol/L; Table 5.5).
5.3.1.7.4 Increased fetal heart rate variability
Multivariate analysis showed entirely different variables associated with increased FHR
variability in the non-embolised and embolised fetuses. In the non-embolised fetuses a
positive association was found between increased FHR variability and diastolic pressure
(p<0.001) and an inverse relationship was found with systolic pressure (p<0.001; Table 5.3).
For each rise in diastolic pressure of 5 mmHg the odds ratio for increased FHR variability
increased by 2.28 (95%CI 1.31-3.95). Conversely, for each rise in systolic pressure of 5
mmHg, the odds ratio for increased FHR variability was reduced by at least 9.44 (95%CI
9.44-7361.80). In embolised fetuses, the opposite effect was apparent with a positive
association between systolic pressure (p<0.001) and an inverse association between diastolic
pressure (p<0.001). The magnitude of these effects was similar to those for non-embolised
251
fetuses except their direction was reversed. ROC analysis demonstrated very different
optimum cut off values for systolic and diastolic pressures for non-embolised and embolised
fetuses (Table 5.5).
In the embolised fetuses multivariate analysis showed a positive association between PCO2
and increased FHR variability and an inverse association with pH, base excess, and lactate
concentration (all p<0.001). The magnitudes of these associations were all greater than those
associated with the arterial pressure changes. No association was found between increased
FHR variability and concentrations of the catecholamines that were measured.
5.3.2 The effect of umbilicoplacental embolisation on the relationship between fetal
scalp blood levels and central levels of lactate and pH
5.3.2.1 Fetal scalp blood lactate levels
Fetal scalp, carotid arterial and jugular venous blood lactate levels for both non-embolised
and embolised fetuses during the experimental phase are presented in Figures 5.13 and 5.14.
In addition to lactate levels from the three sampling sites, these figures present graphically the
differences between fetal scalp and jugular venous lactate levels compared to carotid arterial
lactate levels (∆lactate) based on paired analysis within the experimental group. Comparisons
between the three sampling sites in controls, non-embolised and embolised groups are
presented in Figure 5.15.
5.3.2.1.1 Fetal scalp blood lactate levels and carotid arterial lactate levels
Fetal scalp blood lactate levels were significantly higher than carotid arterial lactate levels in
all four experimental groups (scalp-artery lactate: non-embolised control 1.4±0.5mmol/L,
p=0.017; non-embolised occluded 1.4±0.4mmol/L, p=0.005; embolised control
1.7±0.5mmol/L, p=0.005; embolised occluded 1.7±0.5mmol/L, p=0.002, Figure 5.15A).
The relationship between fetal scalp blood and carotid arterial lactate levels was not
influenced by umbilicoplacental embolisation (p=0.242), cord occlusion (p=0.114),
noradrenaline (p=0.829), adrenaline (p=0.137), glucose levels (p=0.919) or time during the
experimental phase (p=0.161); however, it was modified by arterial lactate levels (p<0.001)
and all of the measures of fetal acid-base status (pH p<0.001; PO2 p<0.001; PCO2 p<0.001).
The effect of arterial lactate and PCO2 levels on the scalp-carotid arterial lactate relationship
was that fetal scalp blood lactate underestimated arterial lactate levels as lactate and PCO2
252
Control Fetuses
-300
2
4
6
8
10
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
Control Fetuses
-300
2
4
6
8
10
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
-300
2
4
6
8
10
0 27 54 81
FetalScalp
JugularVein
Time (min)
Lact
ate
(mm
ol/L
)
-300
2
4
6
8
10
0 27 54 81
FetalScalp
JugularVein
Time (min)La
ctat
e (m
mol
/L)
Scalp Vein
-2
-1
0
1
2p=0.017 p=0.882
∆La
ctat
e (m
mol
/L)
Scalp Vein
-2
-1
0
1
2p=0.005 p=0.575
∆La
ctat
e (m
mol
/L)
NON-EMBOLISED EMBOLISEDA
B
C
D
E
F
Figure 5.13 The relationship between fetal scalp blood, carotid arterial blood and jugular
venous blood lactate levels in control fetuses. Fetal scalp blood lactate levels were
significantly higher than carotid arterial lactate levels in both non-embolised (Figure A,
p=0.017) and embolised controls (Figure D, p=0.005). Similarly fetal scalp blood lactate
levels were significantly higher than jugular venous lactate levels in both non-embolised
(Figure B, p=0.024) and embolised controls (Figure E, p=0.007). ∆lactate in Figures C and F
compares fetal scalp blood lactate and jugular venous lactate to carotid arterial lactate levels;
scalp-artery and vein-artery respectively. In the absence of cord occlusion fetal scalp blood
lactate levels are higher than both carotid arterial and jugular venous lactate levels, which are
similar.
253
Occluded Fetuses
-300
2
4
6
8
10
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
Occluded Fetuses
-300
2
4
6
8
10
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
Lact
ate
(mm
ol/L
)
-300
2
4
6
8
10
0 27 54 81
FetalScalp
JugularVein
Time (min)
Lact
ate
(mm
ol/L
)
-300
2
4
6
8
10
0 27 54 81
JugularVein
FetalScalp
Time (min)La
ctat
e (m
mol
/L)
Scalp Vein
-2
-1
0
1
2p=0.005 p=0.214
∆La
ctat
e (m
mol
/L)
Scalp Vein
-2
-1
0
1
2p=0.002 p=0.066
∆La
ctat
e (m
mol
/L)
NON-EMBOLISED EMBOLISEDA
B
C
D
E
F
Figure 5.14 The relationship between fetal scalp blood, carotid arterial blood and jugular
venous blood lactate levels in occluded fetuses. Fetal scalp blood lactate levels were
significantly higher than carotid arterial lactate levels in both non-embolised (Figure A,
p=0.005) and embolised occluded fetuses (Figure D, p=0.002). Similarly fetal scalp blood
lactate levels were significantly higher than jugular venous lactate levels in both non-
embolised (Figure B, p=0.002) and embolised occluded fetuses (Figure E, p<0.001). ∆lactate
in Figures C and F compares fetal scalp blood lactate and jugular venous lactate to carotid
arterial lactate levels; scalp-artery and vein-artery respectively. This figure suggests that
during cord occlusion scalp lactate levels are higher than carotid arterial levels which
themselves may be higher than jugular venous lactate levels.
253
Figure 5.15 Comparison of three lactate sampling sites in control and occluded non-
embolised (NE) and embolised (E) fetuses. Figure A illustrates that the relationship between
fetal scalp blood lactate and carotid arterial lactate levels is independent of both cord
occlusion (p=0.114) and umbilicoplacental embolisation (p=0.242) with fetal scalp lactate
overestimating carotid arterial lactate levels by 1.6mmol/L. Figure B illustrates that the
relationship between fetal scalp lactate and jugular venous lactate levels is independent of
cord occlusion (p=0.518) but modified by umbilicoplacental embolisation (p=0.006). The
scalp-vein lactate difference was greater in fetuses exposed to embolisation (1.9±0.2mmol/L)
than non-embolised fetuses (1.4±0.2mmol/L, p=0.006). Figure C illustrates the relationship
between jugular venous lactate levels and carotid arterial lactate levels was independent of
embolisation (p=0.385) but modified by cord occlusion with jugular venous lactate levels
significantly lower than carotid arterial lactate levels in occluded fetuses (p<0.001).
254
∆ Lactate (Scalp - Artery)
-2
-1
0
1
2
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆La
ctat
e (m
mol
/L)
∆ Lactate (Scalp-Vein)
-2
-1
0
1
2
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆La
ctat
e (m
mol
/L)
∆ Lactate (Vein - Artery)
-2
-1
0
1
2
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆La
ctat
e (m
mol
/L)
A
B
C
255
levels increased with progressive asphyxia. In contrast, the effect of decreasing pH, PO2 and
base excess levels during asphyxia on the scalp-carotid lactate relationship was for scalpblood
lactate levels to overestimate arterial lactate levels. The analysis of interactions suggested that
the effect of arterial lactate levels on scalp lactate levels was less in the embolised than non-
embolised fetuses and, conversely, the influence of PCO2 levels was greater in embolised than
non-embolised fetuses. The net result of these combined effects were such that scalp lactate
levels reliably overestimated carotid arterial lactate levels by the same amount in all four
experimental groups.
5.3.2.1.2 Fetal scalp blood lactate levels and jugular venous lactate levels
Fetal scalp lactate levels were significantly higher than jugular venous lactate levels in all four
experimental groups (scalp-vein lactate: non-embolised control 1.4±0.6mmol/L, p=0.024;
non-embolised occluded 1.7±0.5mmol/L, p=0.002; embolised control 1.7±0.6mmol/L,
p=0.007; embolised occluded 2.2±0.5mmol/L, p<0.001, Figure 5.15B).
The relationship between fetal scalp blood and jugular venous lactate levels was not
influenced by cord occlusion (p=0.518), time during the experimental phase (p=0.152),
noradrenaline (p=0.374), adrenaline (p=0.361) or glucose levels (p=0.611) whereas it was
significantly modified by umbilicoplacental embolisation (p=0.006). The scalp-jugular lactate
difference in embolised fetuses (1.9±0.2mmol/L) was significantly greater than in non-
embolised fetuses (1.4±0.2mmol/L, p=0.006). Similar to carotid arterial blood, the
relationship between fetal scalp blood and jugular venous lactate levels was influenced by pH
(p=0.002) and lactate levels (p<0.001). The effect of decreasing fetal pH levels during
asphyxia was for fetal scalp lactate levels to overestimate jugular venous lactate levels and,
conversely, as lactate levels increased during UCO, scalp lactate levels underestimated jugular
lactate levels. The net result of these two combined effects was for fetal scalp lactate levels to
reliably overestimate jugular venous lactate levels. The analysis of interactions suggested that
the increased scalp-lactate difference in embolised fetuses compared to non-embolised
fetuses was secondary to a greater effect of pH (p=0.002) and a lesser effect of lactate
(p<0.001) in the embolised fetuses compared to the non-embolised fetuses.
5.3.2.1.3 Fetal carotid artery and jugular venous blood lactate levels
Fetal jugular venous lactate levels were not significantly different from carotid arterial lactate
levels in both groups of non-embolised fetuses and embolised controls (vein-artery lactate:
non-embolised control 0.0±0.2mmol/L,p=0.882; non-embolised occluded -0.3±0.2mmol/L,
p=0.214; embolised control -0.1 ±0.2mmol/L, p=0.575); however, in embolised fetuses
256
exposed to UCO, jugular venous lactate levels were lower than carotid arterial lactate levels
(embolised occluded -0.4± 0.2mmol/L, p=0.066 Figure 5.15C).
The relationship between jugular venous blood and carotid arterial blood lactate levels was
not influenced by embolisation (p=0.385) or time during the protocol (p=0.567); it was
significantly modified by cord occlusion with the jugular venous lactate levels significantly
less than carotid arterial lactate levels in fetuses exposed to UCO (p<0.001). These results
suggest that the fetal brain may be metabolising lactate as a source of fuel during asphyxia.
To investigate this hypothesis, further analysis using the blood sampled from the superior
sagittal sinus (SSS) was performed.
Lactate levels were not significantly different between the SSS and the jugular vein (SSS-
jugular vein, -0.10±0.10mmol/L, p=0.3020) nor were there any significant differences
between PCO2 (p=0.234) or base excess (p=0.274) between the two sampling sites (Table
5.6). Blood from the SSS was significantly more acidaemic (pH -0.010±0.002, p<0.001) and
had slightly higher oxygen levels (PO2 +0.104±0.41mmHg) than blood from the jugular vein.
There was no evidence that umbilicoplacental embolisation or cord occlusion modified the
relationship between lactate levels in blood collected from the SSS or jugular vein.
Table 5.6 Summary of differences between superior sagittal sinus blood and jugular venous blood
∆ SSS-jugular vein Group Effect Occlusion Effect
(mean±SEM) p-value
Lactate -0.10±0.10 0.302 0.212 0.558
pH -0.010±0.002 <0.001 0.559 0.991
PO2 1.04±0.41 0.015 0.351 0.883
PCO2 -2.27±1.88 0.234 0.232 0.933
Base excess -0.70±0.63 0.274 0.489 0.980
To assess fetal brain metabolism during asphyxia, the lactate and PO2 levels in blood collected
from the SSS and the carotid artery have been compared (Figure 5.16). Lactate levels in the
SSS were not significantly different from carotid arterial lactate levels in both control and
occluded non-embolised fetuses (SSS-carotid artery: non-embolised control 0.1±0.2mmol/L,
p=0.540; non-embolised occluded 0.1±0.2mmol/L, p=0.714; Figure 5.16A). However, in
256
Figure 5.16 Lactate and oxygen differences across the fetal cerebral circulation during cord
occlusion. Figure A is a box and whisker plot of ∆lactate levels* across the fetal cerebral
circulation. There was no evidence of a net change in lactate levels across the cerebral
circulation of the non-embolised fetuses; however, in the embolised fetuses SSS lactate levels
were significantly less than carotid arterial lactate levels (p<0.001) suggesting a net uptake of
lactate by the fetal brain. Figure B plots ∆PO2 across the fetal cerebral circulation in absolute
values and Figure C plots ∆PO2 across the fetal cerebral circulation as a fraction of the
available oxygen.
*∆lactate = carotid arterial lactate - SSS lactate
**∆PO2 = carotid arterial PO2- SSS PO2
***Fractional oxygen extraction = (carotid artery PO2 - SSS PO2) / carotid artery PO2
257
-4
-3
-2
-1
0
1
2
Control Occl Control Occl
p<0.001
EmbolisedNon-Embolised
p<0.01∆
Lac
tate
* (m
mol
/L)
0
2
4
6
8 p<0.001
Control Occl Control Occl
EmbolisedNon-Embolised
p<0.001
∆ P
O2*
* (m
mH
g)
Control Occl Control Occl
EmbolisedNon-Embolised
0
10
20
30p=0.003
p=0.071
Frac
tiona
l Oxy
gen
Extra
ctio
n***
(%)
A
B
C
258
fetuses that experienced umbilicoplacental embolisation, SSS lactate levels were significantly
less than carotid arterial lactate levels (SSS-carotid artery: embolised control -
1.4±0.4mmol/L, p=0.011; embolised occluded -1.8± 0.4mmol/L, p=0.002) suggesting that
the fetal brain may be using lactate as a source of fuel in these fetuses (Figure 5.16A). The
oxygen utilisation by the fetal brain was also altered by umbilicoplacental embolisation.
When non-embolised fetuses were exposed to hypoxia during UCO, the difference between
SSS PO2 levels and carotid arterial PO2 levels was significantly reduced in both absolute
(p<0.001) and fractional measures compared to controls (p=0.003; Figure 5.16 B and C). In
contrast, embolised fetuses had reduced oxygen extraction by the fetal brain (p<0.001) in
both control and occluded embolised fetuses when compared to non-embolised controls and
no further reduction in oxygen extraction occurred with the addition of UCO.
5.3.2.2 Fetal scalp blood pH levels
Fetal scalp, carotid arterial and jugular venous blood pH levels for both non-embolised and
embolised fetuses during the experimental phase are presented in an identical series of figures
to those presented for scalp lactate levels (Figures 5.17 – 5.19). In all four experimental
groups it can be seen that scalp blood pH was significantly higher than carotid arterial pH,
which itself was significantly higher than jugular venous blood pH levels (all p<0.001).
5.3.2.2.1 Fetal scalp blood pH levels and carotid arterial pH levels
Fetal scalp blood pH levels were significantly higher than carotid arterial pH levels in all four
experimental groups (scalp-artery pH: non-embolised control 0.058±0.011, p<0.001; non-
embolised occluded 0.048±0.010, p<0.001; embolised control 0.048±0.011, p<0.001;
embolised occluded 0.077±0.011, p<0.001, Figure 5.19A).
The relationship between fetal scalp blood pH and carotid arterial pH was not modified by
embolisation (p=0.895), or time during the protocol (p=0.184) whereas it was modified by
cord occlusion, but only in the embolised fetuses (p=0.034). This resulted in scalp pH levels
underestimating the severity of the acidaemia in the carotid artery by significantly more in
embolised occluded fetuses than non-embolised occluded fetuses. In both non-embolised
and embolised fetuses the relationship between fetal scalp blood pH and carotid arterial pH
was modified by arterial pH (p=0.005), lactate (p=0.003) and adrenaline levels (p=0.036).
The scalp-carotid pH relationship was not modified by any other blood gas variable,
noradrenaline (p=0.934) or glucose level (p=0.934) and no interactions between variables and
groups were identified. The effect of arterial pH and adrenaline levels on the scalp-carotid
259
Control Fetuses
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
CarotidArtery
FetalScalp
Time (min)
pH
Control Fetuses
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
CarotidArtery
FetalScalp
Time (min)
pH
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
FetalScalp
JugularVein
Time (min)
pH
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
FetalScalp
JugularVein
Time (min)pH
Scalp Vein-0.10
-0.05
0.00
0.05
0.10 p<0.001 p<0.001
∆pH
Scalp Vein-0.10
-0.05
0.00
0.05
0.10 p<0.001 p<0.001
∆pH
NON-EMBOLISED EMBOLISEDA
B
C
D
E
F
Figure 5.17 The relationship between fetal scalp blood, carotid arterial blood and jugular
venous blood pH levels in control fetuses. Fetal scalp blood pH levels were significantly
higher than carotid arterial lactate levels in both non-embolised (Figure A, p<0.001) and
embolised controls (Figure D, p<0.001). Similarly fetal scalp blood pH levels were
significantly higher than jugular venous pH levels in both non-embolised (Figure B, p<0.001)
and embolised controls (Figure E, p<0.001). ∆pH in Figures C and F compares fetal scalp
blood pH and jugular venous pH to carotid arterial pH levels; scalp-artery and vein-artery
respectively. This figure suggests that without cord occlusion fetal scalp blood pH levels are
significantly higher than carotid arterial pH levels which are significantly higher than jugular
venous pH levels.
260
Occluded Fetuses
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
FetalScalp
CarotidArtery
Time (min)
pH
Occluded Fetuses
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
CarotidArtery
FetalScalp
Time (min)
pH
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
FetalScalp
JugularVein
Time (min)
pH
-307.0
7.1
7.2
7.3
7.4
7.5
0 27 54 81
FetalScalp
JugularVein
Time (min)
pH
Scalp Vein-0.10
-0.05
0.00
0.05
0.10 p<0.001 p<0.001
∆pH
Scalp Vein-0.10
-0.05
0.00
0.05
0.10 p<0.001 p<0.001
∆pH
NON-EMBOLISED EMBOLISEDA
B
C
D
E
F
Figure 5.18 The relationship between fetal scalp blood, carotid arterial blood and jugular
venous blood pH levels in occluded fetuses. Fetal scalp blood pH levels were significantly
higher than carotid arterial pH levels in both non-embolised (Figure A, p<0.001) and
embolised occluded fetuses (Figure D, p<0.001). Similarly fetal scalp blood pH levels were
significantly higher than jugular venous pH levels in both non-embolised (Figure B, p<0.001)
and embolised occluded fetuses (Figure E, p=0.011). ∆pH in Figures C and F compares fetal
scalp blood pH and jugular venous pH to carotid arterial pH levels; scalp-artery and vein-
artery respectively. The difference between scalp blood pH levels and carotid artery pH
levels is greater in the embolised occluded fetuses than the non-embolised occluded fetuses
(p<0.049).
260
Figure 5.19 Comparison of three pH sampling sites in control and occluded non-embolised
and embolised fetuses. Figure A illustrates the relationship between fetal scalp blood pH and
carotid arterial pH levels is independent of umbilicoplacental embolisation (p=0.895) but
altered by cord occlusion in the embolised fetuses (p=0.034). In fetuses exposed to
embolisation and cord occlusion, scalp pH underestimates carotid arterial pH by 60% more
than non-embolised fetuses exposed to UCO (p=0.049). Figure B illustrates the relationship
between fetal scalp pH and jugular venous pH levels is independent of umbilicoplacental
embolisation (p=0.895) but modified by cord occlusion in the embolised fetuses with
embolised fetuses having a significantly greater difference between scalp and venous pH
levels than non-embolised fetuses (p=0.034). Figure C illustrates the relationship between
jugular venous pH levels and carotid arterial pH levels which was independent of cord
occlusion (p=0.553) but modified by embolisation with the difference between jugular
venous and carotid arterial blood pH levels significantly less in embolised than non-
embolised fetuses (p=0.015).
261
∆ pH (Scalp - Artery)
-0.10
-0.05
0.00
0.05
0.10
p=0.049
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆ p
H
∆ pH (Scalp-Vein)
-0.10
-0.05
0.00
0.05
0.10
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆pH
∆ pH (Vein - Artery)
-0.10
-0.05
0.00
0.05
0.10
NEcontrol
NEoccluded
Econtrol
Eoccluded
∆ p
H
A
B
C
262
pH relationship was to increase the difference between fetal scalp pH and central pH during
asphyxia. Conversely the effect of arterial lactate levels on the scalp-carotid pH relationship
was to decrease the pH difference between the two sampling sites. The net result of these
combined effects was for scalp pH levels to be higher than carotid arterial pH levels (ie. to
underestimate the severity of the acidosis). As the embolised occluded fetuses had greater
falls in pH, larger increases in adrenaline, and the same lactate responses as non-embolised
fetuses during UCO, the greater difference in scalp and carotid arterial pH levels in
embolised fetuses is consistent with this explanatory model.
5.3.2.2.2 Fetal scalp blood pH levels and jugular venous blood pH levels
Fetal scalp blood pH levels were significantly higher than jugular venous pH levels in all four
experimental groups (scalp-vein pH: non-embolised control 0.080±0.011, p<0.001; non-
embolised occluded 0.078±0.010, p<0.001; embolised control 0.073±0.012, p<0.001;
embolised occluded 0.095±0.011, p<0.001, Figure 5.19B). The relationship between fetal
scalp blood and jugular venous blood pH levels was not influenced by embolisation
(p=0.895) or time during the protocol (p=0.184) but it was modified by cord occlusion;
although only in the embolised fetuses (p=0.034). Embolised fetuses exposed to UCO had a
significantly greater difference between scalp and venous pH levels than non-embolised
fetuses exposed to UCO.
5.3.2.2.3 Fetal carotid artery and jugular venous blood pH levels
Fetal jugular venous pH levels were significantly lower than carotid arterial pH levels in all
groups of fetuses (vein-artery pH: non-embolised control -0.022±0.002, p<0.001; non-
embolised occluded -0.029±0.002, p<0.001; embolised control -0.026±0.02, p<0.001;
embolised occluded -0.019 ±0.002, p<0.001, Figure 5.19C). The relationship between jugular
venous blood and carotid arterial blood pH levels was not influenced by occlusion (p=0.553)
or time during the protocol (p=0.154); however it was significantly modified by embolisation
with the difference between jugular venous and carotid arterial pH levels significantly smaller
in embolised than non-embolised fetuses (p=0.015).
263
5.3.3 Prediction of fetal arterial pressure changes during cord occlusion
Given the strong correlation between fetal arterial pressure responses during UCO and
neuronal injury demonstrated in Chapter Three, it may be clinically useful to predict these
arterial pressure responses during cord occlusion from either FHR patterns or fetal scalp
blood biochemistry.
5.3.3.1 Prediction of fetal arterial pressure responses during cord occlusion from
fetal heart rate patterns
The sensitivity, specificity and accuracy for FHR patterns to predict fetal arterial pressure
responses during cord occlusion are presented in Table 5.7 for both non-embolised and
embolised fetuses.
Table 5.7 Fetal heart rate prediction of arterial pressure responses during cord occlusion
Any Arterial pressure Instability †
Biphasic Systolic Response ‡
Biphasic Diastolic Response
FHR Pattern
Sens
itivi
ty
Spec
ificit
y
Acc
urac
y
Sens
itivi
ty
Spec
ificit
y
Acc
urac
y
Sens
itivi
ty
Spec
ificit
y
Acc
urac
y
NEδ 78% 51% 62% 96% 51% 62% 97% 47% 56%Severe Variable Decelerations E§ 81% 45% 66% 85% 38% 55% 98% 43% 62%
NE 36% 94% 71% 44% 91% 80% 45% 88% 81%Late component to decelerations E 65% 95% 78% 84% 87% 86% 98% 88% 92%
NE 34% 89% 68% 38% 85% 74% 30% 82% 73%Increased variability E 18% 98% 51% 14% 91% 62% 16% 91% 67%
NE 88% 29% 51% 89% 26% 41% 85% 24% 35%Overshoot
E 47% 64% 54% 54% 64% 60% 47% 60% 55%† Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ‡ Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns δ NE=non-embolised § E=embolised
264
Severe variable decelerations, increased FHR variability between cord occlusions and FHR
overshoot after decelerations were all poor predictors of fetal arterial pressure responses
during UCO in both embolised and non-embolised fetuses. In embolised fetuses, increased
FHR variability and overshoot after decelerations were less predictive of arterial pressure
instability during UCO than in non-embolised fetuses (p<0.05; Table 5.8).
In contrast, late decelerations were moderate predictors of fetal arterial pressure responses
during UCO in non-embolised fetuses and excellent predictors of systolic and diastolic
hypotension during UCO in embolised fetuses with accuracy of 86% and 92% respectively
(Table 5.7). Embolised fetuses with late components to FHR decelerations were 6.81 times
(95%CI 2.71-17.12, p<0.001) more likely to have systolic hypotension during UCO and 60
times (95%CI 7.36-487.6, p<0.001) more likely to have diastolic hypotension during UCO
than non-embolised fetuses with the same FHR pattern (Table 5.8). The improved accuracy
of late decelerations at predicting hypotension during UCO in embolised fetuses is secondary
to the improved sensitivity in this group which was two fold higher than non-embolised
fetuses (Table 5.7).
Table 5.8 Odds Ratios of abnormal FHR pattern if fetal arterial pressure response is present
FHR Pattern Any Arterial
pressure Instability†
Biphasic Systolic Response‡
Biphasic Diastolic Response
Severe Variable Decelerations
OR§ (95%CI)
1.22 (0.57-2.63)
0.25 (0.05-1.24)
1.56 (0.09-25.90)
Late components to decelerations
OR (95% CI)
3.33*** (1.75-6.35)
6.81*** (2.71-17.12)
60.00*** (7.36-487.6)
Increased variability
OR (95% CI)
0.41* (0.20-0.85)
0.26** (0.10-0.69)
0.43 (0.15-1.23)
Overshoot OR
(95% CI)0.13***
(0.06-0.28) 0.14***
(0.05-0.42) 0.16***
(0.05-0.48)
§ All odds rations calculated embolised: non-embolised † Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ‡ Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns * p <0.05, ** p <0.01, *** p<0.001
265
5.3.3.2 Prediction of fetal arterial pressure responses during cord occlusion from
fetal scalp blood biochemistry
Receiver operator curves (ROC) were used to assess the diagnostic utility of fetal scalp blood
biochemical measures to predict fetal arterial pressure responses during cord occlusion in
both non-embolised and embolised fetuses (Table 5.9). The areas under the ROCs for scalp
blood lactate were marginally higher than for scalp blood pH levels for all three patterns of
arterial pressure response during UCO for both non-embolised and embolised fetuses;
however, the magnitude of these differences did not reach statistical significance (Figure
5.20). Umbilicoplacental embolisation did not alter the areas under the ROCs for both scalp
blood lactate and pH levels for the three arterial pressure response patterns. In both non-
embolised and embolised fetuses the optimum cut off levels for scalp blood lactate increased
as the fetuses progressed through the three stages of cardiovascular decompensation whereas
the optimum cut off values for scalp pH were similar for the three patterns (Table 5.10).
Table 5.9 Biochemical prediction of arterial pressure responses during cord occlusion
Any Arterial pressure Instability †
Biphasic Systolic Response ‡
Biphasic Diastolic Response Biochemical
measure (fetal scalp blood)
Are
a
Sens
itivi
ty
Spec
ificit
y
Are
a
Sens
itivi
ty
Spec
ificit
y
Are
a
Sens
itivi
ty
Spec
ificit
y
NEδ 0.839 85% 69% 0.966 93% 88% 0.983 100% 92% Lactate
E§ 0.874 91% 70% 0.891 83% 83% 0.891 83% 83%
NE 0.721 46% 94% 0.909 93% 76% 0.965 100% 82% pH
E 0.735 71% 66% 0.865 83% 74% 0.856 83% 72% † Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ‡ Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns δ NE=non-embolised § E=embolised
266
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity (false positives)
Sens
itivi
ty (t
rue
posi
tives
)NodiscriminationScalp lactate
Scalp pH
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 11 - Specificity (false positives)
Sens
itivi
ty (t
rue
posi
tives
)
NodiscriminationScalp Lactate
Scalp pH
Figure 5.20 Receiver operator curves for the prediction of any arterial pressure instability
during cord occlusion in non-embolised and embolised fetuses. Umbilicoplacental
embolisation had no effect on the ability of either scalp blood lactate or pH to predict fetal
arterial pressure responses during cord occlusion.
NON-EMBOLISED
EMBOLISED
267
Table 5.10 Accuracy of biochemical prediction of arterial pressure responses during UCO
Any Arterial pressure Instability†
Biphasic Systolic Response‡
Biphasic Diastolic Response Biochemical
measure
(fetal scalp blood) Accuracy Optimum Cut Off Accuracy Optimum
Cut Off Accuracy Optimum Cut Off
NEδ 77% 4.1 88% 4.5 93% 4.9 Lactate (mmol/L) E§ 75% 4.3 77% 4.7 77% 4.9
NE 84% 7.301 78% 7.308 93% 7.295 pH
E 67% 7.329 76% 7.316 76% 7.316 † Sum of decreasing hypertensive response, biphasic systolic response and biphasic diastolic response phases ‡ Sum of biphasic systolic response and biphasic diastolic response as both patterns had biphasic systolic pressure patterns δ NE=non-embolised § E=embolised
5.3.4 Summary of similarities and differences in fetal responses to asphyxia in
embolised and non-embolised fetuses
A summary of the similarities and differences between embolised and non-embolised fetuses
in biochemical, endocrine, cardiovascular and metabolic responses to asphyxia are presented
in Table 5.11.
Table 5.11 Summary of differences and similarities in physiologic responses to asphyxia in embolised and non-embolised fetuses
Similarities Differences
Arterial lactate and base excess responses pH, PCO2, PO2 responses
Relationship between scalp blood lactate and carotid arterial lactate levels
Relationship between fetal scalp blood pH and carotid arterial pH
Arterial pressure responses during cord occlusions
Arterial pressure responses between cord occlusions
Ability of lactate to predict arterial pressure responses during UCO
Ability of FHR patterns to predict arterial pressure responses during UCO
FHR variability and decelerative FHR patterns
Glucose, cortisol and catecholamine responses
Cerebral lactate and oxygen metabolism
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5.4 Discussion
The purpose of the studies performed in this chapter was to investigate fetal lactate
responses during asphyxia after growth restriction induced by uteroplacental insufficiency.
These studies were performed because of the evidence obtained in experiments presented in
Chapters Three and Four of this thesis, and recently by others (Gardner et al., 2002; Gardner
et al., 2003), that previous exposure to an adverse intrauterine environment alters
cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus.
In addition, growth restricted human fetuses are more likely than appropriately grown fetuses
to have non-reassuring FHR patterns (Low et al., 1976; Dashe et al., 2000) and fetal acidosis in
labour (Low et al., 1981; Nieto et al., 1994) and are at increased risk of adverse outcomes
secondary to acute hypoxic intrapartum events (Bukowski et al., 2003). Given that growth
restricted fetuses are more likely than normally grown fetuses to have adverse events during
labour and that they have altered physiologic responses to asphyxia, it seemed important to
specifically investigate the effect of growth restriction on the response to UCO. Such
investigation is necessary to evaluate the potential of fetal scalp lactate monitoring in this
important group of fetuses.
As in previous studies where umbilicoplacental embolisation has been used to induce
placental insufficiency (Clapp et al., 1981; Trudinger et al., 1987; Gagnon et al., 1994; Cock &
Harding, 1997; Murotsuki et al., 1997a; Duncan et al., 2000), I have found significant
reductions in fetal PO2 and pH throughout the duration of the embolisation period and there
was a tendency for the fetuses to be hypoglycaemic. Similar changes have been observed in
blood samples collected by cordocentesis in human growth restricted fetuses (Nicolaides et
al., 1989). Twenty three days of umbilicoplacental embolisation induced a 29% reduction in
fetal weight at 129 days gestational age in my study which is similar to previous reports using
similar embolisation techniques over a period of 25 to 30 days during gestation (Duncan et
al., 2000).
5.4.1 Stability of fetal lactate responses during asphyxia
This is the first experimental study using an animal model to investigate fetal lactate
responses during asphyxia induced by repetitive cord occlusion in growth restricted fetuses.
In this chapter, I have shown that umbilicoplacental embolisation for 23 days alters fetal acid-
base, endocrine, and cardiovascular responses to subsequent asphyxia in the ovine fetus. In
addition, evidence has been provided that suggests that lactate and oxygen metabolism in the
269
fetal brain during asphyxia is altered by previous umbilicoplacental embolisation. Despite the
multiple effects of umbilicoplacental embolisation on fetal responses to asphyxia,
embolisation did not alter fetal arterial lactate responses to asphyxia, the relationship between
fetal scalp blood and carotid arterial lactate levels or the ability of fetal scalp lactate levels to
predict fetal arterial pressure responses during cord occlusion.
5.4.1.1 Arterial lactate responses
The consistent increase in arterial lactate levels seen in this study in response to repeated cord
occlusion in both embolised and non-embolised fetuses suggests that the increase in lactate
levels during asphyxia more closely relate to the severity of the fetal insult than the condition
of the fetuses prior to asphyxia. Similar findings were reported in Chapters Three and Four
of this thesis. In Chapter Four (Section 4.3.3.2, Figure 4.6) both “normal” and
“spontaneously acidaemic” fetuses had the same lactate responses to repeated cord occlusion
whilst having different responses in the other markers of fetal acid-base status. In chronically
instrumented sheep fetuses in Chapter Three, “fast responding” fetuses were noted to have
higher baseline lactate levels than the other fetuses in the study; however, in response to
asphyxia they had lactate levels that increased in parallel to those of fetuses that commenced
the cord occlusion protocol with normal baseline lactate levels (Section 3.3.1.1, Figure 3.2).
The consistent increases in arterial lactate and base excess levels during asphyxia in both
embolised and non-embolised fetuses suggest the same degree of metabolic acidaemia is
present in both groups of fetuses. Conversely the two groups of fetuses had different
patterns of response in pH, PO2 and PCO2 during asphyxia. The more rapid fall in arterial pH
in the embolised fetuses during cord occlusion is likely to be due to the respiratory acidosis
associated with the greater increase in PCO2 in these fetuses. Both the more rapid rise in PCO2
and the greater fall in PO2 with repeated cord occlusion in embolised fetuses may have
resulted from a decrease in placental exchange of respiratory gases associated with the
placental damage induced by prolonged repeated embolisation. Umbilicoplacental
embolisation for 30 days from 110 days has been shown to result in a reduction in
placentome weight and cross sectional area with 20% of the fetal tissue in the placentomes
showing evidence of necrosis (Duncan et al., 2000).
The similar lactate levels during cord occlusion in the two groups of fetuses, despite more
rapid falls in PO2 in the embolised compared with non-embolised fetuses, suggests that the
fetuses that experienced umbilicoplacental embolisation either 1) increased oxygen extraction
to maintain tissue oxygen consumption, and/or 2) increased utilisation of lactate as a
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significant metabolic substrate in embolised fetuses and/or 3) had altered placental handling
of lactate.
It is unlikely that the embolised fetuses in this study increased oxygen extraction when faced
with an asphyxial insult. In the short term (<24hours), the fetus has a considerable buffer to
reduced delivery of oxygen by increasing the efficiency of oxygen extraction from the blood.
Oxygen delivery can be reduced by up to 40-50% for durations between five minutes and 24
hours and a concomitant increase in oxygen extraction from 30% to 50-60% enables
maintenance of tissue oxygen consumption (Itskovitz et al., 1983; Edelstone, 1984; Edelstone
et al., 1985; Bocking et al., 1992), as long as fetal acidaemia does not develop (Rurak et al.,
1990). This ability of the fetus to compensate metabolically for reduced delivery of oxygen
has led to the concept of the margin of safety for fetal oxygenation (Meschia, 1985).
However, in studies in which the period of adverse intrauterine conditions has been
prolonged beyond 24 hours, there is evidence that long term reductions in the delivery of
oxygen to the fetus leads to a breakdown of the oxygen margin of safety and leads to down-
regulation of fetal oxygen-consuming processes (Anderson et al., 1986). In the study by
Anderson et al. (1986) fetal oxygen consumption was assessed in chronically instrumented
ovine fetuses during 10 days of reduced aortic blood flow. A prolonged reduction (≈40%) in
oxygen delivery to the fetus resulted in a strong correlation between total oxygen delivery to
the fetus and oxygen consumption by the fetus (Anderson et al., 1986). In this study fetal
oxygen consumption was not formally assessed; however, a limited assessment of oxygen
consumption in the fetal brain was performed. This suggested that fractional oxygen
extraction was reduced by approximately 30% in embolised fetuses when compared to non-
embolised fetuses (Figure 5.16C) and when challenged with an acute asphyxial insult,
embolised fetuses did not increase fractional oxygen extraction across the fetal brain.
Interestingly, fractional oxygen extraction did not fall when embolised fetuses were exposed
to repeated cord occlusion. At the completion of the embolisation phase PO2 levels in the
embolised fetuses were approximately 12mmHg. When these fetuses were acutely
instrumented to enable the collection of fetal scalp blood samples, fetal PO2 levels increased
to approximately 23mmHg, probably in response to the maternal ventilation. The
subsequent repeated cord occlusion resulted in fetal PO2 levels falling to approximately the
same levels (≈12-13mmHg) as those that were present during the embolisation phase. This
reduction may not have been adequate to stimulate a change in oxygen extraction as the
fetuses had been accustomed to these oxygen levels for at least 20 days prior to the insult.
Alternatively, it may not be physiologically possible for the fetus to decrease oxygen
271
extraction to levels below 15%, the lowest level seen in this study in both non-embolised and
embolised fetuses during cord occlusion.
It is possible that the similar arterial lactate responses in embolised and non-embolised
fetuses during UCO, despite more severe hypoxia in the embolised fetuses, are due to
increased utilisation of lactate as a metabolic substrate in embolised fetuses. In the sheep
fetus lactate metabolism accounts for 40-50% of the total fetal oxygen consumption and it is
the main carbohydrate taken up by the liver and heart for glycogen and lipid synthesis
(Fowden, 2001). Fetuses exposed to umbilicoplacental embolisation have a trend to
hypoglycaemia and it is known that when fetal glucose availability is limited, glucose
utilisation decreases and fetal oxygen consumption is maintained by oxidising more amino
acids resulting in urea production and a reduced rate of protein synthesis by the fetus
(Fowden, 2001). Little is known about lactate metabolism in fetuses exposed to chronic
hypoxia; however I have provided evidence to suggest increased utilisation of lactate by the
fetal brain as a source of metabolic fuel in fetuses exposed to umbilicoplacental embolisation
(Figure 5.16A). Lactate utilisation by other fetal tissues was not formally assessed in this
study.
The similar arterial lactate responses between the two groups of fetuses may be secondary to
altered placental handling of lactate, which was not assessed in this study. It has been shown
that during periods of 24 hours of adverse intrauterine conditions, the placenta acts as a
major site of lactate clearance from the fetal circulation (Hooper et al., 1995). It is not known
if the placenta continues to clear fetal lactate during periods of adverse intrauterine
conditions lasting beyond 24 hours. Placental weight is reduced by umbilicoplacental
embolisation (Duncan et al., 2000) and I have previously suggested that this may impair
oxygen and carbon dioxide transport across the placenta. Lactate transport across the
placenta is probably through facilitated diffusion (Carstensen et al., 1983; Piquard et al., 1990)
and the effect of umbilicoplacental embolisation on placental lactate handling is unknown.
Of the three potential mechanisms responsible for the uniform response in fetal arterial
lactate levels during asphyxia in the two groups of fetuses experiencing cord occlusion in this
study, I favour the theory that embolised fetuses have increased utilisation of lactate as a
metabolic substrate. It is likely that the embolised fetuses have down-regulation of oxygen-
consuming processes in the whole fetal body which results in an increase in tissue lactate
production. The effect of this on circulating lactate levels may be partially minimised by the
30% reduction in fetal body weight in embolised fetuses; however, increased utilisation of
lactate by the fetal heart, brain, liver or other tissues may result in a net lactate response
272
which is similar to normally grown fetuses. To evaluate the mechanisms responsible for the
arterial lactate responses in the growth restricted fetus, further studies using appropriately
instrumented chronically catheterised fetuses are required to formally assess fetal oxygen
utilisation during cord occlusion, fetal lactate metabolism and clearance of lactate by the
placenta.
5.4.1.2 Fetal scalp blood lactate responses
In the studies presented in this chapter, the relationships between fetal scalp blood lactate
levels and carotid arterial lactate levels were independent of embolisation and cord occlusion.
This observation suggests that fetal scalp lactate levels reliably reflect central arterial lactate
levels across a range of fetal conditions, both at rest and during asphyxia. Conversely,
exposure to an adverse intrauterine environment of chronic hypoxia impaired the ability of
fetal scalp blood pH levels to accurately reflect central arterial pH levels during an acute
asphyxial challenge. The addition of asphyxia to prolonged umbilicoplacental embolisation
resulted in the difference in pH levels between the two sampling sites increasing by 60% with
fetal scalp blood pH levels underestimating the severity of changes in central pH levels by a
greater amount in fetuses exposed to embolisation than controls. The effect of pre-asphyxial
umbilicoplacental embolisation on the accuracy of fetal scalp blood pH assessment has not
been reported previously; however, others have reported inaccuracies in fetal scalp blood pH
assessment in the presence of acute hypoxaemia (Shier & Dilts, 1972) and acute acidaemia
induced by hypercarbia (Aarnoudse et al., 1981). Considering the results of this study and
those by other authors (Shier & Dilts, 1972; Aarnoudse et al., 1981) it appears that the
accuracy of scalp pH assessment may be impaired in pregnancies complicated by acute
hypoxaemia, acidaemia and growth restriction. This is a significant limitation to the use of
scalp pH measurement to assess fetal wellbeing as it appears that the ability of scalp pH
measurements to accurately reflect central fetal acid base status is poor at the times when the
most accurate results are required.
The mechanism responsible for the divergent responses in scalp lactate and pH during
asphyxia in the two groups of fetuses in this study is unknown and not immediately apparent;
however, a possible explanation can be obtained from a review of acid-base physiology.
Hydrogen ions are highly reactive and small changes in pH of 0.1 to 0.2 pH units can have
profound effects on metabolic activity and on cardiovascular and central nervous system
function (Gillstrap, 1999). Dramatic changes in fetal pH are minimised via the action of
buffers. The two major buffers are plasma bicarbonate and haemoglobin; other
quantitatively less important buffers include erythrocyte bicarbonate and inorganic
273
phosphates (Blechner, 1993). In the fetus the placental bicarbonate pool may also play a role
in buffering the fetus against changes in maternal pH. In this study, fetal scalp blood lactate
levels were higher than in paired carotid arterial samples suggesting local production of
lactate by tissues in the fetal scalp. Conversely pH levels in the fetal scalp were higher than
carotid arterial pH levels, underestimating the falls in arterial pH by 0.04 to 0.08 pH units.
The difference in pH measures between the two sampling sites may reflect improved tissue
pH buffering in the embolised fetuses in this study. The embolised fetuses were hypoxaemic
and acidaemic for at least 20 days prior to their acute asphyxial insult. It is likely that during
this time the ability of the fetus to buffer acute changes in tissue pH would be improved as
part of the cascade of physiological events designed to protect the fetus in an adverse
intrauterine environment. Therefore, when the embolised fetuses were exposed to an acute
asphyxial insult associated with more dramatic changes in fetal pH (7.323 to 7.111) than that
experienced during the embolisation phase, improved tissue buffering of locally produced
hydrogen ions would result in scalp blood samples underestimating the pH levels in arterial
blood. Other evidence to support the hypothesis that embolised fetuses have improved pH
buffering during acute asphyxia is provided by the comparisons between carotid arterial,
jugular venous and superior sagittal sinus pH levels during asphyxia. Despite the more
dramatic fall in arterial oxygen content in the embolised fetuses, the net output of hydrogen
ions from the fetal brains of embolised fetuses was significantly less than in non-embolised
fetuses again suggesting improved tissue pH buffering in fetuses exposed to prolonged
embolisation.
Therefore, considering fetal scalp blood lactate and pH assessment, scalp blood lactate levels
appear to be the more reliable marker of central arterial acid-base status than scalp blood pH
assessment with inaccuracies in scalp blood pH assessment occurring in fetuses exposed to
prolonged periods of hypoxaemia prior to asphyxia.
5.4.1.3 Lactate and the prediction of arterial pressure responses during cord
occlusion
In this study I have shown that the ability of fetal scalp blood lactate levels to predict arterial
pressure instability during umbilical cord occlusion is not altered by umbilicoplacental
embolisation prior to the insult, suggesting that fetal scalp lactate levels are a relatively robust
marker of fetal brain perfusion during cord occlusion. This observation has not previously
been reported but is consistent with observations made in adult critical care medicine where
lactate levels have been shown to be reliable markers of tissue perfusion across a wide range
274
of conditions including acute trauma, major surgery and septic shock (Perret & Enrico, 1978;
Bakker et al., 1996; Husain et al., 2003).
In Chapter Three, I provided evidence which suggested that the duration of cardiovascular
instability during cord occlusion was the strongest predictor of fetal brain injury 72 hours
after repeated UCO, accounting for 91% of the variability in grading of neuronal injury.
Therefore the ability of scalp lactate levels to reliably predict arterial pressure instability
during cord occlusion across a range of fetal conditions is reassuring if scalp lactate is to be
used as a tool for the assessment of fetal wellbeing during labour where the fetal growth
status prior to delivery is often unknown. The study described in this chapter was not aimed
at determining the link between fetal arterial pressure changes during cord occlusion and the
severity of brain injury in the growth restricted fetus. Further studies are required to address
this issue.
5.4.2 Variation in cardiovascular and endocrine responses during asphyxia
In the studies presented in this chapter I have shown that placental insufficiency induced by
umbilicoplacental embolisation alters cardiovascular, endocrine and metabolic responses to
asphyxia. Similar observations have been made by others using different techniques to
induce an adverse uterine environment (Gardner et al., 2002; Gardner et al., 2003). When
considering the differences identified in cardiovascular and endocrine responses during
asphyxia reported in this chapter, one must remember that the repeated cord occlusions were
performed in an acute fetal preparation. In Chapter Four I demonstrated that the specific
acute preparation used in this study resulted in fetal responses which were similar to those
seen in chronic fetal preparations but differences were noted between the two models in
acid-base and noradrenaline responses to asphyxia and in baseline measures of FHR
variability. Despite these limitations, the alterations in cardiovascular and endocrine
responses described in this chapter warrant discussion and strongly suggest that further
appropriately designed studies are required to investigate the cardiovascular and endocrine
responses of growth restricted fetuses during repeated cord occlusion.
5.4.2.1 Fetal heart rate patterns
I have shown that fetuses exposed to umbilicoplacental embolisation exhibit different FHR
responses during repeated umbilical cord occlusion than non-embolised fetuses. The
differences observed between the two groups occurred both between cord occlusions and
during cord occlusions. Despite the clinical importance of fetal growth disorders and the
increased perinatal morbidity and mortality rates in such fetuses, this is the first experimental
275
study that used an animal model to investigate FHR patterns during asphyxia induced by
repetitive cord occlusion in growth restricted fetuses.
The fetal heart rate patterns in growth restricted fetal sheep during umbilicoplacental
embolisation have been described previously in detail (Murotsuki et al., 1997a). During the
first 48 hours of hypoxaemia secondary to embolisation, the number of accelerations and
decelerations increased compared to controls. In addition, both short- and long-term FHR
variability increased initially which was followed by a return to baseline after 20 hours of
hypoxaemia. After 21 days of embolisation fetuses had a 30% reduction in the number of
FHR accelerations and lower FHR variability than controls (Murotsuki et al., 1997a).
5.4.2.1.1 Fetal heart rate variability
In this study, fetuses exposed to embolisation had reduced FHR variability when compared
to non-embolised fetuses; however, when subjected to repeated UCO, embolised fetuses
were still capable of increasing their FHR variability, although the magnitude of this response
was significantly smaller than seen in non-embolised fetuses during asphyxia. Reduced FHR
variability has previously been reported in both growth restricted sheep (Gagnon et al., 1996;
Murotsuki et al., 1997a) and humans (Gagnon et al., 1988; Snijders et al., 1992) but the
responses in FHR variability during cord occlusion in growth restricted fetuses has not been
described previously.
Fetal sheep normally increase their FHR variability with advancing gestation from 106 to 129
days gestational age (Murotsuki et al., 1997a). Ovine fetuses that were subjected to placental
embolisation over the same gestation did not increase their FHR variability leading the
authors to suggest that the “inherently low” FHR variability in the growth restricted fetuses
may reflect a delay in maturation of the autonomic control of the fetal heart as opposed to a
reduction in FHR variability in response to chronic hypoxaemia (Murotsuki et al., 1997a). A
similar response has been shown in healthy human fetuses, in which there is an increase in
FHR variability between 28 and 30 weeks gestation (Gagnon et al., 1987), suggesting there is
functional maturation of the autonomic control of the fetal heart occurring early in the third
trimester. Longitudinal studies of FHR variation in growth restricted fetuses have shown
that in this group, FHR variability does not change over time and it can be reduced for up to
five weeks before the occurrence of “fetal distress” (Snijders et al., 1992). Theories for the
reduced heart rate variability seen in fetuses that have experienced embolisation include: 1)
alterations in the central control of heart rate variability associated with neuronal hypoxia or
damage and/or 2) decreased number or sensitivity of ß-adrenoreceptors in the fetal heart
276
during chronic placental insufficiency possibly associated with chronically elevated levels of
catecholamines.
The mechanisms which underlie the control of normal FHR variation are not fully
understood (Kozuma et al., 1997). In primates, variability is primarily transmitted via
parasympathetic nerves (Parer, 1999). Variability is likely due to numerous sporadic inputs
from various areas of the cerebral cortex and lower centres to the cardiac integratory centres
in the medulla oblongata which are then transmitted down the vagus nerve (Parer et al.,
1981). It has been hypothesized that hypoxic-tolerant cells such as neurons respond to
oxygen lack by orchestrating a series of molecular defence processes to reduce ATP demand
for ion pumping. These responses include a 90% or greater global decline in protein
biosynthesis (translational arrest), and a generalised decline in membrane permeability
(“channel arrest”) or in firing frequency (“spike arrest”) in nervous tissue (Hochachka et al.,
1996). When these defence mechanisms are overcome tissue injury may occur. The
potentially reversible decrease in firing frequency of the cortical and subcortical neurons, or
the irreversible brain injury associated with prolonged hypoxia may reduce sporadic inputs to
the medulla resulting in decreased fetal heart rate variability. Although I did not examine the
brains of the fetuses in this study, others using the same umbilicoplacental embolisation
protocol as used in this study have demonstrated fetal brain injury associated with the
technique (Mallard et al., 1998; Duncan et al., 2000). Similarly, in humans, severe growth
restriction is associated with fetal brain injury (Gaffney et al., 1994c; Grafe, 1994).
In this study there is evidence that the reduced heart rate variability seen after
umbilicoplacental embolisation may be secondary to alterations in the number or sensitivity
of ß-adrenoreceptors in the fetal heart, possibly associated with chronically elevated levels of
catecholamines. When compared to controls, fetuses exposed to embolisation had reduced
FHR variability prior to UCO, a smaller increase in FHR variability in response to UCO, and
a smaller and less sustained increase in inter-occlusion heart rate with progressive UCO.
These responses occurred despite a two-fold greater increase in noradrenaline levels and a
nine-fold greater increase in adrenaline levels in response to cord occlusion in the embolised
fetuses than in non-embolised fetuses (Figure 5.5).
Both embolised and non-embolised fetuses in this study increased their FHR variability when
exposed to repeated cord occlusion. This response has not been shown before in fetuses
exposed to embolisation. An increase in FHR variability has been shown previously in
studies of inhalational hypoxia in fetal sheep (Dalton et al., 1977; Parer et al., 1980; Lilja et al.,
1984; Field et al., 1991; Kozuma et al., 1997) and monkeys (Ikenoue et al., 1981), placental
277
embolisation in fetal sheep (Gagnon et al., 1996; Murotsuki et al., 1997a) and umbilical cord
occlusion in the ovine fetus (Westgate et al., 1999) and rat (Syutkina, 1988). It is also
consistent with the increase in baseline variability seen in humans in some cases of hypoxia
(Pello et al., 1991). The mechanisms which underlie the increase in FHR variability are not
fully understood but are likely to involve a combination of activation of the carotid
chemoreceptor reflex (Kozuma et al., 1997) and sympathetic stimulation (Dalton et al., 1983;
Rosen et al., 1986; Lindecrantz et al., 1993; Segar et al., 1994). The increase in FHR variability
seen in the embolised fetuses in this study suggests that the carotid body chemoreceptor
reflexes and/or sympathetic stimulation are still capable of responding to a more severe
hypoxic challenge as indicated by increased FHR variability. It is also possible that the
mechanisms responsible for the increase in FHR variability in response to hypoxia may be
different in embolised and non-embolised fetuses. This tenet is supported by the
multivariate logistic regression performed in this chapter which found complex and divergent
associations with increased variability in the two groups of fetuses. The only significant
association identified in the non-embolised fetuses was with inter-occlusion arterial pressure
whereas in the embolised fetuses changes in FHR variability were also associated with fetal
acid-base status.
5.4.2.1.2 Fetal heart rate decelerations
In this study fetuses exposed to embolisation developed late components to their FHR
decelerations and severe variable decelerations earlier and in a more variable manner than
non-embolised fetuses. Human growth restricted fetuses have also been shown to have
twice the incidence of FHR abnormalities in labour (Low et al., 1976; Low et al., 1981). There
are a number of potential mechanisms that may account for the earlier decelerative patterns
in growth restricted fetuses undergoing repetitive cord occlusion. Fetal umbilicoplacental
embolisation results in occlusion of small arterioles of the fetal stem villi and is associated
with a reduction in total placental flow, elevated placental vascular resistance and fetal
hypoxaemia (Gagnon et al., 1996). During umbilical cord occlusion transient falls in fetal
arterial PO2 of approximately 7mmHg are known to occur (Richardson et al., 1996). Given
that the embolised fetuses in this study had lower PO2 levels than the non-embolised fetuses
between the cord occlusions, it is likely that their PO2 levels were also lower during the
occlusions, which may account for the earlier decelerative patterns in the growth restricted
fetuses. Both hypertension and myocardial hypertrophy have also been shown to be induced
by embolisation of the utero-placental vascular bed (Murotsuki et al., 1997b). Increased
cardiac muscle mass may be less tolerant of hypoxaemia, possibly contributing to the
presence of late decelerations earlier in the cord occlusion protocol.
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Late FHR decelerations, with their delay in onset and resolution in relation to uterine
contractions, are one of the sentinel features that clinicians aim to identify during continuous
intrapartum electronic FHR monitoring. Late decelerations are primarily a reflex change that
occurs during non-acidaemic hypoxia (Martin et al., 1979), but when hypoxia is severe enough
to result in acidaemia, the mechanism of late deceleration appears to be non-reflex
myocardial depression (Freeman et al., 1991). The results of the multivariate analysis in this
study suggest that the presence of late components to FHR decelerations was associated with
increases in lactate concentration and systolic pressure, and decreases in base excess in both
normally grown and growth restricted fetuses. The analysis of interactions also found an
additional association between late components to decelerations and arterial pH in the
embolised fetuses suggesting that late decelerations may also be associated with respiratory
acidosis in embolised fetuses.
Both the ROC analyses investigating the optimum cut off points for the significant
associations in the multivariate analysis (Table 5.5) and the analyses comparing late
decelerations and fetal arterial pressure responses during cord occlusion (Table 5.8) suggest
that late components to decelerations in the embolised fetus have more sinister implications
than when they occur in non-embolised fetuses. The results of the ROC analysis suggest that
late decelerations do not reliably begin in embolised fetuses until lower base excess levels
than those in non-embolised fetuses have been reached. In addition, when late decelerations
are present in fetuses exposed to embolisation they are six times more likely than non-
embolised fetuses to have systolic hypotension during umbilical cord occlusion and 60 times
more likely to have systolic and diastolic hypotension during the cord occlusion.
5.4.2.1.3 Fetal heart rate accelerations and overshoot
Accelerations in FHR during intrapartum monitoring have been found to be an indicator of
good perinatal outcome in two cohort studies where the presence of more than two
accelerations in 20 minutes had a sensitivity of 97% for an Apgar score of greater than seven
at five minutes (Powell et al., 1979; Krebs et al., 1982). There are thought to be at least two
physiologic mechanisms responsible for FHR accelerations. The first is the response to fetal
movement and the second mechanism is the response to partial umbilical cord occlusion.
During the 0.5-minute cord occlusions in my study, FHR accelerations were seen in
approximately half the fetuses experiencing cord occlusion (both embolised and non-
embolised) during total cord occlusion confirmed by simultaneous umbilical artery Doppler
assessment. No accelerations were seen in the control fetuses. The multivariate analysis used
in this study suggests that FHR accelerations were present during total cord occlusion when
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there were falls in fetal PO2 prior to the systolic pressure increase and changes in pH, PCO2
and base excess that occur with repeated cord occlusion. Once the latter changes occurred,
they exerted a significant negative effect on the presence of accelerations with cord occlusion.
The net result was that accelerations were no longer present and were replaced by
decelerative FHR patterns. The results of my analysis suggest that recurrent accelerations
during total umbilical cord occlusion may not be as reassuring as the more common type of
acceleration which occurs in response to fetal movement.
In this study, both non-embolised and embolised occluded fetuses displayed FHR overshoot
patterns with the FHR pattern occurring significantly later in embolised fetuses. As discussed
at length in Chapter 3 (Section 3.4.6.2), the aetiology of FHR overshoot is unknown.
Goodlin et al. have suggested that the potential mechanisms for this heart rate pattern may
include a combination of high levels of circulating catecholamines and impaired neural
control of fetal heart rate (Goodlin & Lowe, 1974). No significant associations were
identified in the multivariate analysis between FHR overshoot and fetal biochemical acid-
base status, catecholamines or arterial pressure in this study. The delayed appearance of this
FHR pattern in fetuses exposed to embolisation may suggest that decelerations associated
with hypoxia and acidosis may have a greater impact on the fetal heart rate than the
overshoot in fetal heart rate associated with catecholamine release. Alternatively it may be
another reflection of decreased sensitivity to circulating catecholamines in fetuses exposed to
embolisation.
5.4.2.2 Fetal arterial pressure responses
In this study fetuses exposed to 23 days of umbilicoplacental embolisation had higher arterial
pressures than controls. Elevations in fetal arterial pressure have been reported previously
after as little as one day of umbilicoplacental embolisation at 109 days gestational age
(Murotsuki et al., 1997a) and fetal arterial pressures remain elevated throughout daily
umbilicoplacental embolisation until approximately 130 days gestational age (Cock &
Harding, 1997; Murotsuki et al., 1997a). When umbilicoplacental embolisation has been
continued to 140 days gestational age, arterial pressures in embolised fetuses were lower than
controls (Cock & Harding, 1997). This pattern is probably secondary to the progressive rise
in arterial pressures in control fetuses with advancing gestational age whereas in embolised
fetuses arterial pressure remains relatively constant with advancing gestation after its initial
elevation secondary to embolisation.
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The additional hypoxic challenge of repeated cord occlusion produced an increase in arterial
pressures in non-embolised fetuses whereas in embolised fetuses there were no significant
changes in inter-occlusion arterial pressure during 81 minutes of repeated cord occlusion
despite a significantly larger increase in circulating catecholamines in these fetuses. These
results are different to those reported after a sub-acute hypoxic stress (acute severe
umbilicoplacental embolisation to reduce fetal pH to 7.0) was applied to fetuses that had
undergone daily umbilicoplacental embolisation between 120 and 130 days (Gagnon et al.,
1997). In the study by Gagnon et al. (1997), fetal arterial pressures increased in chronically
embolised fetuses that were exposed to acute severe embolisation. The different results
between this previous study and my own may be secondary to the different durations of
embolisation or the different insults used to induce the acute on chronic hypoxic stress.
It has been suggested that the elevation in fetal arterial pressure during prolonged
umbilicoplacental embolisation results from α-adrenoreceptors remaining responsive to
chronic fetal hypoxaemia and to chronic elevations in fetal plasma noradrenaline (Murotsuki
et al., 1997a). In my study and in others (Gagnon et al., 1994; Murotsuki et al., 1997a;
Murotsuki et al., 1997b), umbilicoplacental embolisation was associated with small increases
in plasma catecholamine concentrations. However, in my study when the fetuses subjected
to umbilicoplacental embolisation were exposed to large increases in noradrenaline levels and
very large increases in adrenaline levels during repeated cord occlusion (Figure 5.5), no
further increase in arterial pressure occurred. This may suggest that with chronic hypoxia the
function of adrenergic nerves in fetal arteries may decline as has been shown to occur with
chronic high-altitude hypoxia (Buchholz & Duckles, 2001). Alternatively it may suggest that
fetal arterial pressure is controlled by different mechanisms in growth restricted fetuses such
as the renin-angiotensin system, which has been found to have an important role in
controlling arterial pressure in fetuses in which growth restriction was induced by
carunclectomy of ewes (Edwards et al., 1999).
5.4.2.3 Fetal endocrine responses
The effects of umbilicoplacental embolisation on fetal glucose, cortisol and catecholamine
concentrations described in this study are similar to those reported previously using similar
embolisation techniques (Gagnon et al., 1994; Cock & Harding, 1997; Murotsuki et al., 1997a;
Murotsuki et al., 1997b). Similarly, the increases in catecholamines and glucose in response to
an acute asphyxial insult in embolised and control fetuses are similar to those previously
described (Gagnon et al., 1994). In my study, when fetuses subjected to 23 days of
umbilicoplacental embolisation were exposed to asphyxia induced by repeated cord
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occlusion, no increase in fetal cortisol concentrations occurred despite a three fold increase in
ACTH. In contrast, non-embolised fetuses exposed to cord occlusion had the same increase
in ACTH but fetal cortisol levels increased two fold (Figure 5.6). This observation suggests
that the adrenal cortex is less responsive to ACTH after prolonged umbilicoplacental
embolisation. Short-term umbilicoplacental embolisation for eight days has been shown to
not alter the responsiveness of the adrenal cortex to ACTH (Carter et al., 1996) and an acute
hypoxic challenge after ten days of umbilicoplacental embolisation resulted in the same
increase in cortisol as controls exposed to acute hypoxia (Gagnon et al., 1994). Fetal adrenal
responses after more prolonged periods of embolisation have not been studied and require
evaluation in future studies using this technique to induce fetal growth restriction.
5.4.3 Influence of the experimental model of fetal scalp blood biochemistry
In this study the relationship between fetal scalp blood lactate levels and carotid arterial
lactate levels in non-embolised controls was different from that observed in controls
described in Chapter Four. In this chapter, fetal scalp blood lactate levels were higher than
carotid arterial lactate levels (≈1.4mmol/L), whereas in Chapter Four there was no significant
difference in lactate levels between the two sampling sites. These results suggest that the
addition of an episode of fetal surgery at 101 days gestational age, the presence of a fetal
arterial catheter for 28 days, and 28 days in a metabolic cage influenced the relationship
between fetal scalp blood and carotid arterial lactate levels.
On careful review of the data from the controls in this study it can be seen that the fetal PO2
levels in controls during the embolisation phase were between 17 and 20mmHg, which is
slightly lower than reported in controls in other studies using umbilicoplacental embolisation
(Gagnon et al., 1994; Cock & Harding, 1997; Murotsuki et al., 1997a). Mild fetal hypoxaemia
of similar levels to those seen in controls in this study (≈17mmHg) has been shown to alter
fetal pressor responses and peripheral vascular resistance (Gardner et al., 2002). In addition,
the adrenaline and noradrenaline levels reported at the end of the embolisation period in
controls in this study, although similar to controls in other similar studies (Murotsuki et al.,
1997a), were higher than those reported in fetuses after five (Gardner et al., 2002) and ten
days of instrumentation (Gagnon et al., 1997). The combined effects of increased peripheral
vascular resistance, increased fetal pressor responses and mildly elevated catecholamine levels
are likely to result in vasoconstriction in the fetal scalp and increased local tissue lactate
production. Similar increases in hind limb lactate production have been described after
transient hypoxaemia (Gardner et al., 2003) and the multivariate regression analyses
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investigating factors influencing the relationship between scalp and carotid arterial blood
lactate levels performed in both this chapter and Chapter Four support the hypothesis that
local scalp tissue production of lactate occurred in this study, but not in the studies presented
in Chapter 4.
Considering the data from both chapters, the control data from Chapter Four where the
fetuses were only instrumented at the time of cord occlusion, is probably the most reliable.
In addition, the differences between fetal scalp blood and carotid arterial lactate identified in
Chapter Four are consistent with the limited human data that are available relating to
sampling from the two sites. The differences identified between fetal scalp biochemistry in
this chapter and Chapter Four highlight the difficulties involved in studies collecting fetal
scalp blood. They also provide strength to studies investigating the relationship between fetal
scalp blood biochemical levels and outcomes directly as the relationships between scalp and
central blood are complex and can be altered by experimental design.
5.4.4 Integration of results into clinical practice
In attempting to integrate the results of the studies performed in this chapter into clinical
practice the question arises as to what extent the events detailed in laboratory animals are
applicable to humans. Although it would be optimal to obtain the data presented in this
chapter from humans, it is clearly not possible to obtain repeated invasive samples in utero in
humans nor is it ethical to induce asphyxia.
The studies performed in this chapter suggest that the relationship between fetal scalp blood
lactate levels and carotid arterial lactate levels is robust and not influenced by fetal growth
status or asphyxia in the ovine fetus. Conversely the accuracy of fetal scalp pH assessment,
our current marker of fetal biochemical status during labour, is adversely affected by asphyxia
in growth restricted ovine fetuses. In addition, the studies presented in this chapter suggest
that fetal growth status does not adversely affect the ability of scalp blood lactate levels to
predict cardiovascular instability during cord occlusion, a marker that I have identified as
being strongly correlated with the severity of neuronal injury after repeated cord occlusion
(Section 3.3.5.3.3). Taken together these observations suggest that fetal scalp blood lactate
levels may be a more reliable predictor of anaerobic metabolism in human fetuses during
asphyxia across a range of fetal conditions and therefore should be a better predictor of fetal
outcome. This conclusion is consistent with a large observational study in humans which
suggested that fetal scalp lactate assessment was a better predictor of moderate-severe
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neonatal encephalopathy and low Apgar scores at five minutes than scalp pH assessment
(Kruger et al., 1999).
The analyses in this study suggest that late FHR decelerations should be considered
particularly ominous in growth restricted fetuses. The multivariate analyses investigating
associations with FHR patterns and the ROC analysis of the significant covariates suggested
that late decelerations were more strongly associated with metabolic acidosis in growth
restricted fetuses than in normally grown fetuses. In addition, in growth restricted fetuses
late FHR decelerations were more closely associated with cerebral hypotension during cord
occlusion than in normally grown fetuses. In humans, late FHR decelerations are one of the
sentinel features that clinicians aim to identify during continuous intrapartum electronic FHR
monitoring and they occur more frequently in growth restricted human fetuses during labour
(Low et al., 1976; Dashe et al., 2000). The results of this study relating to sinister associations
of late decelerations require replication in chronically instrumented growth restricted fetuses
prior to translation into human practice.
5.4.5 Future studies
The studies presented in this chapter suggest that fetal growth restriction alters the fetal
cardiovascular, endocrine and metabolic responses to repeated cord occlusion and it supports
the need for further studies in this area. As described previously, FHR, arterial pressure and
endocrine responses of growth restricted fetuses during repeated umbilical cord occlusion
require evaluation in a chronically instrumented fetal preparation. Although
umbilicoplacental embolisation produces many of the features of human growth restriction,
this may not be the ideal model to use for future studies relating to asphyxia and fetal growth
restriction as the generation of growth restriction requires fetal surgery and prolonged
catheterisation. A more appropriate model may be maternal heat exposure which has been
shown to induce asymmetric growth restriction (Galan et al., 1999a; Regnault et al., 1999),
abnormal fetal umbilical artery Doppler signals (Galan et al., 1998; Galan et al., 1999b), and
impaired placental transport of oxygen and amino acids similar to that seen in human
pregnancy (Battaglia & Regnault, 2001; Regnault et al., 2002; Regnault et al., 2003). This
model of growth restriction does not require surgery or prolonged instrumentation to
generate growth restriction which would allow the growth restricted fetuses to be chronically
instrumented three to five days prior to the cord occlusion studies.
In addition, further studies are required to investigate the ability of FHR patterns and fetal
biochemistry to predict fetal brain injury in growth restricted fetuses exposed to repeated
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cord occlusion. These studies are particularly important given the data presented in this
chapter suggesting that late declarations may be particularly sinister in growth restricted
fetuses, but also because of the recent evidence which suggests that human fetuses with
reduced growth potential are 20 times more likely to develop neonatal encephalopathy
secondary to an acute intrapartum hypoxic event than fetuses with normal growth potential
(Bukowski et al., 2003).
The results presented in this chapter suggest that further studies are required to investigate
fetal oxygen utilisation, lactate metabolism and placental lactate clearance in growth restricted
fetuses and additional studies investigating the hypothalamic-pituitary-adrenal axis are
required given the evidence in this study that the fetal adrenal responsiveness to ACTH is
impaired by prolonged umbilicoplacental embolisation. These studies would need to assess
paraventricular hypothalamic corticotropin-releasing hormone and arginine vasopressin
mRNA levels as well as anterior pituitary pro-opiomelanocortin mRNA levels. Analysis of
ACTH receptor and steroidogenic enzyme levels from the adrenal would provide insight into
adrenal sensitivity to circulating ACTH. Analysis of adrenal medulla phenyl-ethanolamine-
N-methyltransferase levels may provide insight into whether the observed increase in
cathecholamine levels in this study was due to increased production secondary to endocrine
responsiveness or increased sympathetic nervous stimulation or due to decreased clearance.
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5.5 Conclusion
In this chapter, I have shown that umbilicoplacental embolisation for 23 days alters fetal acid-
base, endocrine, and cardiovascular responses to subsequent asphyxia in the ovine fetus. In
addition, evidence has been provided that suggests that lactate and oxygen metabolism in the
fetal brain during asphyxia are altered by umbilicoplacental embolisation. Despite the
multiple effects of umbilicoplacental embolisation on fetal responses to asphyxia,
embolisation did not alter fetal arterial lactate responses to asphyxia, the relationship between
fetal scalp blood lactate levels and carotid arterial lactate levels or the ability of fetal scalp
lactate levels to predict fetal arterial pressure responses during cord occlusion. These results
suggest that scalp lactate levels are a robust marker of fetal anaerobic metabolism and a
reliable predictor of arterial perfusion during umbilical cord occlusion in both appropriately
grown and growth restricted fetuses.
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C h a p t e r 6
CLINICAL APPLICATION:
IS THERE EVIDENCE FOR CHANGE?
Science warns me to be careful how I adopt a view which jumps with my pre-conceptions
and to require stronger evidence for such belief.
My business is to teach my aspirations to conform with fact,
not to try and make facts harmonise with my aspirations.
Thomas Huxley (1860)
Worldwide there are approximately 130 million births annually and of these it has been
estimated that four million suffer from birth asphyxia, one million die and a similar number
develop sequelae including cerebral palsy, seizures, or developmental delay (WHO, 1991,
1995). Although it is widely accepted that a significant proportion of cases of neonatal
encephalopathy (Nelson & Leviton, 1991; Adamson et al., 1995; Badawi et al., 1998b, a) and
cerebral palsy (Blair & Stanley, 1988; Nelson, 1988; Yudkin et al., 1994; MacLennan, 1999)
have an antenatal origin, recent studies suggest that hypoxic-ischaemic events in the perinatal
period are the main cause of early neonatal encephalopathy, seizures or both (Biagioni et al.,
2001; Maalouf et al., 2001; Cowan et al., 2003) and perhaps play a more important role in the
origin of cerebral palsy than postulated over the last two decades (Hagberg et al., 2001).
The two techniques that currently are used widely for the assessment of fetal wellbeing
during labour in the developed world are continuous electronic fetal heart rate monitoring
(EFM) and fetal scalp blood pH measurement. Electronic fetal heart rate monitoring has
never been shown to be associated with a reduction in neonatal encephalopathy, cerebral
palsy or perinatal death (Grant, 1989; Vintzileos et al., 1995b; Thacker et al., 2001). The last
three decades of research into FHR monitoring suggest that EFM should be considered as a
screening test for the prediction of intrapartum fetal asphyxia in preterm and term pregnancy
(Low, 1998; Low et al., 2001; Low et al., 2002) and that EFM should be supplemented by fetal
scalp blood pH measurement. Assessment with the combined techniques has been shown to
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limit the increase in intervention associated with EFM alone and are associated with a 50%
reduction in neonatal seizures (Grant, 1989; Thacker et al., 2001). Although fetal scalp blood
pH measurement has been advocated to improve the specificity of FHR monitoring (Clark &
Paul, 1985; Low et al., 2001; Low et al., 2002), its utilisation varies widely, partly due to the
invasive nature of the testing but also due to the restricted availability of the expensive
equipment required to perform acid-base measurement on micro volume blood samples
(Greene, 1999). In addition, scalp blood pH measurement, although offering additional
benefit to EFM, is a poor predictor of low Apgar scores (Banta & Thacker, 1979; Kruger et
al., 1999) and moderate to severe hypoxic-ischaemic encephalopathy (Kruger et al., 1999).
Therefore, there are significant limitations with our current techniques for the intrapartum
assessment of fetal wellbeing. In order to reduce the burden of birth asphyxia on families,
health care systems and the community, new techniques that are inexpensive, widely available
and accurate are required to allow recognition of early intrapartum asphyxia so that timely
obstetric intervention can avoid asphyxia-induced brain damage or death.
In this thesis I have evaluated the role of lactate measurement in intrapartum assessment of
fetal wellbeing. Specifically, I have addressed the question, “Is fetal lactate measurement
better than the assessment of FHR patterns or the measurement of fetal pH at predicting
fetal brain injury after intrapartum asphyxia?” Using an ovine model of repeated umbilical
cord occlusion designed to mimic events which may occur during human labour, I have
shown that the measurement of fetal lactate levels after repeated cord occlusion is
significantly associated with the severity of brain injury after the asphyxial insult. No
significant associations were identified with fetal pH or with the duration of decelerative or
compound FHR patterns; however, mine is the first study to describe an association between
the duration of both increased FHR variability and FHR overshoot with the severity of
subsequent brain injury. Although no significant association was identified between fetal
arterial pressure between umbilical cord occlusions and the grade of brain injury, the studies
performed in this thesis are the first to show a strong correlation between the duration of
specific arterial pressure responses during cord occlusions and the grade of brain injury,
accounting for approximately 90% of the variability seen in the severity of injury.
The mechanism responsible for the improved ability of lactate measurement to predict fetal
brain injury compared to other measures of fetal acid-base status is unknown. It may be
because lactate is a more stable marker of anaerobic metabolism in the fetus than fetal pH.
During hypoxia, lactate is the end product of anaerobic metabolism of glucose and it
equilibrates slowly over the placenta (Daniels, 1966) probably through facilitated diffusion
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(Carstensen et al., 1983; Piquard et al., 1990) making it a stable marker of anaerobic
metabolism in the fetus. Conversely, changes in fetal pH can result from anaerobic
metabolism of glucose, elevated carbon dioxide levels resulting in respiratory acidosis, and
also from anaerobic metabolism of non-sulphur containing amino acids and fatty acids
(Gillstrap, 1999). The multiple potential sources of changes in fetal pH, and the fact that pH
can normalise rapidly via placental exchange of carbon dioxide, make this traditional marker
of fetal asphyxia less stable than lactate levels.
In this thesis I have provided evidence for two potential mechanisms that may explain why
lactate is a better predictor of fetal brain injury than fetal pH assessment. First, fetal lactate
may predict fetal brain injury through its association with fetal blood pressure responses
during umbilical cord occlusion. This was illustrated by the significantly greater area under
receiver operator curves for lactate than the traditional markers of acid-base status for
predicting arterial pressure responses during cord occlusion. The association was also
illustrated by the progressive increase in optimum cut off values for lactate predicting the
stages of cardiovascular decompensation during progressive asphyxia. The traditional
markers of acid-base status had stable optimum cut off scores as the stages of cardiovascular
deterioration progressed, suggesting that acid-base status may function as a threshold for
injury, whereas optimum lactate cut off scores progressively increased parallel to
cardiovascular decompensation with asphyxia. The second potential mechanism responsible
for why lactate is a better predictor of fetal brain injury than traditional acid-base assessment
may be through the association between fetal lactate levels and oxygen free radical activity in
the fetal brain. There is evidence from animal and human studies showing that levels of lipid
peroxidation measured in both brain and plasma, correlate with both the timing and the
severity of brain injury after hypoxic-ischaemic insults (Russell et al., 1992; Tan et al., 1998;
Ikeda et al., 1999; Candelario-Jalil et al., 2001; Marumo et al., 2001; Yu et al., 2003). In this
thesis, evidence has been provided which suggests that fetal blood lactate levels are
significantly better than pH, PO2 and base excess levels at predicting lipid peroxidation in the
fetal brain.
In human labour the fetal scalp is the window for biochemical assessment of fetal wellbeing.
In this thesis I have thoroughly investigated the relationship between fetal scalp blood lactate
levels and central blood lactate levels, considering both the effects of fetal asphyxia on this
relationship and also the effects of fetal growth restriction secondary to placental
insufficiency. The relationship between fetal scalp blood and carotid arterial lactate levels
was influenced by asphyxia but not by fetal growth restriction whereas the accuracy of fetal
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scalp pH assessment was impaired in growth restricted fetuses with scalp pH levels
underestimating the severity of changes in central blood pH. Despite the complex
relationships between fetal scalp blood lactate levels and central blood lactate levels, scalp
lactate levels were significantly better at predicting lipid peroxidation in the fetal brain than
central markers of fetal acid-base status. In addition, fetal growth restriction did not impair
the ability of fetal scalp blood lactate levels to predict cardiovascular instability during cord
occlusion.
One of the major limitations with our current technique of biochemical assessment of fetal
wellbeing during labour is the restricted availability of expensive equipment required to
perform acid-base measurement on small volumes of blood, which limits this technique to
tertiary obstetric centres. In this thesis I have assessed the accuracy and precision of a
handheld lactate meter which is commercially available in Australia. The meter costs
approximately AUS$900 with analysis of each 15µL sample costing $1. I have found this
meter to be both accurate and precise in lactate measurement in human blood micro samples
across the gestational age range of 26 to 42 weeks. Therefore if the assessment of fetal
lactate levels were to be introduced into clinical practice, unlike fetal scalp blood pH
measurement, lactate measurement would not necessarily be limited to large obstetric centres
based on the cost of purchase and maintenance of measuring equipment.
Is it time to change? What is the optimum method of fetal monitoring in labour to prevent
hypoxic-ischaemic brain injury? Published human data (Low et al., 1976; Low et al., 1981;
Nieto et al., 1994; Dashe et al., 2000; Grant & Glazener, 2001; Bukowski et al., 2003) and data
presented in this thesis relating to growth restricted ovine fetuses suggests that growth
restricted fetuses respond differently than normally grown fetuses to asphyxial stress that can
occur in labour. Therefore, when considering the optimum method of fetal monitoring
during labour, I will address fetuses with appropriate growth and those with growth
restriction separately.
For fetuses with appropriate growth for gestational age, based on the body of currently
published literature, there is little doubt that the minimum standard of care is good quality
intermittent FHR auscultation, every 15 minutes during the first stage of labour and every
five minutes during the second stage of labour (ACOG, 1995; NICH, 1997; Parer & King,
2000; RCOG, 2001; Thacker et al., 2001; Freeman, 2002; Liston et al., 2002a; Liston et al.,
2002b; RANZCOG, 2002). In addition, it is considered by most clinicians and professional
obstetric societies that if a pregnancy is considered high risk then the woman should be
offered continuous EFM for her fetus during labour (NICH, 1997; RCOG, 2001; Liston et
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al., 2002a; Liston et al., 2002b; RANZCOG, 2002), although this opinion is not based on any
data from randomised clinical trials in humans.
If non-reassuring FHR patterns are identified by intermittent auscultation then it is
considered optimal that continuous EFM is undertaken. If the non-reassuring FHR pattern
continues, randomised clinical trial evidence suggests that fetal scalp blood sampling for acid-
base assessment should be performed (Grant, 1989; Vintzileos et al., 1993; Thacker et al.,
2001) and further obstetric management should be based on the pH result and the overall
clinical picture including parity, progress in labour, proximity to delivery and the presence of
meconium stained amniotic fluid. Electronic fetal heart rate monitoring in the absence of
fetal blood sampling should not be advocated as it has never been shown to be better than
intermittent auscultation in preventing adverse neonatal outcomes. Furthermore, it is
associated with increased rates of instrumental vaginal delivery and caesarean section (Grant,
1989; Vintzileos et al., 1993; Thacker et al., 2001), both of which have small but real risks to
the mother and fetus .
Is there currently enough evidence to recommend introduction of measurement of lactate
levels in fetal scalp blood samples rather than, or in addition to, pH assessment? In addition
to the data presented in this thesis there have been a number of observational studies in
humans (Eguiluz et al., 1983; Smith et al., 1983; Nordstrom, 1995; Nordstrom et al., 1995b;
Kruger et al., 1999; Pennell et al., 2002) and one randomised clinical trial (Westgren et al.,
1998) addressing this question. In the published human data comparing the two techniques,
no differences were identified between the two techniques in rates of instrumental vaginal
delivery or caesarean section, nor was there any difference in neonatal outcome, umbilical
artery acid-base status or umbilical artery lactate levels (Westgren et al., 1998). Four
differences were identified between the two techniques. First, the process of fetal blood
sampling to assess fetal biochemical status was 16 times less likely to fail if blood was
collected for lactate measurement rather than pH measurement, probably due to the smaller
samples required for lactate measurement (Westgren et al., 1998). Second, fewer scalp
lacerations were performed if the fetuses were assigned to lactate measurement rather than
pH measurement (Westgren et al., 1998). Third, fetal scalp blood sampling for lactate
measurement was associated with a shorter testing time than scalp pH measurement and
fourth, scalp lactate measurement was significantly cheaper than scalp pH measurement
(Westgren et al., 1998). In an observational study performed in my obstetric unit whilst I was
undertaking my doctoral candidature, in addition to confirming the observations previously
described, we found that fetuses with both an abnormal scalp pH measurement (pH<7.20)
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and an abnormal scalp lactate measurement (>4.8mmol/L) were more likely to have an
umbilical artery pH<7.1 than fetuses with abnormal scalp pH measurement but normal
lactate measurement (Pennell et al., 2002). In addition, fetuses with both an abnormal scalp
lactate level and an abnormal scalp pH level were more likely to be admitted to the neonatal
intensive care unit for longer than three days than babies who only had an abnormal scalp
pH measurement (Pennell et al., 2002). The only study which has compared scalp lactate
measurement with scalp pH measurement in humans with an adequate sample size (n=1709)
to investigate neonatal encephalopathy reported that scalp lactate measurement was
significantly better than scalp pH measurement at predicting moderate to severe neonatal
encephalopathy with a scalp lactate level of 4.8mmol/L having a sensitivity of 100% and
specificity of 73%. Therefore, considering the fetal physiological studies presented in this
thesis and the published literature regarding fetal scalp pH and scalp lactate assessment, there
probably is adequate data to change from scalp pH assessment to scalp lactate assessment, or
to change from EFM with no biochemical assessment to EFM combined with scalp lactate
assessment; however, it would be ideal if another large observational study such as the one
reported by Kruger et al. (1999) was performed to further evaluate the two techniques with
regard to the prevention of important adverse outcomes of neonatal encephalopathy and
cerebral palsy.
Is a randomised clinical trial (RCT) comparing scalp pH assessment to scalp lactate
assessment required prior to the introduction of scalp lactate assessment into clinical
practice? Although it is widely accepted that randomised clinical trials are the gold standard
for the assessment of clinical interventions (Woodward, 1999), they are particularly difficult
in the situation where one is attempting to prevent rare outcomes such as neonatal
encephalopathy and cerebral palsy. To perform a RCT comparing scalp pH assessment to
scalp lactate assessment aiming to identify a 25% reduction in intrapartum acquired neonatal
encephalopathy would require a total sample size of approximately 11,500 women (80%
power, α=5%, calculated on rates presented in Table 1.8, assuming 50% of cases of neonatal
encephalopathy are acquired during labour). If the RCT was designed to investigate similar
changes in cerebral palsy rates a total sample size of 200,000 women would be required. In
addition to the sample size issues, performing RCTs on women in labour with non-
reassuring FHR patterns has important ethical and recruiting issues. If the measurement of
fetal scalp blood pH and fetal scalp blood lactate were assessing two different biochemical
pathways, I believe that it would be optimal to perform a RCT at least addressing the issue of
reducing the risk of neonatal encephalopathy. However, scalp lactate assessment and scalp
pH assessment are both measures of fetal metabolic acidaemia and lactate measurement is a
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more accurate way of assessing metabolic acidaemia than pH alone. In addition, fetuses born
with a severe respiratory acidosis (pH<7.0, base excess 4mmol/L to -8mmol/L) have been
shown to have neonatal outcomes that are no different from babies with normal acid-base
status at birth (Low et al., 1994). Therefore, as both techniques are assessing the same
biochemical pathway and lactate measurements appears a more accurate way of measuring
metabolic acidaemia than pH measurement, I propose that there is no need to perform a
RCT comparing scalp lactate to scalp pH measurement.
Once a baby is born, it is considered optimal for umbilical artery and vein acid-base
measurement to be performed (MacLennan, 1999). This procedure is not universally
performed, partly because of blood-gas equipment issues but also due to unfounded fears of
medico-legal retribution. Should we be performing umbilical artery lactate assessment at
birth instead of or in addition to acid-base assessment? A lack of association between fetal
acid-base status and the severity of fetal brain injury has consistently been reported in animal
studies since 1972 (Myers, 1972; Ting et al., 1983; Gunn et al., 1992; Mallard et al., 1992; de
Haan et al., 1993; Ikeda et al., 1998a; Fujii et al., 2003). In addition, human studies have
suggested that despite defining a group of neonates at risk, fetal umbilical artery acidaemia at
delivery has a poor correlation with outcome as most term infants with severe acidaemia at
delivery have an uncomplicated course (Winkler et al., 1991; Farrell et al., 1998; Huang et al.,
1999). Taken collectively, the animal and human data suggest that pH assessment identifies a
threshold for injury. If the pH level is higher than this threshold, fetal brain injury is unlikely
and if is lower than the threshold, fetal brain injury is possible. In this thesis, I have shown
that in sheep, fetal lactate levels after repeated cord occlusion are associated with the grade of
resultant fetal brain injury, accounting for approximately 60% of the variation in the severity
of brain injury. Similar findings have been reported by others in fetal sheep (Ikeda et al.,
1998a) and there is evidence from humans suggesting that measurement of lactate-creatinine
ratios in neonates at 6 hours of age (Huang et al., 1999) and the measurement of fetal brain
lactate levels using proton magnetic resonance spectroscopy (MRS) are predictive of poor
outcome in the neonatal intensive care unit (Hanrahan et al., 1996; Hanrahan et al., 1998), at
two months of age (Malik et al., 2002) and at 12 months of age (Amess et al., 1999).
Therefore, there are sufficient data to suggest that lactate assessment in umbilical arterial
blood may be beneficial; however, I believe further large human studies are required because
umbilical arterial lactate assessment at birth, although potentially valuable, may not be as
useful as lactate assessment performed in other ways such as MRS. Umbilical arterial lactate
assessment may define a subgroup of babies that could be evaluated by MRS prior to the
initiation of new experimental rescue therapies such as hypothermia (Yager et al., 1993;
293
Edwards et al., 1995; Laptook et al., 1995a; Laptook et al., 1995b; Thoresen et al., 1995;
Thoresen et al., 1996a; Thoresen et al., 1996b; Gunn et al., 1997; Gunn et al., 1998a; Gunn &
Gunn, 1998; Gunn et al., 1998b; Gunn et al., 1999; Gunn, 2000; Battin et al., 2001; Battin et al.,
2003), erythropoietin (Dame et al., 2001; Chong et al., 2002; Eid & Brines, 2002; Juul, 2002;
Kumral et al., 2003; Solaroglu et al., 2003), and antioxidants (Halks-Miller et al., 1986; Mishra
& Delivoria-Papadopoulos, 1999). This may reduce the number of babies requiring MRS,
which is expensive, and it would allow early identification of babies at risk of adverse
outcomes because early patient selection is important to gain the maximum benefit from
these new rescue techniques.
Growth restricted fetuses pose many challenges when considering labour. These fetuses are
more likely than appropriately grown fetuses to have non-reassuring FHR patterns (Low et
al., 1976; Dashe et al., 2000) and fetal acidosis in labour (Low et al., 1981; Nieto et al., 1994)
and are at increased risk of adverse outcomes secondary to acute hypoxic intrapartum events
(Bukowski et al., 2003). One of the first issues which require addressing is whether fetuses
with growth restriction should undergo labour or be delivered by an elective caesarean
section? A recent meta-analysis of published studies has addressed this issue and concluded
that currently there is not enough evidence to evaluate the use of elective caesarean section
for growth restricted fetuses (Grant & Glazener, 2001). The authors did note that growth
restricted fetuses exposed to labour were more likely to have neonatal seizures and death
than those delivered by elective caesarean section, but these differences did not reach
statistical significance (Grant & Glazener, 2001).
In this thesis I have presented the first experimental study using an animal model to
investigate fetal lactate responses during asphyxia induced by repetitive cord occlusion in
growth restricted fetuses. I have shown that growth restricted ovine fetuses have different
acid-base, endocrine, and cardiovascular responses to subsequent asphyxia than appropriately
grown fetuses. In addition, evidence has been provided which suggests that lactate and
oxygen metabolism in the fetal brain during asphyxia is altered by fetal growth restriction.
Despite the multiple effects of growth restriction on fetal responses to asphyxia, it did not
alter fetal arterial lactate responses to asphyxia, the relationship between fetal scalp blood and
carotid arterial lactate levels, arterial pressure responses during cord occlusion or the ability of
fetal scalp lactate levels to predict fetal arterial pressure responses during cord occlusion. The
data that I have presented suggesting that abnormal FHR patterns may have altered
significance in growth restricted fetuses are clinically vitally important but require replication
in appropriately instrumented fetuses. The data presented suggest that in growth restricted
294
fetuses late components to FHR decelerations are particularly sinister. In growth restricted
fetuses late components to decelerations did not reliably begin until lower base excess levels
than in appropriately grown fetuses and they were associated with cardiovascular instability
during umbilical cord occlusion with sensitivity of 98% and specificity of 88% for predicting
systolic and diastolic hypotension during cord occlusion. In growth restricted fetuses, FHR
overshoot after variable decelerations or changes in FHR variability had weaker associations
with changes in arterial pressure during cord occlusion than in normally grown fetuses. If
these observations are replicated in future studies, it may be that clinical decisions during
labour of growth restricted human fetuses can be made based on the duration of repeated
late decelerations whilst changes in FHR variability and overshoot may still require
biochemical assessment of fetal wellbeing. The responses of the growth restricted fetus to
labour is a challenging area which to date has undergone little scientific evaluation and in the
future requires detailed and thorough assessment both in animal models of asphyxia and in
human studies.
The suggestion that lactate levels are better predictors of outcome than traditional acid-base
status, although relatively recent in perinatal medicine, has been known for a number of years
in adult critical care medicine (Perret & Enrico, 1978; Bakker et al., 1996; Husain et al., 2003).
In adults, lactate level has been shown to be a reliable marker of tissue perfusion across a
wide range of conditions including acute trauma, major surgery and septic shock (Perret &
Enrico, 1978; Bakker et al., 1996; Husain et al., 2003) and lactate assessment is now widely
used in accident and emergency departments and in intensive care units around the world.
The assessment of fetal lactate levels both during and after labour is a simple, inexpensive
technique that can easily be integrated into clinical obstetric practice. It is probably superior
to our current techniques for the assessment of fetal wellbeing during labour; however, it is
not the final solution to intrapartum fetal monitoring. Abnormal lactate levels, whilst having
high sensitivity and specificity for perinatal brain injury, have poor positive predictive value
because the incidence of severe adverse outcomes is rare. Data presented both in this thesis
and by others (Myers, 1972; Ting et al., 1983; de Haan et al., 1993; de Haan et al., 1997b; Ikeda
et al., 1998a; Fujii et al., 2003) suggest that the key factor precipitating cerebral damage is
hypotension and the consequent compromise in cerebral oxygen delivery. I propose that the
next challenge for scientists, engineers and clinicians is to develop a technique to assess fetal
blood pressure non-invasively during labour because this measurement appears to be one of
the fundamental observations that we as clinicians are currently lacking when attempting to
make decisions relating to care in the hours before birth.
295
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