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Dr. Shiuli MukherjeeMD, Fellow in Reproductive Medicine
Institute of Reproductive Medicine, Kolkata, India
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Introduction
Recurrent spontaneous miscarriage (RSM) represents most common complication of reproductive failure
60–70% remains unexplained
Impaired immunoregulatory atmosphere of endometrium may be a contributing factor
Raghupathy R et al, Immunology Today,1997
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Impaired immunoregulatory atmosphere
of endometrium
Hampers endometrial receptivity
Altered embryo-endometrial cross talk
Improper implantation
Unexplained recurrent first trimester miscarriage
HYPOTHESIS
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SUMMARY OF EVENTS IN ENDOMETRIAL RECEPTIVITY
Physiological Biochemical Immunomodulatory
Genetic expression
PINOPODES
Adhesion Molecules Cytokines
NK cells
HOXA 10Trophinin
‘E2’ stimulates ‘P’ activates
Receptive endometrium Window of implantation ( 7th – 9th post ovulation day)
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“Implantation is biomarkers’ interplay”
Co-expression with integrin & LIF
Nardo & Nikas et al and Aghajanova L, Stavreus-Evers et al Fert.Stert 2003
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Future and Facts
Fact Future
Failure of implantation due to deficient expression of different biomarkers
Pinopode αvβ3 Integrin LIF L-selectin
Acosta et al, 2000
To establish their role in pathogenesis of unexplained RSM in 1st trimester via alteration of endometrial receptivity
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Objectives
To asses the expression level of different implantation markers in endometrium of women with Idiopathic Recurrent Miscarriage
αvβ3 Integrin
Leukemia Inhibitory Factor (LIF) L-Selectin Pinopode
and compare them with that of fertile women
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Materials and methods
• Prospective Case-control study
• August 2009 to July 2010
• Institute of Reproductive Medicine, Kolkata and IIMST, Kharagpur, India
• Approval obtained from the institutional ethical committee and written consent taken.
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Patient selection
Case ( n = 47)
Women with past history of idiopathic spontaneous recurrent miscarriage
Control ( n = 39)
Fertile women without previous pregnancy loss
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Inclusion criteria
Age between 24-35 years
Previous ≥ 2 miscarriages (<12wk)
Not on any kind of medication
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Exclusion criteria
Autoimmune defects (APLA-Anti cardiolipin and anti lupus antibody)
Endocrine causes ( thyroid function test) Chromosomal defect (karyotyping) Coagulopathy ( P/H or F/H of thrombophilia) Infectious disease Uterine malformation ( HSG/hysteroscopy) Sperm defects (DNA fragmentation)
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Method
TVS - folliculometry from 10th day of natural menstrual cycle
Endometrial biopsy done between 7th and 9th day of post ovulation
Serum Progesterone level estimation on day of biopsy
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Tissue preparation & assessment
Immunohistochemistry to asses cell surface expression of αvβ3 Integrin, LIF and L-selectin
Pinopodes observed under scanning electron microscope.
Endometrial dating by histopathology.
Statistical analysis by Statistica version 6
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Pinopode Expression
Qualitative Quantitative
Developing -Small, smooth, naked, with short microvilli.
Fully developed – large, smooth, with/without small wrinkles.
Regressing - wrinkled surface, deflated ball
Percentage of endometrial surface occupied
Abundant - > 50% Moderate – 20-50% Scanty - < 20%
Mean values of 30 randomly selected fields taken
Nikas et al, 1995,1999.
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Qualitative estimation of Pinopode
Developing Fully developed
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Quantitative estimation of Pinopode Scanty (< 20%) Abundant (>50%)
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Result analysis
Clinical characteristics Patient with RSM( n= 47 )
Control( n= 39 )
Age ( years ) 29.97±3.0 28.61±3.5
BMI (kg/m2 ) 26.83±4.17 25.27±4.1
Day of EB 22.08±1.09 21.84±1.03
Histopathology dating Mid-secretory Mid-secretory
Progesterone (ng/ml) 11.28±1.27 11.81±1.39
Unpaired student t test, mean ± SD
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Comparison of biomarkers in endometrium
P < 0.001 P < 0.001 P < 0.001
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Comparison of pinopode morphology
Fisher’s exact test with Freeman-Halton extension P =.051
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Comparison of pinopode numbers
Fisher’s exact test with Freeman-Halton extension P < 0.01
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Discussion
Few studies have demonstrated correlation between unexplained recurrent miscarriage and expression of cytokines.
Cork et al 1999, Laird & Tuckerman et al,2003
Relation with pinopode not been discussed thoroughly
Aghajanova L et al,2003, Nardo LG et al, 2003
Nothing conclusive had yet been achieved
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Discussion continued..
Present study shows statistically significant difference in expression levels of
αvβ3 Integrin ( P <0.001 vs NS in Tuckerman et al,
2006)
LIF ( P <0.001 similar to Cork et al, 1999)
L-selectin ( P <0.001 ≈ Kruse C.et al 2003, Foulk R.A. et al, 2007)
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Discussion continued..
Pinopode expression in RSM
Morphological comparison – no significant difference ( P = 0.051)
Comparison of numbers – had significant difference ( P < 0.001)
Other studies have shown correlation in unexplained infertility, endometriosis, PCOs
Aghajanova L et al,2003
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Conclusion
Expression of cytokines, pinopodes and adhesion molecules seem to be deficient in recurrent miscarriage group.
A combined protocol of biomarkers like integrin, pinopode and LIF could have a good predictive value of endometrial receptivity in idiopathic recurrent miscarriage.
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Acknowledgement
Prof. (Dr.) B.N.Chakravarty Prof. K. Chowdhury Prof. ( Dr.) A.Hazra Dr. S. Ghosh Ms. P. Banerjee Dr. R. chatterjee Dr. S. Sharma Dr. S. Rajani
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Hope for a new beginning……. Thank you.