Vol: 6, No. 1, July 2011

In the name of Allah, the most Beneficent, the most Merciful

JIIMC JOURNAL OF ISLAMICINTERNATIONAL MEDICAL COLLEGE

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PATRON

ADVISOR

CHIEF EDITOR

Maj. Gen. (R) Muhammad Zulfiqar Ali Khan, TI (M), SBtManaging Trustee, Islamic International; Medical College Trust

Mr. Hassan Muhammad KhanPro Chancellor Riphah International University

Prof. Dr. Anis AhmedVice Chancellor Riphah International University

Maj.Gen. (R) Masood Anwar, HI (M)Dean Faculty of Health & Medical Sciences (RIU)Principal Islamic International Medical CollegeRiphah International University

MANAGING EDITORS

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NATIONALLt. Gen. (Retd) Najam Khan HI (M)Prof. Brig (Retd) M. SalimProf. Muhammad Iqbal KhanBrig (R) Prof. Danyal RashidProf. Muhammad Tariq BaqaiBrig (R) Prof. Wahid Bakhsh SajidProf Brig (Retd) Ahsan Ahmad AlviProf Col (Retd) Abdul Bari KhanProf. Samiya Naeema UllahProf. Fareesa WaqarProf. Sohail Iqbal SheikhProf. Imran Azam ButtProf Shah SattarProf Muhammad TahirDr. Naveed Ahmed KhanBrig (R ) Dr. Shahid Javaid Dr Muhammad Azam ZiaProf. Syed Touqeer Abbas

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CONTENTS

EDITORIAL

Dengue Fever Outbreak

Dr Maqsood-ul- Hassan 1

ORIGINAL ARTICLES

Results of Myringoplasty in Chindren:

A Comparison with an Adult Population

Muhammad Azeem Aslam,Danyal Rashid, Ashfaq Ahmed

3

External Cephalic Version (ECV) for Breech

Presentation at Term -Experience at

Railway Hospital

Shamsunnisa Sadia, Shamsa Rizwan,Saadia Sultana, Fareesa Waqar,Raazia Rauf, Shaheena Owais

8

Outcome of Early Versus Delayed Oxytocin

Augmentation, in Nulliparous Women on the

Duration of Labour and other Obstetric and

Neonatal Outcome - A Randomized Controlled

Trial

Azra Saeed Awan, Umber Bakhtiar,Ghazala Amin

14

The Reno-Protective effect of Ethanolic Extract

of Cassia Fistula (Amaltas) Leaves on

Streptozotocin Induced Diabetic Nephropathy

in Rats

Farhana Adnan, Soban Sadiq,Adnan Jehangir

20

Effects of Lead Toxicity on Serum Testosterone

and LH levels in Adult Male Rats

Fatima Riaz, Umar Ali Khan,Saffia Shaukat, Ayyaz Ahmed

28

Effects of Lead Toxicity on Spermatogenesis

in the Testes of Albino Rats

Dr. Saffia Shaukat, Dr. Shabana Ali,Dr. Fatima Riaz

33

INSTRUCTIONS FOR AUTHORS 37

Volume 6 Number 1 July 2011

1

Dengue fever is fast emerging as one of the

common arthropode borne diseases. Viral th

aetiology of dengue was established in 20

century but dengue like illness could be

traced back 200 years. In 1950s the infection

was being reported by 9 countries, today a

wide array of countries, report regular

outbreaks. Pakistan is one of those countries

which have been in the grip of outbreaks

since 1990s. First reported epidemic could

be traced back to 1994. It is evident that

dengue fever is not a very recent phenomena

in Pakistan. It has had its history but 2011

witnessed hyperbole and press hype of

gigantic magnitude. This created scare and

irrational response to the outbreak.

Somehow the outbreak was pinned on or

attributed to collapse of health services,

whereas established wisdom attributes this

emergence of dengue fever to uncontrolled

population growth, rapid urbanization and

slum formation, inappropriate and

inadequate waste water management,

refugee movement and to erosion of vector

control program, non of this falls in the

domain of health sector. The hype created by

the press this year was a cause of daily

headlines on the news channels, resultant

response to this outbreak could at best be

described as misdirected.

Lot of fallacy and misplaced response was

undertaken. For example, cases of fever

were subjected to virological diagnoses with

resultant increase in cost and unnecessary

effort. Similarly isolation wards were put up

in hospitals and a pressure created by fever

patients to get admitted in them. A

misplaced efficacy of platelet transfusion in

every case was advocated with the result

that demand for cell separators and platelets

skyrocketed. Health education on the lack of

specific treatment was not common

knowledge and this outbreak was somehow

thought of as fault of medical and health

professionals and a cause of consternation

for the profession in the country in general

and Lahore in particular.Dengue fever outbreak was reported not for the first time and surely it will not be for the last time. As we square upto the fact that dengue fever epidemics are here to stay, it is already recognized that the countries reporting dengue fever outbreaks suffer from the same every five to six months in different regions and report major outbreaks every three years. It is hoped that the experience gained in 2011 will give rise to saner voices and prudent response after all the mayhem created by the press. A judicious response would have to be tailored for the long term and most of the action has to be taken by the government functionaries other than the health and medical professionals. Some of the steps that need to be advocated are, firstly population management and population control and policies to curb rapid urbanization and slum formation, secondly gearing up of municipal services in the cities and slums to

EDITORIAL

Dengue Fever OutbreakMaqsood-ul- Hassan

-------------------------------------------------DR. MAQSOOD-UL-HASSANDPH, MCPS, MSC(Med Adm)

Islamic International Medical CollegeRawalpindi

2

avoid waste water accumulation and ensuring appropriate drainage in and around human habitations.In case of outbreak the press will also have to be tutored to play its role of educating the general public on general and personal protective measures rather than be a harbinger of scare. People will have to be told before and during outbreaks to manage water collections in and around their residences, be it waste water or fresh water, and role of press can be invaluable. There is also a need to have a robust vector control program in terms of mosquito surveillance and surveillance of its larvae to map out areas with high density of vector, so as to undertake selective spraying of locales with high density so as to be aware of areas at risk of outbreaks and avoid consequences of environmental degradation and insecticide resistance of universal spraying. These are measures to be taken by sectors other than health and medical professionals, in case of outbreak through an important role is to be played by medical professionals in managing the cases. Health/ medical professionals will have to propagate

conventional wisdom that not every case needs virological confirmation once epidemiological data and testing of index and initial cases have confirmed the outbreak. Primary health care physician will have to be trained on risk management of cases, in that most of the cases will be treated outdoors, but high index of suspicion on bleeding diathesis due to platelet lowering and or evidence of leaking plasma causing dengue shock syndrome be readily recognized. As these merit hospital handling of the cases. The public and medical profession will have to be made aware of screening fever cases so as to avoid mosquito bites both in the houses as well as hospitals. Use of Aspirin during the outbreak will have to curbbed/stopped.A proactive approach though may not eliminate dengue fever, dengue shock syndrome, dengue hemorrhagic fever but can minimize its consequences. Society as a whole, needs to make concerted efforts to prevent outbreaks of the disease. A political will to undertake measures prior to outbreak is imperative.

3

ORIGINAL ARTICLE

Results of Myringoplasty in Chindren: A Comparison withan Adult PopulationMuhammad Azeem Aslam, Danyal Rashid, Ashfaq Ahmed

IntroductionMyringoplasty in adults is now considered

to be well established and rewarding 1procedure with high success rate but

controversy still surrounds the advisability 2of tympanic membrane repair in children.

The main concern regarding paediatric

myringoplasty is that the child has an

increased susceptibility to upper respiratory

tract infections and eustachian tube

dysfunction as compared to an adult leading 3to recurrent middle ear infection. This may

jeopardize the results of tympanic

membrane grafting.Moreover, tympanic

membrane perforation may favor the child

with poor eustachian tube function as it

provides ventilation to middle ear resulting

in decreased likelihood of development of 4,5effusion in it. If tympanic membrane

grafting is done in these children with poor

tubal function, negative pressure will

develop in middle ear resulting in graft

medialization or retraction minimizing the

benefits of surgery. All these reservations

about paediatric myringoplasty appear to

be valid but we must take into account the

effects of recurrent middle ear infections and

decreased hearing associated with tympanic

membrane perforation. Childhood is a 6period of active learning and socialization.

Impaired hearing at this stage of active

intellectual growth can be detrimental to 6child education and socialization. At the

same time, repeated infections in the middle

ear will further damage the hearing

ABSTRACT

Objective: To find out validity of myringoplasty in children by comparing its anatomical and functional results with adult myringoplasty.Study Design: A descriptive cross sectional studyPlace and Duration of Study: Department of Otolaryngology and Head and Neck Surgery at a tertiary care teaching hospital in Rawalpindi from January 2007 to December 2010.Materials and Methods: A total of 89 patients having dry central tympanic membrane perforation of various sizes secondary to chronic tubotympanic suppurative otitis media were included in the study. The patients were divided in two groups. First group comprised of children upto the age of 14 years whereas those above the age of 14 years were included in second group. Myringoplasty was performed in all patients with temporalis fascia utilizing underlay technique. The two groups were compared regarding graft success rate and hearing improvement at the end of 6 months of follow up. Results: Out of 89 patients included in the study, 37 (41.6%) belonged to child group whereas 52 (58.4%) were included in adult group. Among 37 patients in child group, 34(91.8 %) had successful graft take at the end of 6 months of follow up whereas 47 (90.3 %) patients out of 52 in adult group had successful graft. Average air bone gap closure of 13.5 dB was noted in child group whereas in adult group it was 12.5 dB. The difference between graft success rate and average air bone gap closure was found to be statistically insignificant (P>0.05). Conclusion: Results of myringoplasty in paediatric and adult age group are comparable in terms of graft success rate and hearing improvement.

Key words: Myringoplasty, tympanic membrane, paediatric, middle ear

----------------------------------------------------Correspondence:Dr. Muhammad Azeem AslamHouse No. 57, Main Road, Sector G-8/2, Islamabad.E-mail drazeemaslam@gmail.comPhone: 0333 5131237

4

mechanism.

In this study, we tried to solve the

controversy regarding advisability of

paediatric myringoplasty by comparing the

results of tympanic membrane grafting in

children to that of adults.

This study was conducted at the

Department of Otolaryngology and Head &

Neck surgery at a tertiary care teaching

hospital in Rawalpindi during the period of

January 2007 to December 2010. A non

probability purposive sampling technique

was adopted by including all patients

coming to outpatient clinic of our

department with dry central tympanic

membrane perforation secondary to chronic

tubotympanic suppurative otitis media,

irrespective of age and gender. A detailed

history was taken and thorough ear nose

throat examination was done. All patients

were examined under otomicroscope and

findings were recorded in a pre-designed

performa. The information obtained was

age and sex of patient, duration of disease,

size of tympanic membrane perforation,

status of middle ear mucosa and ossicles,

previous ear surgery and nasal complaints.

Patients with chronic rhinosinusitis, prior

ear surgery, atticoantral type of disease were

excluded from the study. Pure tone

audiometery with air and bone conduction

threshold was done in all patients. Patients

selected according to the above mentioned

criteria were divided in two groups

according to age. First group comprised of

children up to the age of 14 years and the

second group consists of patients above 14

years of age. All the patients underwent

myringoplasty under general anesthesia

Materials and Methods

using post aural approach and underlay

technique of tympanic membrane grafting.

Post operative antibiotic cover was given for

5 days. Patients were discharged on second

postoperative day and were followed up

after three weeks when bismuth iodoform

paraffin paste pack was removed from the

external auditory canal. Status of graft (full

take or failure) was noticed on that visit.

They were then followed up at monthly

interval for up to at least 6 months. During

that period, they were observed for graft

success. At the end of 6 months of follow up,

pure tone audiogram with air and bone

conduction thresholds was repeated. Both

groups of patients were compared in terms

of graft success rate (anatomical gain) and

hearing improvement (functional gain).

Graft take was defined as full, intact healing

of tympanic membrane graft at 6 months

postoperatively. Hearing improvement was

reviewed as the change in air-bone gap at the

end of six months follow up period. Gap

change was defined as the difference

between the pre and postoperative air-bone

gap. Air-bone gap was calculated as the

average difference between air conduction

and bone conduction at 0.5, 1 and 2 kHz.

Results were statistically analyzed using

SPSS version 10.0. Chi square test was

performed to find out the difference

between graft success rates of both the

groups. Pre and postoperative air-bone gap

change in dB between the two groups was

analyzed by using t test. Statistical

significance was accepted as p<0.05.

A total of 89 patients fulfilled the inclusion

criteria between January 2007 to December

2010. Thirty seven of them, up to the age of

Results

5

14 years, were included in child group

whereas 52 having more than 14 years of age

w e r e i n c l u d e d i n a d u l t g r o u p .

Demographics of both the groups are shown

in Table I.

Average follow up duration was 8.8 months

(range 6 to 29 months). Graft success rates

and hearing improvement (air-bone gap

closure in successful graft) in both the

groups are shown in Table II. The difference

between graft success rate and air-bone gap

closure in two groups were found to be

statistically insignificant (P >0.05).

Overall hearing improvement was noted in

all patients of both groups except those who

met with graft failure.

Table-I: Demographics of Child and Adult Group

Child Group Adult Group

Total No. ofPatients in each group

37(41.6%)

52(58.4%)

Male

Female

Mean Age (Years)

Age Range (Years)

Average Duration ofDisease (Years)

Size of Tympanicmembrane perforation

19(51.4%)

18(48.6%)

13

6 to 14

7.6 (1 to 13

Small = 3Medium = 25

Large = 9

16(30.8%)

36(69.2%)

28.5

15 to 57

12.5 (0.5 to 35)

Small = 1Medium = 32

Large = 19

Table-II: Graft Success Rates and Air Bone Gap Closure in Both Groups

Graft Success rate

Child Groupn = 37

34(91.8%)

47(90.3%)

13.5(Range 5 to 33.3

12.5(Range 3 to 24)

Adults Groupn = 52

Average Air-Bone gapclosure (dB)

6

DiscussionMyringoplasty in children is still a

controversial issue. Although a number of

studies appeared in literature on this subject

in the recent past but opinions still differ on

this subject. In the present study, we tried to

solve this controversy by comparing the

results of paediatric myringoplasty with

that of same procedure in the adults. We

obtained graft success rate of 91.8% in

children between the ages of 6 to 14 years.

Comparable graft success rate (90.3%) were

achieved in adult patients (> 14 yrs) utilizing

the same underlay technique and graft i.e.,

temporalis fascia. The difference between

graft success rates was statistically not

significant. These results are similar to

certain other studies in which adult and

pediatric myringoplasty results were 7compared .

In literature, we find wide variations

between the graft success rates of paediatric

myringoplasty ranging from 35 to 8,9,10,11100%. One local study reported graft

success rate of 72.96% at six months of

follow up in a study of thirty cases of

paediatric myringoplasty in children less 12than 18 years of age. Another study

reported graft success rate of 88% at one year

of follow up, in 100 cases of myringoplasty

performed in children less than 14 years of 13age. In another study of 231 cases of

paediatric myringoplasty, 93.5% graft

success rate was achieved at one year of 14follow up.

Few studies reported delayed perforations

reducing the overall initial success rate. In

one study, overall graft success rate of 87%

was reported with 3 years of follow up

whereas in another with longer follow up

period, 80.5% graft success rate was

reported in 41 cases of peadiartic

Myringoplasty. The mean follow up period 9in that study was 39 months. We reported

91.8% graft success rate in children at 6

months of follow up. We suggest studies

with long term follow up of at least three

years to evaluate the issue of delayed graft

failures in paediatric myringoplasty.

The functional results of paediartic

myringoplasty are not only promising but

are also comparable to same procedure in

adults. All of our patients in both child and

adult group had hearing improvement after

the surgery except those with graft failures.

We noted an average air-bone gap closure of

13.5 dB and 12.5 dB in child and adult group

respectively. The difference in air bone gap

closure in both groups was statistically

insignificant. These results showed that the

functional results of paediatric and adult

myringoplasty are comparable. This view is

shared by number of other studies reported 13,15in literature.

We believe that the results of our study may

be of considerable importance in resolving

the controversy of paediatric myringoplasty

in favor of early surgical intervention. This

will confer considerable benefits to children

with tympanic membrane perforation in

terms of improved hearing and decrease in

number of recurrent ear discharge, which in

turn leads to better overall academic and

intellectual performance and socialization.

In spite of our study results, we suggest that

further studies should be carried out with

larger number of patients and longer follow-

ups to resolve the controversy regarding

paediatric myringoplasty.

The success rate of myringoplasty in

Conclusion

children is comparable to that of same in

adults both in terms of anatomical and

functional gains.

1. Ashfaq M, Aasim MU, Khan N. Myringoplasty:

anatomical and functional results. Pak Armed

Forces Med J 2004; 54:155-8.

2. Sheahan P, Dwyer T, Blayney A. Results of type 1

tympanoplasty in children and parental

perceptions of outcome of surgery. J Laryngol

Otol2002 ;116: 430-34.

3. Kessler A, Potsic WP, Marsh RR. Type 1

tympanoplasty in children. Arch Otolaryngol

Head Neck Surg. 1994;120: 487-90.

4. Isaacson G. Tympanoplasty in children.

Otolaryngol Clin North Am1994; 27: 593-605.

5. Koch WM, Friedman EM, McGill TJ, Healy GB.

Tympanoplasty in children. Arch Otolaryngol

Head Neck Surg 1989; 116:35-40.

6. Mak D, MacKendrick A, Bulsana M et al.

Outcomes of myringoplasty in Australian

Aboriginal children and factors associated with

success: a prospective case series. Clin

Otolaryngol Allied Sci. 2004; 29:606-11.

7. Gersdorff M, Garin P, Decat M, Juantequi M.

Myringoplasty : long- term results in adults and

References

children. Am J Otol 1995;16: 532-5.

8. Bluestone CD, Cantekin EI, Douglas GS.

Eustachian tube function related to the results of

tympanoplasty in children. Laryngoscope 1979;

89: 450-8.

9. Pignataro L, Berta L, Capaccio P, Zaghis A.

Myringoplasty in children: anatomical and

functional results. J Laryngol Otol 2001; 115: 369-

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10. Chandrasekhar SS, House JW, Devgan U.

Pediatric tympanoplasty. A 10 years experience.

Arch Otolaryngol Head Neck Surg 1995; 121: 873-

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11. Pots i c WP, Winawer MR, Marsh RR.

Tympanoplasty for the anterior- superior

perforation in children. AmJ Otol 1996; 17:115-8.

12. Rafi T. Tympanoplasty in children-A study of 30

cases. J Surg Pakistan2001; 6:11-12.

13. Umapathy N, Dekker PJ. Myringoplasty: is it

worth performing in children? Arch Otolaryngol

Head Neck Surg.2003;129:1053-5.

14. Denoyelle F, Roger G, Chauvin P, Garabedian EN.

Myringoplasty in children: predictive factor of

outcome. Laryngoscope. 1999;109:47-51.

15. Alberna R, Riontino E, Giordano L, Gervasio CF et

al. Myringoplasty in children:a comparison with

an adult population. Acta Otorhinolaryngol Ital

1998; 18: 295-9.

7

8

ORIGINAL ARTICLE

External Cephalic Version (ECV) for Breech Presentation atTerm -Experience at Railway HospitalShamsunnisa Sadia, Shamsa Rizwan, Saadia Sultana, Fareesa Waqar, Raazia Rauf, Shaheena Owais

ABSTRACTObjective: To assess the effectiveness of ECV in singleton breech presentation at term and to determine its effect on maternal, delivery and perinatal outcomes in women to whom the procedure was offered.Study Design: A quasi experimental study Place and Duration of Study: The study was carried out in the Department of Obstetrics and Gynaecology, Railway hospital, Rawalpindi, from August 2006 to December 2008.Material and Methods: Eligible women, presenting with uncomplicated breech, between 37-41 weeks gestation, underwent ECV on day care basis. Fifteen minutes before the procedure, injection salbutamol 0.5 mg was administered. Cases with contraindication to ECV or salbutamol injection were excluded from the study. Success rate of ECV (in terms of conversion from breech to cephalic presentation at the completion of procedure confirmed through ultrasound) along with maternal, delivery and perinatal outcomes were assessed. Maternal and fetal demographic characteristics were also recorded as secondary outcome measures. For statistical analysis, SPSS version 10 was used and Chi-square test applied with a p<0.05 taken as significant.

Results: Of the 42 ECV procedures, 25 (59.5%) were successful. None of the patient suffered from serious maternal complications. Seven (16.7%) parturients complained of severe palpitations and 4 (9.5%) of marked discomfort during the procedure. Reversible fetal bradycardia was seen in 1 (2.4%) patient. Reversion to a non cephalic presentation occurred in two cases. Vaginal delivery was carried out in 21 patients out of the 25 who successfully underwent external cephalic version while all the patients with failed ECV underwent caesarean delivery. The 5 minute Apgar score was more than 8 in all except one baby.Conclusion: Adverse maternal and fetal outcomes of breech presentation at term are rare and there was no increased risk of complications after external cephalic version. Findings provide important data to quantify the frequency of adverse outcomes that will help facilitate informed decision-making and ensure optimal management of breech presentation.

Keywords: External cephalic version, breech presentation, pregnancy outcome

IntroductionExternal cephalic version at more than or

equal to 37 weeks gestation in suitable

women with breech presentation was

introduced in 1991 as a new management 1option. The rate of breech presentations in

the general population of parturients at term

has remained unchanged at 3-4%. However,

the recent finding that the fetus has an

increased morbidity during a vaginal

delivery when compared with Cesarean 2section has driven obstetricians towards the

decision that all breech presentations will be

delivered surgically .Thus, the Term Breech

Trial (2000) has impacted management with

3-4% increase in overall C/S rate. This is

further supported by the finding of survey

of centre collaborators (2003) 92.5% report

change in practice to planned C/S .The

morbidity of the mother with a breech

presentation is not increased with a vaginal

delivery; in fact the maternal morbidity

associated with surgery is higher than after a 3, 4 vaginal delivery. Subsequent pregnancies

are automatically deemed high risk due to 5the presence of a uterine scar.

In an attempt to reduce the need for surgery

with a breech presentation, the only option

available in the current climate, where a

vaginal delivery is out of the question, is to

atempt to convert the fetal presentation from

---------------------------------------------------------Correspondance:Dr. Shamsunnisa Sadia Associate Professor,Gynae Obs Unit-IPakistan Railway Hospital IIMC-T Rawalpindi

9

a breech to a vertex (head) presentation. The

success rates of ECV vary from 30% in

nulliparous women, to 67% in multiparous 6women. The use of tocolytic agents has

been shown to increase the success rate of 7ECV.

This technique may result in the premature

onset of labor, which would require

emergent surgery, and there is also a risk of

umbilical cord entanglement, prelabour

rupture of membranes as well as placental 8abruption. Following ECV, the fetus may

spontaneously return to the breech position.

Overall complication rates have ranged

from 1-2 % & in recent studies with strict

inclusion criteria no significant fetal or

maternal morbidity occurred as a result of 9ECV. In spite of these risks most women

wish to avoid CS, preferring ECV - the only

effective intervention to convert a breech

fetus to cephalic presentation with the

potential to help women avoid CS. The

Royal College of Obstetricians and

Gynecologists recommends that all women

with an uncomplicated breech presentation

at term should be offered ECV. Survey of

centre collaborators (2003) further support

this rationale by reporting a change in

practice with 13.8% more practitioners

offering/performing ECV. Unfortunately,

the fact that versions are not practiced in all

obstetrical departments is partly due to the

embarrassing lack of expert knowledge on

the part of some practitioners / clinicians

and more importantly failure in adequately

disseminating information regarding

management options available to patients

presenting with breech near term.

Probabilistic information on outcomes of

breech presentation is important for clinical

decision-making. The aim of the study was

to assess the effectiveness of ECV in

singleton breech presentation at term and to

determine its effect on maternal, delivery

and perinatal outcomes in women to whom

the procedure was offered.

It was a prospective study, carried out in the

department of gynaecology and obsterics,

Islamic International Medical College Trust,

Railway hospital, Rawalpindi. A total of 42

patients with singleton breech presentation

but otherwise uncomplicated pregnancies,

between 37-41 weeks gestation, were offered

ECV from August 2006 to December 2008.

Type of breech was not considered a factor

for suitability. Exclusion criteria included

m u l t i p l e p r e g n a n c y, a n t e p a r t u m

haemorrhage, ruptured membranes,

p l a c e n t a p r e v i a , k n o w n u t e r i n e

malformation, oligohydramnios (AFI < 8),

fetal abnormality, previous caesarian

section, active labour, intra uterine growth

restriction, severe proteinuric hypertension,

bad obstetric history, need for LSCS for any

other indication & any contraindication to

tocolysis such as maternal diabetes mellitus,

cardiac & thyroid disease and patient's

wishes after thorough counseling.

Each patient was fully explained regarding

the procedure, its possible complications

and an informed consent was taken.

Immediately prior to the ECV procedure,

the woman was reassessed to ensure she

was still eligible for ECV, duration of

pregnancy was between 37-41 weeks and

the fetal presentation was confirmed by

ultrasound. This ultrasound was used along

with clinical assessments to determine any

contraindications to ECV prior to each

procedure. Fetal well-being was assessed

Materials and Methods

10

prior to the procedure by continuous fetal

heart rate monitoring for 20 minutes. Fetal

heart rate assessment was required to reveal

a normal baseline rate, good variability, and

no evidence of decelerations prior to ECV.

All ECV procedures were undertaken by

exper ienced c l in ic ians and in an

environment where any complication could

b e a p p r o p r i a t e l y m a n a g e d . Tw o

obstetricians, at least one of whom had

experience with the procedure, performed

the ECV, with the aid of ultrasound

surveillance. The ECV procedure had to be

halted if any of the following occurred: fetal

bradycardia, placental abruption, and

failure of ECV.

I n j e c t i o n s a l b u t a m o l 0 . 5 m g w a s

administered intravenously to 22 and

subcutaneous to 20 patients.

After detecting rise in maternal pulse,

softening of uterus & easy palpation of fetal

parts, external cephalic version was

attempted under ultrasound guidance.

Continuous uterine pressure was limited to

5 minutes while total uterine manipulation

was limited to a maximum of 10 minutes. No

more than 2 attempts were tried. After

successful version, attitude was maintained

manually for a few minutes. Fetal heart

monitoring was done every 2 minutes

throughout the procedure. The woman

remained under supervision for at least one

half hour following the procedure. Fetal

well-being was assessed following the

procedure by confirming fetal movement on

ultrasound and by recording a reactive fetal

heart rate on continuous fetal heart rate

monitor ing for 20 minutes . Fetal

presentation was confirmed by ultrasound

immediately following the procedure. Since

Rhesus isoimmunization is a risk of ECV,

non-sensitized Rh negative women were

required to be provided with anti-D

immunoglobulin following the procedure.

Clinical parameters including age, height in

centimeters, parity, duration of pregnancy in

weeks, estimated fetal birth weight and

placental location were recorded. The

occurrence of any complication such as

PROM, APH, fetal distress, severe maternal

tachycardia (>120 beats/min) with

palpitation, sweating with hypoglycaemia

and reversion of fetus to breech presentation

were noted. Failure of ECV was followed by

elective lower section caesarean section on

completion of 38 weeks of gestation. On

success of ECV women were discharged

with weekly antenatal follow up to await

spontaneous labor up to 41 weeks. Care after

external cephalic version including mode of

delivery was determined by the attending

obstetrician. The care in labour ward was

guided by departmental protocols. Mode of

delivery, weight & sex of the baby was

recorded at time of delivery. Fetal outcome

was measured in terms of Apgar score at 5

mins.

The principal outcome measures of the

study were success rate of ECV, its overall

effect on maternal/ perinatal outcome and

mode of delivery. Maternal and fetal

demographic characteristics were also

recorded as secondary outcome measures.

For statistical analysis, SPSS version 17 was

used and student t- test applied with a

p<0.05 taken as significant.

In our study 42 patients with singleton

breech presentation between 37-41 weeks

gestation but otherwise uncomplicated

pregnancies fulfilled the inclusion criteria

Results

11

and were offered external cephalic version

(ECV). Overall success rate was 59.5%

(25).Reversion to breech occurred in 1

patient (2.4%) who was diagnosed on follow

up visit and repeat ECV was successful but

the patient had caesarean delivery due to

intrapartum fetal distress. Another patient

had reversion to transverse lie after

successful ECV but presented in labour and

was found to have a bicornuate uterus on

caesarean section.

The mean age of our patients was 26.2 years

(SD=5.9) while the mean height was 157cm

(SD=4.7). The parity of patients is shown in

table 1. The mean parity of the patients with

successful ECV was 1.8 while that of

unsuccessful ECV was 0.3.The mean

gestation at which ECV was performed was

38 weeks (SD=1.2). Intravenous tocolysis

was done in 22 women (52.4%) while

subcutaneous salbutamol was given to 20

patients (47.6%) prior to attempting ECV.

Similar frequencies had successful ECV's in

both the groups.

The ultrasonography carried out prior to the

procedure revealed that the placenta was

posterior / fundoposterior in 23 (54.8%),

anterior / fundoanterior in 16 (38.1%) and

fundal in position in only 3 (7.1%) cases. The

mean estimated fetal birth weight (EFBW)

was 3 kg (SD0.33gm). The mean EFBW of

patients with successful ECV was 2.96kg

while that of unsuccessful ECV was 3.1 kg.

There was only 1 (2.4%) case of transient

fetal bradycardia which recovered within 5

minutes. Seven (16.7%) parturient

complained of severe palpitations and 4

(9.5%) of marked discomfort during the

procedure. About 28 (66.7%) did not have

any complaint.

Vaginal delivery was carried out in 21

patients out of the 25 who successfully

underwent external cephalic version. One

patient was lost to follow up after successful

ECV. Caesarean delivery was necessary in

rest of the three women, two of whom had

reversion to non-cephalic presentation. All

the patients with failed ECV underwent

caesarean section.

The 5 minute Apgar score was more than 8 in

all except one baby delivered by LSCS due to

fetal distress in whom it was 6, but the score

improved to 10 at 10 mins of birth. Twenty

two (52.4%) babies were male while 19

(45.2%) had female sex. The mean weight at

birth was 3.25 kg, minimum & maximum

being 2.8 & 4 kg respectively.

Breech presentation is the most common

malpresentation affecting at least 20 000

babies per year in the UK alone. It is the third

most common indication for caesarean

section (C/S) following previous caesarean

section and labour dystocia. Approximately

87% of breech presentations in the United 10States result in cesarean delivery. External

cephalic version (ECV) has been clearly

shown to decrease the incidence of breech

presentation at term, thereby reducing the

elective caesarean section rate. The Royal

Discussion

12

College of Obstetricians and Gynaecologists

(RCOG) and American College of

Obstetricians and Gynecologists currently

recommend that all women with an

u n c o m p l i c a t e d s i n g l e t o n b r e e c h

presentation at term should be offered ECV.

Trial of ECV is cost-effective when

compared to a scheduled cesarean for

breech presentat ion provided the

probability of successful ECV is at least more 11than 32%.

This is in line with our case series where the

success of ECV was 59.5%.

In spite of this as well as recommendations

by professional bodies, acceptance of ECV

among both clinicians and consumers

appears to be limited, apparently because of 12fears over safety.

Apart from only one case of reversible fetal

bradycardia, there was no remarkable

perinatal complication in our study. About

17% of patients complained of severe

palpitations and 10% of marked discomfort

during the procedure. The low frequency of

complications in our series is matched by 13other studies which suggest that women

should be counseled that ECV is extremely

safe but has a 0.5% risk of emergency

caesarean section at the time of the 14procedure. In fact women with a breech-

presenting fetus at term and previous

caesarean delivery, who desire a trial of

labor, should be counseled regarding the

accumulating evidence about the efficacy

and apparently safety of this procedure and 15may be offered an ECV attempt.

External cephalic version at 3435 weeks

versus 37 or more weeks of gestation

increases the likelihood of cephalic

presentation at birth but does not reduce the

rate of caesarean section and may increase 16the rate of preterm birth. So in our study,

ECV was offered only to patients between

37-41 weeks.

In our case series there was no significant

d i f f e rence in materna l and fe ta l

demographic features. However, multipara

tended to have a better chance of a 17successful ECV as in other studies.

Twenty one out of the 24 patients who were

followed up after a successful ECV had a

normal vaginal delivery in our study. This is

in concordance with other studies exhibiting 18successful pregnancy outcome after ECV.

However, certain other studies show that

pregnancies after a successful external

cephalic version at term are not the same as

those with cephalic presentation. They are at

higher risk of both dystocic labor and fetal

distress and therefore require close 19intrapartum monitoring.

In our case series there was no difference in

the success of ECV whether tocolysis was

carried out by subcutaneous or intravenous

salbutamol. Currently, various options to

improve the success rate of ECV are being

explored in research trials. Moxibustion 20treatment due to its simplicity and

combined spinal-epidural analgesia due to 21its efficacy are being extensively studied.

Adverse maternal and fetal outcomes of

breech presentation at term are rare and

there was no increased risk of complications

after external cephalic version in our study.

Findings provide important data to quantify

the frequency of adverse outcomes that will

help facilitate informed decision-making

and ensure optimal management of breech

presentation.

Conclusion

13

References1. Hannah ME, Hannah WJ, Hewson SA. Planned

caesarean section versus planned vaginal birth for breech presentation at term: a randomized multicentre trial Term Breech Collaborative Group. Lancet 2000 ;356:1368-9

2. Rauf B, Ayub T.Maternal and perinatal outcome in term singleton breech presentation. J Postgrad Med Inst 2004;18:373-9

3. Sanchez-Ramos L, Wells TL, Adair CD. Route of breech delivery and maternal and neonatal outcomes. Int J Gynecol Obstet 2001 ;73:7-14

4. Jackson N, Paterson-Brown S. Physical Sequelae of caesarean section. Best Pract Res Clin Obstet Gynecol 2001; 15 :49-61

5. Mozurkewitch EL, Hutton EK. Elective repeat cesarean section versus trial of labor: a meta-analysisof the literature from 1989-1999. Am J Obstet Gynecol 2000;183: 1187-97

6. Ezra Y, Elram T, Plotkin V, Elchalal U. Significance of success rate of external cephalic versions and vaginal breech deliveries in counseling women with breech presentation at term. Eur J Obstet Gynecol Reprod Biol 2000;90 :63-6

7. Yanny H, Johanson R, Baldwin KJ, Lucking L, Fitzpatrick R, Jones P.Double blind randomized controlled trial of glyceryl trinitrate spray for external cephalic version. Br J Obstet Gynecol 2000; 107:562-4

8. Kasule J, Chimbria TH, Brown IM. Controlled trial of external cephalic version. Br J Obstet Gynaecol 1985;92:14-18

9. Devendra K. introducing routine external cephalic version for the management of the malpresenting fetus near term. Med J Malaysia 2002;57: 454-9

10. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Munson ML.Births: final data for 2002. Natl Vital Stat Rep 2003, 52:1-113

11. Jonathan M Tan, Alex Macario, Brendan Carvalho, Maurice L Druzin, Yasser Y El-s. Cost-effectiveness of external cephalic version for term breech presentation. BMC Pregnancy and Childbirth 2010, 10:3doi:10.1186/1471-2393-10-3

12. Thomas J, Callwood A, Brocklehurst P, Walker J. The National Sentinel Caesarean Section Audit. Br J Obstet Gynaecol 2000;107:579-80.

13. Grootscholten K, Kok M, Oei SG, Mol BW, van der Post JA. External cephalic version- related risks: a meta-analysis. Obstet Gynecol. 2008; 112:1143-51.

14. Collins S, Ellaway P, Harrington D, Pandit M, I m p e y L . S h o r t c o m m u n i c a t i o n : T h e complications of external cephalic version: results from 805 consecutive attempts. Br J Obstet Gynaecol 2007;114: 636-8.

15. Sela HY, Fiegenberg T, Ben-Meir A, Elchalal U, Ezra Y. Safety and efficacy of external cephalic

version for women with a previous cesarean delivery. Eur J Obstet Gynecol Reprod Biol 2009; 142:111-4.

16. Hutton EK, Hannah ME, Ross SJ, Delisle MF, Carson GD, Windrim R,et sal. The Early External Cephalic Version (ECV) 2 Trial: an international multicentre randomised controlled trial of timing of ECV for breech pregnancies. Br J Obstet Gynaecol. 2011;118: 564-77.

17. Tasnim N, Mahmud G, Khurshid M.External cephalic version with salbutamol - success rate and predictors of success. J Coll Physicians Surg Pak. 2009;19:91-4.

18. Siddiqui D, Robert J, Stiller MD, Collins J, Steven A, Laifer MD. Pregnancy outcome after successful external cephalic version. Am J Obstet Gynecol 1999;181:1092-5.

19. Lau TK, Kit Lo KW, Michael Rogers M. Pregnancy outcome after successful external cephalic version for breech presentation at term. Am J Obstet Gynecol 1997;176:218-23.

20. Manyande A, Grabowska C. Factors affecting the success of moxibustion in the management of a breech presentation as a preliminary treatment to external cephalic version. Midwifery 2009;25: 774-80.

21. Sullivan J.T , Grobman W.A, Bauchat J.R , Scavone B.M, Grouper S, McCarthy RJ et al. A randomized controlled trial of the effect of combined spinal-epidural analgesia on the success of external cephalic version for breech presentation. 2009 Intl JObstetAnesth;18 : 328- 34.

14

IntroductionThe active phase of labor may be augmented

routinely as in active management of labor

or selecting when progress of labor is 1considered to be inadequate.

Prolonged labor is most common among

nulliparous women and is related to

increased caesarean section rate i.e. nearly

25% birth in England (Pakistan-40-50% in

some private institutes) of which 30% are 2due to failed progress. WHO recommend

best outcome of mothers and babies to occur

with c/section rates of 5-10%.The world

health organization recommends the use of

partogram with an alert line indication

normal progress, defines as a cervical

dilatation of 1cm/hr and an action line 4 hrs

to the right to detect and treat prolonged 3labour.

It has been argued for long time that active

management reduces the duration of labour

and incidence of labour lasting more than 12

hrs, as well as c/section rate however this is

ORIGINAL ARTICLE

Outcome of Early Versus Delayed Oxytocin Augmentationin Nulliparous Women on the Duration of Labour and otherObstetric and Neonatal Outcome - A Randomized ControlledTrialAzra Saeed Awan, Umber Bakhtiar, Ghazala Amin

ABSTRACTObjectives: To determine the outcome of early versus delayed Oxytocin augmentation in nulliparous women on the duration of labor and other obstetric and neonatal outcomes. Study Design: A prospective randomized trial.Place and Duration of study: The study was conducted at Pakistan Railways Hospital, for the duration of 1 year with effect from January 2010 January, 2011. It is 400 bedded teaching hospital affiliated with IIMC-T of Riphah International University, IslamabadMaterials and Methods: In healthy nulliparous women with normal pregnancy,the progress of labor was thoroughly monitored and documented every 23 hours. If there was still no progress 1 hour after amniotomy, the woman was randomly allocated to either labor augmentation by oxytocin infusion or to postponement of oxytocin augmentation for 3 hours (expectant group, n = 158). Women whose labors had progressed satisfactory (1 cm/hour) after amniotomy were not randomized. Participants were managed according to a standard protocol entailing continuous documentation of the progress of labour, the amount of oxytocin administered, and obstetrical and neonatal outcomes. Oxytocin infusion was started at 6 mU/minute and was raised by 6 mU/minute every 30 minutes until efficient contractions were established in the early oxytocin group. In the expectant group, if no progress occurred after 3 hours, the women were reassessed regarding the need for oxytocin augmentation. Data were analyzed with SPSS 15.0. The MannWhitney U test was used to compare means. Proportions of events were compared with Fisher's exact test or chi-square analysis. Statistical significance was set at a P value of <0.05.Results: The caesarean section rate was 9% in the early oxytocin group and 10.7% in the expectant group (OR 0.8, 95% CI 0.51.4), and instrumental vaginal delivery 17% in the early oxytocin versus 12% in the expectant group (OR 1.5, 95% CI 0.972.4). Early initiation of oxytocin resulted in a mean decrease of 85 minutes in the randomization to delivery interval.Conclusion: Early administration of oxytocin did not change the rate of caesarean section or instrumental vaginal delivery but shortened labor duration significantly in women with a 2-hour arrest in cervical dilatation. No other clear benefits or harms were seen between early and delayed administration of oxytocin.

Key words: Oxytocin augmentation, prolonged labor, fetal distress.

---------------------------------------------------Correspondence:Dr. Prof Azra Saeed AwanHOD Gynae Obs Unit-IIPakistan Railways HospitalRawalpindi

15

4not supported in recent Meta analysis.

Aim of our study was to study effects of

early augmentation versus post ponemant

of oxytocin administration on obstetrical

and neonatal outcomes in nulliparous

women with spontaneous but prolonged

labour.

This prospective randomized trial was

conducted Pakistan Railways Hospital,

IIMC-T, Riphah International University,

Islamabad.

Healthy nulliparous women with normal

pregnancies received written information

about the study in the third trimester at their

antenatal clinics. Inclusion criteria were

nulliparity, a singleton fetus in cephalic

presentation, spontaneous onset of active

labour with regular contractions and an

effaced cervix dilated between 4 and 9 cm, at

a gestational age between 37 + 0 and 41 + 6

weeks. The choice of a minimum of 4-cm

dilatation was to ensure that the active

phase of labour was established at the time

of inclusion into the study. The latent phase

was defined as the interval between start of

contractions (women's report) until active

labour was established at inclusion into the

study. Women with spontaneous rupture of

the membranes together with regular

contractions were also included in the study.

Exclusion criteria were non-cephalic

presentation, prelabour rupture of

membranes, serious maternal disease and

fetal death.

During the study period 350 women

fulfilled the inclusion criteria. At admission

to the delivery ward, only the women given

the study information antenatally were

asked to participate in the study.

Material and Methods

Participation into the study was accepted by

315women who were included in active

labor.

The progress of labour was thoroughly

monitored and documented every 23 hours

according to the departmental guidelines.

Slow progress of labour was defined as an

arrest in cervical dilatation for two hours or

a dilatation less than 1 cm for 3 hours in the

first stage of active labour. If labour was

slow and the membranes were intact

amniotomy was first performed. If there was

still no progress 1 hour after amniotomy, the

woman was randomly allocated to either

labour augmentation by oxytocin infusion

within 20 minutes (early oxytocin group, n =

157) or to postponement of oxytocin

augmentation for 3 hours (expectant group,

n = 158). Women whose labours had

progressed satisfactory (1 cm/hour) after

amniotomy were not randomized.

Participants were managed according to a

standard protocol entailing continuous

documentation of the progress of labour, the

amount of oxytocin administered, and

obstetrical and neonatal outcomes.

Oxytocin infusion was started at 6

mU/minute and was raised by 6

mU/minute every 30 minutes until efficient

contractions were established in the early

oxytocin group. Maximum dose of oxytocin

infusion during the opening phase was 40

mU/minute. In the expectant group, if no

progress occurred after 3 hours, the women

were reassessed regarding the need for

oxytocin augmentation. . Data were

analyzed with SPSS 15.0. The MannWhitney

U test was used to compare means.

Proportions of events were compared with

Fisher's exact test or chi-square analysis.

Statistical significance was set at a P value of

16

<0.05.

During the study period 315 consenting

women were randomised. Primary analysis

was done according to intention to treat with

all cases as randomized. The two study

groups were well balanced against each

other in re lat ion to the basel ine

characteristics, for example, maternal age,

gestational age, proportion of women with

spontaneous rupture of membranes before

inclusion, intensity of labour pain and

cervical dilatation at inclusion and at

randomisation. In accord with the study

protocol, oxytocin augmentation was

started earlier, administered more often and

in higher doses in the early oxytocin group.

There were no significant differences in the

primary outcome, mode of delivery. The

instrumental vaginal deliveries were all

vacuum extractions except two forceps

deliveries in the early oxytocin group. The

interval from randomisation to delivery was

85 minutes shorter in the early oxytocin

group. There was no difference in duration

of the second stage between the groups.

Shows the percentages of women giving

birth from time of randomisation in both

groups. The rate of postpartum blood

transfusion did not differ significantly

between the two groups.

No neonatal outcome variable differed

significantly between the study groups.

Birth weight is similar. There were no

significant differences between the study

groups in 5-minute Apgar score below 7,

admission to NICU or phototherapy for

jaundice. There were no perinatal deaths.

Results

17

DiscussionSome or all components of active

management of labor have been adopted 1since 1970's. Several randomized control

studies have been carried out different

components of active management of labour

are: strict diagnostic criteria for labour, early

amniotomy, early use of oxytocin and 2continuous professional support.

The rise of operative delivery particularly

ceasarean section continous to be an

obstetric concern. Continuous increased

caesarean section rate may have influence

on maternal and neonatal mortality & 3morbidity. Prolong labour has been

described one of the leading indications for

18

4caesarean section.

Our study is relates to the oxytocin

augmentation of labor. Since the

formulization of high dose protocol for

oxytocin administration several papers

have published on ability of oxytocin to

fasten labor and decrease caesarean section 6, 11rate. In this protocol oxytocin infusion is

started at equalent of 6miu/min and

increasing 6miu every 30 minutes to

maximum 40 miu/min. Evidence of fetal

distress on CTG or FBS is the only

c o n t r a i n d i c a t i o n f o r o x y t o c i n

administration.

Satin et al demonstrated that duration of

labour may be decreased by 3 hours to lead

less ceasarean section but his study included 6,both nulliparous and multiparous women.

13 Other randomized controlled trial has not

realized the decreased rate of ceasarean

section but site similar reduction in duration

of labor.

Our study has shown that although early or

delayed use of oxytocin did not make any

difference in mode of delivery but it is very

obvious from results that every oxytocin

group had a significant shortened length of

labour and there was also a clear reduction

in rate of operative vaginal deliveries. There

was no significant difference in rate of PPH,

Apgar score of neonatal and need for NICU

admission.

This study had the following weakness.

Obstetrician was not blinded to the

treatment group. The decision to perform

caesarean section or operative vaginal

delivery may be tailored by knowledge of

treatment arm. There is no current Cochrane

review of effect of oxytocin used as a single

intervention in labour. There are only a few 10, 13, 15small trials for oxytocin alone.

A Meta analysis suggested that there was no 7, 13effect on caesarean section rate. Our

study also supported that a Meta analysis by

observing lack of any statistical difference in

caesarean section rate.

Although the shortening of labour has long

been widely assured with ac t ive 8, 14, 13management of labour. Our study has

reliably shown an 85 minutes reduction in

total duration of labour, which is similar to

previous studies which show 50-160minute

reduction.

In this study no significant differences were

seen between early versus expectant

administration of oxytocin stimulation in

nulliparous women with slow labour in

relation to the mode of delivery, postpartum

h a e m o r r h a g e , p o s t p a r t u m b l o o d

transfusions, neonates with a 5-minute

Apgar score below 7 or neonatal admission

to NICU. The interval from randomization

to delivery was 85 minutes shorter in the

early oxytocin group. No other clear benefits

or harms were seen between early

administration and expectancy of oxytocin

1. Waldenstrom U, Hildingsson I, Rubertsson C,

Radestad I. A negative birth experience:

prevalence and risk factors in a national sample.

Birth2004; 31:17-27.

2. Nystedt A, Hogberg U, Lundman B. Some

Swedish women's experiences of prolonged

labour. Midwifery 2006; 22:56-65.

3. Swedish National Board of Health and Welfare.

The Swedish Medical Birth Register, Centre of

Epidemiology

4. Florica M, Stephansson O, Nordstrom L.

Indications associated with increased cesarean

section rates in a Swedish hospital. Int J Gynaecol

Obstet 2006; 92:181-15.

Conclusion

References

19

5. Bugg GJ, Stanley E, Baker PN, Taggart MJ,

Johnston TA. Outcomes of labours augmented

with oxytocin. Eur J Obstet Gynecol Reprod Biol

2006; 124:37-41.

6. Satin AJ, Levan KJ, Sherman ML et al High VS

Low dose oxytocin for labour stimulation.

Obstetric Gynae 1992” 80: 111-16.

7. Selin L, Wallin G, Berg M. Dystocia in labour risk

fac tors , management and outcome: a

retrospective observational study in a Swedish

setting. Acta Obstet Gynecol Scand 2008; 87:216-

21.

8. Fraser W, Vendittelli F, Krauss I, Breart G. Effects

of early augmentation of labour with amniotomy

and oxytocin in nulliparous women: a meta-

analysis. Br J Obstet Gynaecol 1998; 105:189-94.

9. Sadler LC, Davison T, McCowan LM. A

randomised controlled trial and meta- analysis of

active management of labour. BJOG 2000;

107:909-15.

10. Hodnett ED, Gates S, Hofmeyr GJ, Sakala C.

Continuous support for women during

childbirth. Cochrane Database Syst Rev

2007;(3)CD003766.

11. Lavender T, Alfirevic Z, Walkinshaw S. Effect of

different partogram action lines on birth

outcomes: a randomized controlled trial. Obstet

Gynecol 2006; 108:295-302.

12. Rogers RG, Gardner MO, Tool KJ et al. Active

management of labour: a cast analysis of a

randomized controlled trail west J Med 2000”172:

240-3

13. Hodnett ED, Gates S, Hofmeyr GJ, et al;

Continuous support for women during

childbirth. Cochrane Database Syst Rev. 2003

;(3):CD003766.

14. Hofmeyr GJ; Evidence-basedintrapartum care.

Best Pract Res Clin Obstet Gynaecol. 2005;

19(1):103-15.

15. Wei S, Wo BL, Xu H, et al; Early amniotomy and

early oxytocin for prevention of, or therapy for,

delay in Cochrane Database Syst Rev 2009

;(2):CD006794.

20

ORIGINAL ARTICLE

The Reno-Protective Effect of Ethanolic Extract of CassiaFistula (Amaltas) Leaves on Streptozotocin Induced DiabeticNephropathy in RatsFarhana Adnan, Soban Sadiq, Adnan Jehangir

ABSTRACTObjective: To determine the Reno-protective effect of ethanolic extract of Cassia fistula (Amaltas) leaves on streptozotocin induced diabetic nephropathy in rats.Study Design: An animal experimental study Place of Study: The study was done in Biochemistry department, Islamic International Medical College,Rawalpindi and NIH.Islamabad.Materials and Methods: Single injection of STZ was given intraperitoneally to rats and those rats that showed fasting blood glucose level over 280mg/dl were included in the study. After induction of diabetes all rats were divided into, normal control group (A), diabetes positive control group (B), and the two groups (C and D) served as experimental groups while group E served as standard as it received glibenclamide. Group C and D diabetic experimental rats received ethanolic extract of Cassia fistula leaves at 400 mg/kg and 500mg/kg of body weight orally for eight weeks on daily basis. On the other hand group E rats received glibenclamide at 0.5 mg/kg body weight orally for eight weeks. Blood samples were collected after eight weeks to find reno-protection against STZ induced diabetic nephropathy.Results: The diabetic positive group rats showed variable increase in levels of serum glucose, serum urea, serum creatinine, total urinary protein and microalbuminuric levels. Body weight decreased and urine volume increased in diabetic groups. Cassia fistula ethanolic extract of 400mg/kg and 500mg/kg body weight dose and glibenclamide significantly decreased the levels of these parameters in rats. On comparison Cassia fistula ethanolic extract of 500mg/kg dose reduced levels of biochemical parameters more effectively than the 400mg/kg dose of Cassia fistula and glibenclamide. Cassia fistula constituents, especially polyphenols and flavonoids have strong anti-oxidant activity which might be involved in reno-protection. Conclusion: Cassia fistula ethanolic leaf extract showed reno-protection against STZ induced diabetic nephropathy in rats.

Key words: Reno-protection, streptozotocin , Cassia fistula, diabetic nephropathy, oxidative stress

3of diabetes mellitus.

Diabetic nephropathy is the leading cause of

end stage renal diseases (ESRD) in western

societies and accounts for 30 to 35% of

patients on renal replacement therapy in

North America. Type 1 and type 2 DM affect

approximately 0.5 and 4% of the population,

respectively.

Nephropathy complicates 30% of cases of

type 1 DM and approximately 20% of cases

of type 2 DM. However most diabetic

patients with ESRD have type 2 DM because

of the greater prevalence of type 2 DM 4worldwide. The level of hyperglycaemia

seems to be quantitatively linked to risk of

developing renal lesions. Hyperglycaemia

IntroductionDiabetes mellitus (DM) is a syndrome

characterized by chronic hyperglycaemia

and relative insulin deficiency, resistance or 1both. Diabetes mellitus is not a single

disease but basically a group of disorder

which is characterized by hyperglycemia,

hyperlipidemia, glycosuria, ketonemia and

if prolonged leads to diabetic complications

such as nephropathy, neuropathy and 2retinopathy. Nephropathy is one of the

important microangiopathic complications

---------------------------------------------------Correspondence:Dr. Farhana AdnanAssistant ProfessorDepartment of BiochemistryIIMCT Rawalpindi

21

enhances the non-enzymatic glycosylation

of proteins; hence advanced glycosylation

end-products (AGE) are formed. Increased

serum levels of AGE seem to predict changes

in kidney morphology such as expansion of

mesangial cell matrix and glomerular 5basement membrane thickening. The

activation of hyperglycaemia-induced

secondary mediators, such as protein kinase

C and mitogen-activated protein kinase, and

cytokine production are also responsible for

oxidative stress induced renal injury in the 6diabetic condition.

Various experimental animals have been

utilized to investigate diabetic nephropathy,

however STZ induced hyperglycaemic rats

have been used in this model of diabetic

nephropathy. STZ is synthesized by

streptomycetes achromogenes and is used 7to induce both type 1 and type 2 DM.

Cassia fistula belonging to the family

Leguminosae Casesalpinaceae is commonly

called as Amaltas, an Indian Labernum and

is native to India, Sri Lanka and is 8extensively diffused in various countries.

Its main property being that of a mild

laxative suitable for children and pregnant

women. It has been used to treat many 9-10intestinal disorders like ulcers. The plant

has a high therapeutic value and it exerts 11antipyretic and analgesic effects. Cassia

fistula leaf extract is also used for its anti-12-13tussive and wound healing properties. It

has been concluded that plant parts could be

used as a therapeutic agent in the treatment

of hypercholesterolemia partially due to 14their fibre and mucilage content. It has

15been reported to possess antitumor 16,hepatoprotective, antifertility and

17antioxidant properties. The Cassia fistula

leaves are known to be an important source

of secondary metabolites, notably phenolic

compounds.

The purpose of the present experimental

model was to observe the effect of Cassia

fistula on streptozotocin induced diabetic

nephropathy when given together to wistar

rats.

The study was done in Biochemistry

department, Islamic International Medical

College,Rawalpindi and NIH.Islamabad.

The present study was carried out on 50

male wistar rats, each weighing 200 250g

and were divided into 5 groups (10 each).

Each group was kept in a separate cage, in

the same room and under similar

physiological conditions in animal house of

National Institute of Health (NIH)

Islamabad.

Initially all groups were fed on normal rat

chow (consisting of wed, starch, choline,

methionine, vitamin, mineral mixture and

fat) and water for a period of one week for

acclimatization before starting the

experiment The care and handling of rats

were in accordance with the internationally

accepted standard guidelines for use of

experimental animals.

Commercially available kits (Randox ) for

biochemical analysis of glucose, urea,

c rea t in ine , to ta l ur inary prote in ,

microalbuminuria, 95% ethanol, pre-coated

TLC(Thin Layer Chromatography) plate

silica gel GF254 and toluene. The standard

compounds used are ellagic acid, gallic acid

and protocatechuic acid. The instruments

used were soxhlet and rotary evaporator,

centrifuge (Germany), clinical chemistry

Material and Methods

Animals:

Chemicals/Instruments:

22

analyzer and TLC scanner III (Camag,

Switzerland) with win CATS software.

Leaves of Cassia fistula were collected from

Jinnah Park, Rawalpindi and authenticated

from a botanist. Ethanolic leaf extract (ELE)

was prepared from Cassia fistula. Freshly

collected leaves were washed in tap water

and then distilled water and were shade

dried for about 1 week. The dried leaves

were crushed into a coarse powder. One

hundred gram of the powder was soaked in

1 liter of ethanol for 30 days with occasional

shaking. After 30 days this ELE was filtered

and evaporated to dryness over a water bath 18at 60°C .The yield of ethanolic leaf extract

was 19-20%.This (ELE) of Cassia fistula was

got standardized from NIH, Islamabad.

TLC (Thin Layer Chromatography) was

used for standardization. TLC was

performed on a pre-coated TLC plate silica

gel GF254. Sample was applied on the plate

as 8 mm wide bands with an automatic TLC

sampler. The development was carried out

in trough chamber (20 cm × 10 cm),which

w a s p re - s a t u r a t e d w i t h m o b i l e

phase(solvent system, toluene-ethyl acetate-

formic acid-methanol ( 30:30:8:2), for 20 min

at room temperature (25 ± 2°Cand 40%

relative humidity). Subsequent to the

development, TLC plates were dried under

stream of hot air and then subjected to

densitometric scanning using a TLC scanner

III (Camag, Switzerland) with win CATS

software (version 1.4.1) in the absorbance-

reflectance scan mode. Quantitative

evaluation of the plate was performed in

absorption-reflection mode at 338 nm. The

Plant materials and preparation of the extract

Standardization of plant extract:

standard compounds used are ellagic acid,

gallic acid and protocatechuic acid.

After acclimatization, 10 rats were labeled as

control. All other rats were starved for 16

hours and diabetes was induced using a

single intraperitoneal injection of freshly

dissolved streptozotocin (60 mg/kg) in

0.01M citrate buffer (pH 4.5). One week after

the streptozotocin injection, rats were

assessed for diabetes and those with fasting

blood glucose over 280 mg/dl were 19included in this study. Thereafter, all rats

were divided into five groups each having

10 animals. The control rats (Group A) were

fed on standard diet with tap water and

received no drug. Group B i.e. diabetic

control rats received 60 mg/kg of STZ as a

single intraperitoneal injection and were fed

on standard diet and tap water.

Group C i.e. experimental group rats

received 60 mg/kg of STZ as a single

intraperitoneal injection and ELE in a daily

oral dose of 400 mg/kg for a period of sixty

days. Group D i.e. experimental group rats

received 60 mg/kg of STZ as a single

intraperitoneal injection and ELE using a

dose of 500 mg/kg body weight daily

(orally) for a period of sixty days.16, 18

Group E i.e. standard group rats received 60

mg/kg of STZ as a single intraperitoneal

injection and glibenclamide 0.5mg/kg body

weight orally for a period of sixty days.

Timings: (day 0, day 60th). Blood samples

were taken at day 0 to establish that all rats

had normal glucose and renal profile.

Twenty four hour after administration of the

last dose of extract i.e. on 60th day and after

overnight fasting, the animals were weighed

and anaesthetized under ether vapours. A

Experimental Procedure

Sample collection:

23

sample of 2ml blood was drawn from tail

vein from all animals. Blood was transferred

to the sterile vacuotainers with gel and

allowed to clot at room temperature for one

hour. It was then centrifuged for ten minutes

at a speed of 3000 rpm. Serum was separated

and stored in sterile eppendorf tubes at -

20°C for analys is of b iochemical 20parameters.

Twenty four hour urine samples were

collected using metabolic cages and

analyzed. The animals were kept

individually in metabolic cages, they were

given only water. The urine was then

collected in 24 hours and measured. Body

weight also measured initially and at the

end of experiment.

Glucose levels were estimated using

commercially available kit (Randox, UK) 21based on glucose oxidase method. Serum

urea was estimated by commercially

available kits (Randox, UK), based on 22enzymatic colorimetric method while

serum creatinine using Jaffe alkaline picrate 23method (Randox, UK). Total urinary

protein was estimated by colorimetric

method and microalbumin level based on 24-25immunoturbimetric assay (Randox, UK).

The data was entered and analyzed using

SPSS 17.0 (Statistical Package for Social

Sciences). All data are shown as mean ±

S.E.M. One way ANOVA was applied to

observe group mean differences. Post Hoc

Tukey test was applied to observe mean

differences among the groups. A p-value of

<0.05 was considered as statistically

significant.

Biochemical Analysis

Statistical Analysis

Results

The biochemical parameters showed that

the injection of STZ caused a significantly

(p<0.05) increased serum glucose, serum

creatinine and total urinary protein levels in

the rats of group B, C, D and E as compared

to control group. On the other hand,

simultaneous administration of ethanolic

extract of Cassia fistula to group C and D

and glibenclamide to group E resulted in a

significant (p<0.01) decrease in the serum

glucose, serum creatinine and total urinary

protein levels in groups C ,D and E when

compared with that of group B.

The urea levels showed that the injection of

STZ caused a significantly (p<0.05)

increased urea levels in the rats of group B,

C, D and E as compared to the control. On

the other hand, the simultaneous

administration of ethanolic extract of Cassia

fistula resulted in a significant (p<0.01)

decrease in urea levels in the rats of group C

and D when compared with that of group B.

When group B was compared with group E,

no significant difference was observed in

urea levels (p = 0.05).

The microalbuminuric levels showed that

the injection of STZ caused a significantly

(p<0.05) increased microalbuminuric levels

in the rats of group B, C, D and E as

compared to the control. On the other hand,

the simultaneous administration of

ethanolic extract of Cassia fistula resulted in

a significant (p<0.01) decrease in the

microalbuminuria in the rats in group D

when compared with that of group B. When

group B was compared with group C

(p=0.07) and E (p=0.06), no significant

d i f f e r e n c e w a s o b s e r v e d i n

microalbuminuric levels.

The urine volume showed that the injection

of STZ caused a significantly (p<0.05)

24

increased levels of urine volume in the rats

of group B, C, D and E as compared to the

control group. On the other hand, the

simultaneous administration of ethanolic

extract of Cassia fistula resulted in a

significant (p<0.01) decrease in the levels of

urine volume of the rats in group C and D

when compared with that of group B. When

group B was compared with group E, no

significant difference was observed in urine

volume levels (p =0.08).

The body weight showed that the injection

of STZ caused a significant (p<0.05) decrease

in body weight in the rats of group B, C, D

and E as compared to the control.

On the other hand, the simultaneous

administration of ethanolic extract of Cassia

fistula resulted in an increase in the body

weight of the rats in group D. When group B

was compared with group C (p= 0.63) and

E (p=0.55), no significant difference was

observed in body weight.

The ear l i es t c l in ica l ev idence o f

nephropathy is the appearance of low but

a b n o r m a l l e v e l s ( = 3 0 m g / d a y o r

20mcg/min) of albumin in the urine referred

as microalbuminuria and patients with

microalbuminuria are referred to as 2 6nephropathy. Oxidat ive s t ress i s

c o n s i d e r e d t o b e t h e m a j o r

pathophysiological mechanisms involved

in the development of diabetic nephropathy.

High glucose directly increases hydrogen

peroxide production by mesangial cells and

also causes lipid peroxidation of glomeruli 27and glomerular mesangial cells. Effective

control of blood glucose level is a key step in

reversing diabetic complications and

improving the quality of life in diabetic

Discussion

patients. Currently available drugs for the

treatment of DM have a number of

limitations including adverse effects and

high rate of secondary failure. A number of

plants are being assessed for their

therapeutic potential as there is a growing

trend towards the use of natural remedies as 28adjuncts to conventional therapy.

25

The present study showed a significant

elevation observed in the levels of serum

glucose, urea, creatinine, total urinary

protein and urinary albumin excretion in

group B diabetic rats as compared to group 29A normal rats. Urine volume was also

elevated in group B rats while body weight

reduced in group B rats as compared to

group A rats. Elevated levels of these

parameters in serum and urine are

presumptive markers of diabetes associated

lesions in kidneys of rats. Co-administration

of ethanolic extract of Cassia fistula leaves

brought the levels of these diagnostic

parameters in the serum and urine of group

C, D and E animals towards normal as

compared to group B rats (Table I).

When we compared mean values of group C

with group D, although both decrease

glucose and renal parameters levels, but

group D reduced the levels more as

compared to group C. When we compare

mean values of group C and D with group E,

although glibenclamide decrease levels, but

cassia fistula ethanolic leaf extract reduced

t h e l e v e l s m o re a s c o m p a re d t o

glibenclamide. Cassia fistula also improved

the body weight as compared to

glibenclamide (Table I), showing better

effectiveness of cassia fistula ethanolic leaf

extract over glibenclamide. Our results are

in accordance with the reports by others

who used chemical antioxidants and diet of 30-31natural antioxidant plants.

The main constituents in Cassia fistula are

polyphenols (quercetin), anthraquinones,

flavonoids and flavan-3-ol derivatives. The

proposed mechanism of Cassia fistula in

renoprotection could be due to the

antioxidant mechanism. Anjaneyulu and

Chopra observed that quercetin is a strong

27antioxidant. It is likely that the scavenging

effects of quercetin on free radicals and the

inhibition of lipid peroxidation may play a

role in improving renal dysfunction in 32diabetes. Robards and Antolovich in 1997

have critically reviewed the analytical

chemistry of bioflavonoid and it was found

that flavonoids possess antioxidant activity,

they are potent free radical scavengers and

metal chelators and they also inhibit lipid 33oxidation .Therefore, in our study

flavonoids and quercetin in Cassia fistula

might have a role in the recovery in STZ

induced diabetic nephropathy in rats.

Further studies are needed to observe

histopathological examination and also to

see effect of higher doses and variable routes

of administration for its protective effect on

the kidneys of diabetic nephropathy

subjects.

The results of the present study show that

the co-treatment of Cassia fistula ethanolic

leaf extract prevents STZ induced diabetic

nephropathy in rats. The high dose

(500mg/kg) Cassia fistula leaf extract,

showed better results as compared to low

dose (400mg/kg) Cassia fistula leaf extract

and glibenclamide.

The authors would like to thank the

Principal IIMC Maj.Gen(Retd) Dr. Masood

Anwar for his continuous supervision and

NIH animal house incharge Mr.Hussain Ali

for helping in instrument handling and

biochemical analysis.

1. Gale EA, Anderson JV. Diabetes mellitus and

Conclusion

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28

Effects of Lead Toxicity on Serum Testosterone and LHlevels in Adult Male Rats

ORIGINAL ARTICLE

Fatima Riaz,, Umar Ali Khan, Saffia Shaukat, Ayyaz Ahmed

ABSTRACTObjective: To find out lead toxicity affects all levels of reproductive axis including both testosterone and Luteinizing Hormone production in adult male rats.Study Design: An experimental study on animalsPlace of Study: The study was carried out at Islamic International Medical College, Rawalpindi from January 2010 to November 2010.Materials and Methods: Thirty adult male Sprawgue Dawley rats, purchased from NIH, Islamabad were divided into two groups A and B each containing 15 rats. Group A served as normal control receiving plain tap water. Group B received 0.3% lead acetate in drinking water. Five rats from each group were sacrificed at the end of 2nd, 4th and 6th weeks. Serum testosterone and LH levels were analyzed using ELISA technique. Results were analyzed using SPSS version 13. Results: Serum testosterone level was significantly decreased in lead treated group as compared to control group whereas LH levels showed no significant change. Conclusion: Lead toxicity in male rats suppressed serum testosterone levels at all duration whereas LH levels at all durations manifested no significant change. This might be a result of direct testicular toxicity acting at testicular enzyme level alone or involving the hypothalamic-pituitary axis as well.

Key words: Lead toxicity, testosterone, luteinizing hormone, male rats.

Lead toxicity imposes adverse effects on

male reproduction and fertility both in 4clinical and animal studies. Testosterone

production by testicular Leydig cells is

essential for reproductive function and

vitality of male. Lead causes reduction in

semen volume and density, sperm count and

increased morphological abnormalities of 5spermatozoa both in humans and

6experimental animals . It has also resulted in

reduction of serum LH and FSH along with

serum testosterone levels, thus showing that 7it also targets hypothalamic-pituitary axis.

It may have direct testicular toxicity thus

decreasing testosterone levels or indirect

effects through targeting the endocrine 8control of reproduction or both. Studies in

male rats have shown that lead intoxication

disrupts testicular steroidogenesis by

inhibiting the activities of testicular 8steroidogenic enzymes.

The present work was conducted to study

the impact of lead intoxication on plasma

IntroductionLead (Pb) is one of the oldest and

commonest environmental pollutant, which

is reported to cause damage in multiple 1body systems. In a developing country like

Pakistan, people are specially exposed to 2lead pollution through air, water and soil.

Studies which have been conducted in

Pakistan, have revealed that major

population have blood lead levels above the 3internationally acceptable limits. It can

cause serious health problems including

high blood pressure, damage to the brain,

nervous system, kidneys and reproductive 1system.

Like other toxic metals, lead causes

oxidative damage and disrupts the

prooxidant/ antioxidant balance which has 4been demonstrated in multiple studies.

-------------------------------------------------

Dr. Fatima Riaz,Assistant Professor Physiology Department, IIDCEmail: Fatimaehsan76@yahoo.com

Correspondence:

29

testosterone and LH levels of male rats to

evaluate its mechanism of action.

Thirty adult male Sprawgue Dawley rats

between the ages of 60-90 days, weighing

130-200 gms were randomly selected. They

were divided into two groups with fifteen

rats in each group. Group A served as

normal control whereas group B was given

0.3% lead acetate dissolved and added in

drinking water, which was given in clean,

inverted bottles specific for the rat cages.

Both groups were fed on standard pellet diet

and water ad libitum in the Animal House of

NIH and kept in separate standard cages

designed accordingly. Drinking water

consumption in both the groups during the

previous 24 hours was recorded daily and

rats were weighed on weekly basis.

Treatment in both the groups continued for

42 days with 5 rats being sacrificed in each 9group at the end of 2nd, 4th and 6th weeks.

They were sacrificed 24 hours after the last

experimental day by an overdose of ether.

Three to five ml blood was drawn by

intracardiac catheterization. Samples were

immediately transferred into labeled gold

top vacutainers without anticoagulant kept

in an ice packed rack. Samples were then

shifted in an hour from NIH to Riphah

Diagnostic and Research Lab at Riphah

College Islamabad. Serum was separated by

centrifugation, transferred into labeled 1.5

ml eppendorf tubes, frozen and stored at -

80o C till assayed. Testosterone and LH

levels in each group were quantitatively

determined using solid phase Enzyme-

Linked Immunosorbent Assay (ELISA) as

described in the kit instructions leaflets 1 0p ro v i d e d b y t h e m a n u f a c t u re r .

Materials and Methods

Testosterone and LH ELISA kit were

purchased from DRG International, Inc. (Lot

# 16k096). Testosterone ELISA kit is based on

the principle of competitive binding. All

reagents and samples were brought to room

temperature. Desired number of micro titer

wells were placed in the holder. 25 µL of each

standard control and samples were pipetted

into appropriate wells of the strips. 200 µL of

HRP testosterone conjugate was added to

each well. After though mixing, it was then

incubated for 60 minutes at room

temperature. The contents were then briskly

shaken out and rinsed with wash solution

and then 200 µL of substrate solution was

pipetted to each well. After incubation the

enzymatic reaction was stopped by

pipetting stop solution. It was then read at

450 nm with the help of an ELISA reader

(strip reader das, model C serial #961, 2005).

Using semi-algorithmic graph paper a

standard curve was constructed by plotting

the mean absorbents obtained from each

standard against its concentration with

absorbance va lues a t Y-ax i s and

concentration on the horizontal X-axis. Thus

the corresponding concentration for each

sample was determined from the standard

curve. The DRG LH ELISA kit is based on the

sandwich principle, all instruction provided

in the leaflets was followed and the

corresponding concentration of LH levels

were determined from the standard curve

constructed.

Statistical analysis was performed by using

SPSS version 13. The arithmetic mean and

standard deviation of all observations were

calculated. Difference in mean among

control and treated groups was calculated

by 'independent t-test'. The difference was

considered significant if p-value was found

30

less than 0.05.

At the end of day 14, serum testosterone

level in lead treated (group B) was

1.5200±.49699 ng/ml as compared to control

(group A) (3.0200±.19235 ng/ml) with a

statistically significant decrease in p value <

0.001. Similarly at the end of day 28,

testosterone level in group B (1.3200 ±.42071

ng/ml) manifested a significant decrease in

p-value (<0.001) as compared to group A

(2.9200±.55857 ng/ml). This statistically

significant decrease in serum testosterone

was persistent even at the end of day 42 (p-

value <0.022) when lead treated group

(1.9400±.23022 ng/ml) was compared to

control group (3.0800 ±.72938 ng/ml) as

shown in figure 1.

.

Comparison of mean serum LH levels

between group B (lead treated group) and

group A (control group) revealed

statistically insignificant difference (p>0.05)

at all durations as shown in figure 2.

Results

Fig 1: Comparison of mean serum testosterone level in control (group A) and lead treated group (group B) at 14, 28 & 42 days respectively. Values are expressed as mean ± SD.

Fig 2: Comparison of mean serum LH level in control (group A) and lead treated group (group B) at 14, 28 & 42 days respectively. Values are expressed as mean ± SD.

DiscussionThe present study was conducted on adult

male rats to study the effects of lead toxicity

on serum testosterone and LH levels.

Throughout the experiment lead treated

group showed significant reduction in

serum testosterone levels at all time periods

as compared to control group. LH levels

however, exhibited no statistically

significant change. As it is known that serum

testosterone levels are regulated via

hypothalamic-pituitary-testicular axis,

hence suppression in serum testosterone

level indicate either a direct action of lead on

testes, or an indirect action affecting the

hormonal milieu at hypothalamic-pituitary

level.

These observations are in agreement with

other studies7,8 reporting similar effects on

serum testosterone levels. The toxic

manifestations of lead on testes resulted in

degeneration of both spermatogenic and

Leydig cells.6 The results presented herein

are in agreement with another study

reporting similar effects of lead on serum

testosterone level in rats ( Sokol 1990). She

verified that increased duration of exposure

31

after 14 days did not further suppress serum 11testosterone levels or spermatogenesis.

Another study reported lower expression of

enzymes 3 and 17â HSD involved in

testosterone biosynthesis as a result of lead 8intoxication. This might be the reason of

decreased serum testosterone level in our

study as well. According to Roy Chowdhury

lead adversely affects steroidogenesis either

directly or through endocrinological 12system.

However, in contrast to our study serum

testosterone level remained unaffected in 13another study while some others observed

an increase in testosterone level after lead 14administration . Their data could be

viewed as a result of a mixture of specific

lead toxicity (at the enzyme level) with other

more general actions (at the level of

hypothalamic-pituitary-testicular axis).

In present study serum LH levels in lead

treated group, revealed no significant

change when compared to the control group

showing that the major target of lead

intoxication are the Leydig cells with only a

modest effect on pituitary axis. A rise in LH

levels after lead intoxication showed that the

negative feedback mechanism is trying to

overshadow the toxic manifestation of lead.

The non-significant increase in LH levels in

the study after lead intoxication was similar

to another study conducted by Allouche and

her co-workers. In that study chronic

exposure to lead acetate did not significantly

affect LH levels along with impairment of

testosterone levels again emphasizing that

lead may target testicular function at Leydig 15cell level.

No changes in serum LH was observed by 14Kempinas, as mentioned above. Sokol

reported that the signals between

hypothalamus and pituitary gland are

disrupted by lead exposure in an intact 16animal. Hence, we cannot rule out the

possibility of involvement of hypothalamic

or supra hypothalamic area in our study.

There could be a reduction in serum GnRH

levels resulting in decreased serum

testosterone synthesis by the Leydig cells

and a secondary increase in pituitary LH

secretion as observed in this study.

Contrary to our study of LH results, a

decrease in LH was observed recently by

some studies alongwith suppressed 6,17testosterones . This difference in LH levels

could be attributed to the longer duration of

lead exposure, difference in technique of

measuring hormones or the route of

administration of lead. A study conducted

on lead exposure in an occupationally

exposed population suggested that lead

toxicity initially produced a direct testicular

toxicity followed by hypothalamic or

pituitary disturbance with longer periods of 18exposure.

The pathogenicity of lead toxicity on

reproductive system can be explained by

multiple mechanisms of action. Lead along

with other commonly found persistent toxic

metals like mercury, arsenic and cadmium

damages cellular material and produce

alteration in genetic material.12 The

mechanism underlying in all these metals is

common, involving oxidative damage.

Studies have shown that lead poisoning

disturb the normal balance of pro oxidants

to antioxidants, thus affecting membranes,

DNA and antioxidant defense systems of 19cells.

Based on the findings of study presented

Conclusion

32

herein, it is concluded that lead intoxication resulted in testicular dysfunction as mani fes ted by suppressed serum testosterone level with a modest effect on LH levels. Hence it seems that lead toxicity has a direct toxic action on Leydig cells along with some indirect effect by disturbing the hormonal milieu at hypothalamo-pituitary axis. The underlying mechanism of action of lead on reproductive system needs to be further evaluated to define new preventive measures against it.

1. Meyer PA, Brown MJ, Falk H. Global approach to reducing lead exposure and poisoning. Mutation Research E Pub 2008;659:166-75.

2. Kazmi T, Omair A. Control of lead poisoning in Pakistan. J Pak Med Assoc 2005; 55: 410-3. Khan DA, Malik IA, Saleem M, Hashim R, Bashir R. Screening for chronic lead poisoning in lead factory workers. J Pak Med Assoc Oct 1994; 44:239-41.

3. M a rc h l e w i c z a M , M i c h a l s k a b T a n d Wiszniewskaa B. Detection of lead-induced oxidative stress in the rat epididymis by chemiluminescence. Chemosphere 2004; 57:1553-62.

4. A. Mahmoud, P. Kiss, M. Vanhoorne, D.Bacquer, F. Comhaire. Is inhibin B involved in the toxic effect of lead on male reproduction? Int J Androl 2005; 55:150-5.

5. Ahmad I, Sabir M, Yasin K F. Study of the effects of lead poisoning on the testes in albino rats. Pak J. Med 2003;42:97-101.

6. El-Sayed Y.S,El-Neweshy M S. Impact of lead toxicity on male rat reproduction at “hormonal and histopathological levels. Environ Chemistry 2010; 92:765-74.

7. Biswas N M, Ghosh P K; Protection of adrenal and male gonadal functions by androgen in lead-treated rats. Kathmandu University Med J 2006;14:1218-21.

8. Biswas N M, Ghosh P K. Effect of lead on male gonadal activity in Albino Rats. Kathmandu

References

University Medical Journal 2004; 2: 43-6.

9. Wang C, Zhanga Y, Lianga J, Shana G, Wanga Y and Shi Q. Impacts of ascorbic acid and t h i a m i n e s u p p l e m e n t a t i o n a t d i f f e r e n t concentrations on lead toxicity in testis. Wei

Sheng Yan Jiu Clin Chim Acta Aug 2006; 370:82-8.

10. Naveen Tbeileh, Ahmed Elbetieha, Homa Darmani, Wael Khamas. Effects of Long Term Exposure to Cadmium Chloride on Fertility in Adult Male Mice. Medwell Journals 2007; 1:40-8.

11. Sokol RZ. The effect of duration of exposure on the expression of lead toxicity on the male reproductive axis. Americ J of Androl 1990; 11:521- 26.

12. Chowdhury A R. Recent Advances in heavy metals induced effect on male reproductive function. Al Ameen J Med Sc 2009; 2:37-42.

13. Suzan A. Wadi, Ghayasuddin Ahmad. Effects of lead on the male reproductive system in mice. Journal of Toxicology and Environmental Health 1999; 56:513-21.

14. W. G. Kempinas, A. L. V. Favaretto, V. R. Melo, T. L. Lamano Carvalho, S.O. Petenusci, R.M. Oliveira-Filho. Time-dependent effects of lead on rat reproductive functions. Journal of Applied Toxicology 1994; 14: 427-33.

15. Allouche L, Hamadouche M, Touabti A. Chronic effects of low lead levels on sperm quality, gonadotropins and testosterone in albino rats. Experimen and Toxicol Path 2009;61: 503-10

16. Sokol RZ, Berman N, Okuda H, Raum W. Effects of lead exposure on GnRH and LH secretion in male rats: response to castration and alpha- methyl-p-tyrosine (AMPT) challenge. Reprod Toxicol. 1998;12:347-55.

17. Hamadouche N, Slimani M , Merad-Boudia B, Zaoui C. Reproductive Toxicity of Lead Acetate in Adult Male Rats. American J of Scientific Res 2009; 3: 38-50.

18. M.Rodamilans. Lead toxicity on endocrine testicular function in an occupationally exposed population. Human & experimental toxicol 1988;125-8.

19. Yasir Farooq, Muhammad Mazhar Hussain, Shoaib Bin Aleem, Muhammad Asif Farooq. Lead Intoxication: The Extent of Problem and its Management. Pak J Physiol 2008;4:14-18

33

ORIGINAL ARTICLE

Effects of Lead Toxicity on Spermatogenesis in the Testes ofAlbino RatsDr. Saffia Shaukat, Dr. Shabana Ali, Dr. Fatima Riaz

ABSTRACTObjective: The objective of this study was to observe changes in spermatogenesis in the testes of albino rats exposed to lead acetate. Study Design: An experimental animals study Place of Study and Duration: This study was carried out in the Department of Anatomy, Islamic International Medical College Rawalpindi and at National Institute of Health Islamabad from January to April, 2009. Materials and Methods: Male adult rats were exposed to lead acetate with intraperitoneal dose of 4mg/Kg body weight for 5 days a week for 6 weeks. The animals in group A were used as control. The animals of groups B were treated with lead acetate with specified dose for 6 weeks. After 6 weeks the animals of subgroup B were sacrificed. The results of these two groups were then compared. Results: After six weeks, it was observed that the number of spermatogenic cells had decreased in the test groups as compared with control group (p<0.05).Conclusion: Lead is toxic for cells of spermatogenic series, injurious to heaith and plays a significant role in reducing male fertility.

Key words: Spermatogenesis, Lead toxicity, Rat fertility

3around 60 µgms/dl (Khan, 1994) . Lead is

found in almost all agricultural food. Lead

present in air falls onto crops or soil which is

absorbed by plants, thus entering the food

chain and finally consumed by humans

gaining entry into human blood (George, 41993). Following are the major sources of

lead:-

! I n s e c t i c i d e / P e s t i c i d e s p r a y s 5(Tollestrup, 1995).

! Lead can enter the water supply from

lead solder in plumbing, lead service

connections or lead pipes in home.

Lead is resistant to corrosion and

hence old lead used as drains in the

baths are still in service (Lanphear, 62005).

! Lead based indoor and outdoor 7paints (Lanphear, 1998).

The study was carried out in the Department

of Anatomy, Islamic International Medical

College Rawalpindi and National Institute

Materials and Methods

IntroductionLead is a bluish-grey metal present in the

earth's crust. It is one of the most ancient and

commonest environmental pollutants,

which has been reported to have caused

damage in multiple body systems. It can be a

cause of severe health problems such as high

blood pressure, renal damage, nervous

system breakdown, and anomalies in 1reproductive system (Meyer, 2008).

Pakistan is a developing country. People

here are specially exposed to lead pollution

through three main sources i.e., air, soil, and 2water (Rajendra, 1997).

Various researches carried out in Pakistan,

have stated that the majority of population

has blood lead levels much above the

internationally acceptable limits. Another

survey conducted in lead factory workers in

Pakistan showed blood lead levels hovering -------------------------------------------------

Dr. Saffia Shaukat,Assistant Professor Anatomy Department,IIMCT Rawalpindi

Correspondence:

34

of Health, Islamabad. It was an analytical

trial. The duration of study was 6 weeks.

Eight weeks old healthy male albino rats of

Sprague Dawley strain weighing 200 ±10

gm. were selected for the study. A total of 30

adult male albino rats of Sprague Dawley

strain were used in this study. These animals

were randomly divided into two groups;

one Control (A)and one Experimental group

(B). The animals in group A (control) were

given normal diet ad libitum. The animals in

group B were treated with intraperitoneal

lead acetate in the dosage of 4 mg / Kg body

weight / day, 5 days a week for a period of 6

weeks. These animals were sacrificed at the

end of six weeks. All animals were kept in

the animal house under standard conditions

at a room temperature (180 C to 260 C) for six

weeks. They were maintained on 12 hours

light and dark cycle. The rats were fed ad

libitum. Day 0 was considered to be the

starting day of experiment. Pure lead

acetate, taken from Pharmaceutical

Laboratory in Islamic International Dental

College (IIDC), was used. It was purchased

from the Merck Company (Lot No. 107372).

The temperature was maintained between

18°C to 26°C. The animals were sacrificed by

an overdose of ether anesthesia. Cotton

soaked in ether was placed into the jar. The

animal to be sacrificed was lifted by the tail

and dropped into the jar. After sacrifice, the

animal was taken out of the jar and placed

on the dissecting board. The scrotal sac was

then opened with the help of forceps and

scissors. After fixation, histological sections

were made and staining was done by

Haematoxylin and Eosin.

Results

Fig.1: Photomicrograph-Section of testis of experimental group (Group B) animal number 15, showing reduced germ cell count and reduced height of germinal epithelium. PAS stain. x 420.

Fig. 2: Photomicrograph; Section of testis showing seminiferous tubule with normal germ cell count in group A, Animal Number 13, PAS Stain, x 400

Data was entered in a data base using

Statistical Package for Social Sciences (SPSS)

for window version 13. The results were

analyzed and considered significant with P

value less than 0.05 (P <0.05) applying t test.

Only three cross sections of seminiferous

tubules (randomly selected in different

fields), were observed. Total numbers of

germ cells in the selected tubules were

counted.

Histological Examination: The following

observations were made: Number of

spermatogenic cells/cross section of

seminiferous tubule at 40 x.

35

Germ Cell Count / Cross Section Of Seminiferous Tubule)

Discussion

The average germ cell count in group A was

304.53 cells/cross section of seminiferous

tubule (SD + 28.46), in group B it was 116.91

cells/unit (SD + 32.66).

The germ cell count was significantly higher

in group A as compared to groups B (p <

0.001). (Fig No. 1). The germ cell count was

significantly lower in group B as compared

to groups A (Fig. 1) (p < 0.001).

The present study was conducted to

evaluate whether or not, lead has toxic

effects in the rat's testes. For this purpose,

albino rats of Sprague Dawley strain were

selected. The animals were randomly

divided into a control A and experimental

group B. The animals of experimental group

(B) were treated with intraperitoneal lead

acetate in the dosage of 4 mg / Kg body

weight / day, 5 days a week for a period of 6

weeks. After 6 weeks, the animals of group B

were sacrificed to see the toxic effects of lead

in their testes. In lead toxic testes the

seminiferous tubules decreased in size and

had a wavy outline. The germ cell count was

significantly reduced in group B, as

compared to the control group A, which

indicated the intensity of harm caused by

lead on the cells of spermatogenic series. No

effect on Sertoli cells was observed.

The population of Leydig cells had become

dispersed and they were sparingly seen in

clumps. These findings are similar to those

of Chowdhury et al, Saxena et al and

Hilderbrand et al.8, 9, 10. All these scientists

described variety of toxic changes induced

by lead in the testes depending upon dose

and duration of the treatment which are in

accordance with present study. At the end it

is concluded that lead is injurious to the cells

of spermatogenic series and the interstitial

cells of Leydig, while the Sertoli cells are

spared.

Chapin (2002) described in his study that

CDC defines blood lead levels exceeding 10

micrograms/dl as elevated in children11.

This may lead to cognitive and behavioral

developmental changes. The researcher

reported that lead induced reproductive

abnormalities may occur in male rats at a

dose as low as 10 µg/dl 11. Manlay (1995)

administered lead acetate to rats in the dose

of 8mg/kg body weight, 5 days a week for 35

days 12.

During past three decades, the decline in

male reproductive health and fertility has

been linked with environmental toxicants

and xenobiotics. (Sikka and Wang, 2008)13.

My present study validated the previous

studies?, results and further added a new

dimension to the existing literature by

investigating lead-induced toxicity in the

testes of Sprague Dawley rats.

1. Meyer PA, Brown MJ, Falk H. Global approach to

reducing lead exposure and poisoning. Mutation

Research E Pub 2008;50:166-75.

2. Ramlogan A. Environment and human health: a

threat to all. Environmental Management and

Health, 1997; 8: 51- 66.

3. Khan DA, Malik 1A, Saleem M, Hashmi R, Bashir

R. Screening for chronic lead poisoning in lead

factory workers. JPMA1994;239-41.

4. George A. Gellert GA. Wagner, Roberta M.

Maxwell, Douglas M. Faster I. Lead Poisoning

Among Low-Income Children in Orange County,

California. JAMA 1993; 69-71.

5. Tollestrup K, Baling Jr., Allard J. Mortality in a

cohort of orchard workers exposed to lead

References

36

arsenate pest ic ide spray. Archives of

Environmental health; 1995; 221-9.

6. LanphearBP. Low-level environmental lead

exposure and children's intellectual function: an

international pooled analysis. Environ. Health

Percept 2005; 113: 894-99.

7. Lanphear BP. Matte TD, Rongers J. The

contribution of lead-contaminated house dust

and residential soil to children's blood lead levels.

Environment research 1998; 51-68.

8. Eyden BP, Maisin JR and Mattelin G. Long term

effect of dietary lead acetate on survival, body

weight and seminal cytology in mice. Bull

Environ Contam Toxicol 1978; 19:266-72.

9. Saxena DK, Srivastara RS, Lal B and Chndra SV.

The effects of lead exposure on the testis of

growing rats. Exp Pathol 1987; 31: 240-52.

37

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In an original article of JIIMC Vol 5, No. 2, 2010 titled “Assessment of Stress Related Hypertension (white coat hypertension) in a Primary Health Care Setting. A Growing Concern for Community Health Physicians” the name of one of the author has been spelled wrongly as SABINA M HAQ, The correct name is “SABIHA M HAQ”.