Dr. Shiuli MukherjeeMD, Fellow in Reproductive Medicine

Institute of Reproductive Medicine, Kolkata, India

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

Impaired immunoregulatory atmosphere

of endometrium

Hampers endometrial receptivity

Altered embryo-endometrial cross talk

Improper implantation

Unexplained recurrent first trimester miscarriage

HYPOTHESIS

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)

“Implantation is biomarkers’ interplay”

Co-expression with integrin & LIF

Nardo & Nikas et al and Aghajanova L, Stavreus-Evers et al Fert.Stert 2003

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

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

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.

Patient selection

Case ( n = 47)

Women with past history of idiopathic spontaneous recurrent miscarriage

Control ( n = 39)

Fertile women without previous pregnancy loss

Inclusion criteria

 Age between 24-35 years

Previous ≥ 2 miscarriages (<12wk)

Not on any kind of medication

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)

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

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

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.

Qualitative estimation of Pinopode

Developing Fully developed

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

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

Comparison of biomarkers in endometrium

P < 0.001 P < 0.001 P < 0.001

Comparison of pinopode morphology

Fisher’s exact test with Freeman-Halton extension P =.051

Comparison of pinopode numbers

Fisher’s exact test with Freeman-Halton extension P < 0.01

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

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)

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

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.

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

Hope for a new beginning……. Thank you.