Changes in cell proliferation, but not in vascularisation are characteristic for human endometrium in different reproductive failures - a pilot study

Reproductive Biology and Endocrinology MCehthaodnologgeys in cell proliferation, but not in vascularisation are characteristic for human endometrium in different reproductive failures - a pilot study Ariane Germeyer 0 Michael von Wolff 2 Julia Jauckus 0 Thomas Strowitzki 0 Tanuj Sharma 1 Anna T Grazul-Bilska 1 0 Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital Heidelberg , Heidelberg , Germany 1 Department of Animal Sciences and Cell Biology Center, North Dakota State University , Fargo, North Dakota , USA 2 Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital Berne , Berne , Switzerland Background: Reproductive failure, determined as recurrent spontaneous abortions (RSA) or recurrent implantation failure (RIF) in women is not well understood. Several factors, including embryo quality, and cellular and molecular changes in endometrium may contribute to the insufficient feto-maternal interaction resulting in reproductive failure. Prior clinical studies suggest an inadequate endometrial growth and development of the endometrium, leading to a lesser endometrial thickness. Methods: We therefore aimed to determine the cellular proliferation using Ki67, and the expression of markers of vascularisation, such as factor VIII (a marker of endothelial cells) and smooth muscle cell actin (SMCA; a marker of pericytes and smooth muscle cells) in endometrium of healthy women and women with RSA or RIF. LH-dated midsecretory endometrial biopsies of seven healthy women and twenty women with reproductive failure were examined via immunohistochemistry followed by image analysis. Results: Cellular proliferation but not expression of factor VIII or SMCA was decreased (P < 0.0004) in endometrium of women with RSA and RIF compared to healthy controls. Conclusion: Our data indicate that reproductive failure is due to insufficient cell proliferation/tissue growth rather than inadequate vascularisation in the endometrium. - Background Reproductive failure comprises idiopathic recurrent spontaneous abortions (RSA) as well as recurrent implantation failure (RIF) [1]. One of the current RIF definition is a failure to conceive after three transfers of one or more good quality embryos; however, this definition may vary depending on the center [2]. On the other hand, idiopathic RSA is defined as three or more consecutive pregnancy losses within the first 20 weeks of gestation after exclusion of known contributing factors including uterine malformations, bleeding abnormalities, hormone disequilibrium, parental chromosomal defects, infections and others [3]. Adequate implantation is a limiting factor in human reproduction. Despite major improvement in assisted reproduction techniques (ART) the clinical pregnancy rate per embryo transfer in fresh ART cycles is 31%, and up to 41% in oocyte donation programs [4]. A number of etiologies, including decreased embryonic quality, endometrial receptivity, feto-maternal communication, endocrine, genetic and other factors have been suggested as causes for reproductive failure [5-7]. Even though intrauterine endometrium is not essential for embryo implantation, since ectopic pregnancies occur [8], several studies indicated an altered endometrial function in women with reproductive failure [9,10]. Changes at the cellular and molecular level in endometrium from women with reproductive failure have been reported [8,11,12]; suggesting an impact of specific genes on the success of implantation. However, these studies determine mostly gene expression profiles and only partially examine the transcribed products. Although inadequate regulation of endometrial growth has been recognized as a possible factor in reproductive failures [11,13-15], limited data is available concerning endometrial cell proliferation and its regulation. Furthermore, inconsistent results were obtained from studies of the role of endometrial vascularisation, angiogenesis, blood flow and/or endometrial thickness in women with implantation failure; while some studies demonstrate differences between normal and reproductive failures subjects others do not [16-19]. We hypothesized that reproductive failures are due to altered uterine growth caused by changes in cell proliferation and vascularisation/angiogenesis in the endometrium. Therefore, the aim of this study was to examine cellular proliferation and expression of markers of vascularisation (factor VIII and smooth muscle cell actin [SMCA]) at the protein level in endometrial biopsies from the mid-secretory phase of healthy women and women with reproductive failure. Methods In this observational, non-therapeutic pilot study, endometrial biopsies were taken after informed consent according to the Ethical Committee of the University of Heidelberg, Germany. A total of 27 women, at age 25 to 42 years, who were not on any hormonal treatments were included in this study. All women exhibited regular 28 1 day cycles. Pipelle biopsies were taken in the mid-secretory phase on day 8-9 after the LH surge (LH +8/+9) from 11 women with RIF, 9 women with RSA, and 7 healthy woman. RIF was defined by failure to detect a positive serum hCG after three transfers of two or three good quality embryos created through in vitro fertilization. RSA was defined as the appearance of three or more spontaneous abortions of unknown reasons during the first trimester. None of the women with RIF or RSA had ever had a successful term pregnancy. Control women delivered healthy babies at term, with one woman having had a preterm delivery due to premature rupture of membranes. None of the women included in the study had known contributing factors for pregnancy failure, including thrombophilia (Factor V Leiden mutation, prothrombin mutation, antithrombin III, as well as protein C or S deficiency), antiphospholipid syndrome (anticardiolipin antibodies, lupus anticoagulans, ANAs), uterine anomalies detected by ambulatory hysteroscopy, polycystic ovarian syndrome, hormone abnormalities (thyroid, prolactin etc.) and other endocrinopathies (e.g., diabetes). On the day of biopsy blood samples were collected for determination of estradiol-17 (E2) and progesterone concentration in serum using a competitive immunoassay by the University of Heidelberg core facility. The LH surge was determined using a commercially available urine LH kit (Clear blue, WICK PHARMA/Procter & Gamble GMBH, Schwalbach am Taunus, Germany). Furthermore, histology of the biopsies typical for the secretory phase was confirmed by two individual researchers according to the modified Noyes' criteria [20]. Immediately after the biopsies were performed, tissues were immersed in OCT, frozen in liquid nitrogen and stored at -80C. Tissues were then sectioned at 8 m, mounted onto SuperFrost Plus slides (Menzel GmbH & Co, Braunschweig, Germany), fixed with 100% acetone at 4C for 10 min, and stored at -70C before immunohistological procedure. Cell pr (...truncated)