Abnormal Pinopode and Cytokine Expression in Recurrent Miscarriage

1. Dr. Shiuli Mukherjee MD, Fellow in Reproductive Medicine Institute of Reproductive Medicine, Kolkata, India Abnormal pinopode and cytokine expression affecting endometrial receptivity in idiopathic recurrent miscarriage

2. 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

3. HYPOTHES IS Impaired immunoregulatory atmosphere of endometrium Hampers endometrial receptivity Altered embryo-endometrial cross talk Improper implantation Unexplained recurrent first trimester miscarriage

4. SUMMARY OF EVENTS IN ENDOMETRIAL RECEPTIVITY ‘E2’ stimulates ‘P’ activates Physiological Biochemical Immuno modulatory Genetic expression Adhesion Molecules HOXA 10 Trophinin PINOPODES Cytokines NK cells Receptive endometrium Window of implantation ( 7th – 9th post ovulation day)

5. “Implantation is biomarkers’ interplay” Co-expression with integrin & LIF Nardo & Nikas et al and Aghajanova L, Stavreus-Evers et al Fert.Stert 2003

6. 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

7. 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

8. 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.

9. Patient selection • Case ( n = 47) Women with past history of idiopathic spontaneous recurrent miscarriage • Control ( n = 39) Fertile women without previous pregnancy loss

10. Inclusion criteria • Age between 24-35 years • Previous ≥ 2 miscarriages (<12wk) • Not on any kind of medication

11. 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)

12. 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

13. 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

14. 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.

15. Qualitative estimation of Pinopode Developing Fully developed

16. Quantitative estimation of Pinopode Scanty (< 20%) Abundant (>50%)

17. Result analysis Unpaired student t test, mean ± SD

18. Comparison of biomarkers in endometrium Mean fluorescing pixel area (%) P < 0.001 P < 0.001 P < 0.001

19. Comparison of pinopode morphology Patientsnumber Fisher’s exact test with Freeman-Halton extension P =.051

20. Comparison of pinopode numbers Fisher’s exact test with Freeman-Halton extension P < 0.01

21. 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

22. 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)

23. 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

24. 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.

25. 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

26. Hope for a new beginning……. Thank you.