POSTER VIEWING SESSION - ENDOMETRIOSIS, ENDOMETRIUM, IMPLANTATION AND FALLOPIAN TUBE

Abstract

Introduction: Degradation of basement membrane (BM) is reported to be involved in the metastatic process of cancers. BM components, collagen four (COLIV) and laminin, have also been shown to be aberrantly expressed in the benign metastatic disease of endometriosis. Cytokeratin 5/6 (Ck5/6) and podocalyxin-like protein (PODXL) have been proposed as markers of stem cells. We aim to investigate if an alteration of BM integrity is associated with differential expression of PODXL and Ck5/6, in endometrium collected from endometriosis patients. Material and Methods: Eutopic endometrium was collected from 16 patients with endometriosis; n = 8 in the window of implantation (WOI, day 21 {+/-} 2), and n = 8 in the late secretory phase (LSP, day 26 {+/-} 2) when remodelling of BM components is known to peak. The inclusion criteria for this group were: (1) reproductive women with regular menstrual cycles; (2) surgically diagnosed active peritoneal endometriosis; and (3) history of dysmenorrhoea and/or chronic pelvic pain. Exclusion criteria included hormonal treatment in the previous three months. Samples were also obtained from 18 normal fertile controls; n = 10 during the WOI and n = 8 during the LSP. These women had surgically excluded endometriosis and at least one successful pregnancy. In each specimen the expression of COLIV, laminin, PODXL and CK5/6 were studied using immunohistochemistry. The thickness and staining intensity of immuno-located BM components supporting glandular and luminal epithelium was separately measured with a computer-assisted image analysis system and a semi-quantitative scoring system. A previously reported semi-quantitative, modified "quickscore" method which consolidates both staining intensity and the percentage coverage in glandular secretions (Schiessl et al. Placenta 2009) was used to analyse PODXL immuno-staining in at least 10 glands per section. Additionally, a semi-quantitative scoring system was used to evaluate PODXL staining intensity in vascular endothelium and Ck5/6 staining intensity in luminal epithelium. Furthermore, the number of positive glands in a low power field for each section was counted to compare the CK5/6 staining pattern. Non-parametric Mann-Whitney test was used to compare the non-related endometriosis and fertile control samples. Results: In normal fertile women, the thickness and staining intensity for both COLIV (p < 0.0001 and p < 0.0001 respectively) and laminin (p < 0.0001 and p = 0.004 respectively) in the glandular BM increased towards the LSP. Conversely, PODXL staining in glandular secretions decreased to virtually absent staining in the LSP (p = 0.001). Eutopic endometrium from endometriosis patients did not show a similar thickening of COLIV in the glandular BM in the LSP. Instead, there was a significant reduction in the intensity of COLIV in glandular BM (p = 0.01) and decrease in laminin staining [thickness (p < 0.03) and intensity (p = 01)] in the endothelial BM in the LSP when compared with the WOI. Furthermore, when compared with normal fertile controls, endometrium from endometriosis patients showed thinner BM (WOI p = 0.04 for laminin; LSP p = 0.01 for COLIV) and a significantly decreased staining intensity (WOI p = 0.01 for COLIV; LSP p = 0.03 for COLIV). The glandular PODXL quickscores showed a negative correlation with COLIV thickness (r = -.428, p = 0.01). We could not identify a significant difference in the staining for PODXL or Ck5/6 between endometriosis and fertile control groups. Conclusion: In fertile women there is an increase in BM components at the end of the secretory phase and this is altered with endometriosis. This may suggest that the disruption of the endometrium at menstrual shedding is more extensive in women with endometriosis and this may contribute to the pathogenesis. The increase in PODXL expression at WOI is likely to reflect the involvement of this protein possibly in the implantation process. The negative correlation between PODXL and COLIV stained BM thickness may suggest that disruption of BM may up-regulate this stem cell marker in the endometrial epithelium. Introduction: Urocortin 1 (UCN1), member of the corticotropin-releasing hormone family, is a neuropeptide produced by the human endometrium, and also important for promoting implantation. Human urocortin was found to bind with high afinity to corticotropin-releasing factor (CRF) receptors. The activation of CRF1 receptor pathway stimulates the growth of human endometrial cells and the decidualization of human endometrium. Moreover, it also promotes blastocyst implantation and early maternal tolerance primarily by killing activated T cells. Initial dose-finding studies for antagonist used during IVF have suggested that increasing antagonist doses may have negative impact on the implantation rate. The aim of the study is to determine plasma UCN1 levels in women who is in long agonist cycle and antagonist cycle during IVF. Material and Methods: Sixty women with infertility and an indication for IVF were randomly assigned to either an IVF treatment with antagonist protocol (n = 30) or that with a long IVF protocol (n = 30), during which the agonist has been started midluteally. Plasma urocortin 1 level was measured by ELISA in all women just before the OPU procedure. Serum samples from each subject were assayed for Urocortin 1 by a commercially available enzyme-linked immunosorbent assay (ELISA Kit for Human Urocortin 1, Uscn Life Science Inc. Wuhan, China). The ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of human UCN1 in serum. The minimum detectable dose of human UCN1 is typically less than 1.2 pg/mL. This assay has high sensitivity and excellent specificity for detection of human UCN1. No significant cross-reactivity or interference was observed. Results: There were no differences between agonist and antagonist groups with respect to age. UCN1 concentrations were significantly higher in agonist group than in those antagonist group (146.21 {+/-} 49.11 pg/ml vs 79.10 {+/-} 41.59 pg/ml; P = 0.001). There were no significant difference with respect to endometrial thickness between agonist and antagonist groups (8.93 {+/-} 0.18 mm vs 8.43 {+/-} 0.13; P = 0.818 mm). Metaphase II oocyte number after OPU were similar in each group (5.77 {+/-} 0.24 vs 5.56 {+/-} 0.19; P = 0.507). After embryo transfer, 21 patients in agonist group and 18 patients in antagonist group became pregnant. In agonist group UCN1 concentrations were significantly higher in women who became pregnant than in those who did not (228.57 {+/-} 38.6 pg/ml vs 2.07 {+/-} 4.68 pg/ml; P = 0.001). Similarly in antagonist group UCN1 concentrations were significantly higher in women who became pregnant than in those who did not (182.93 {+/-} 38.9 pg/ml vs 3.50 {+/-} 2.67 pg/ml; P = 0.001). UCN1 concentration was significantly correlated with endometrial thickness in agonist (r = 0,823; P = 0.000) and antagonist groups (r = 0.490; P = 0.001). In agonist and antagonist groups UCN1 concentrations were significantly correlated with metaphase II oocyte number (r = 0.834; P < 0.000, r = 0.588; P < 0.000). Conclusions: UCN1 concentrations were significantly higher in the agonist cycle than the antagonist cycle and women who subsequently achived pregnancy had higher UCN1 concentrations before IVF. Introduction: UDP-glucose is released from damaged or dying cells via lytic and nonlytic pathways. P2RY14, a G protein-coupled P2Y purinergic receptor for UDP-glucose, plays a role in the regulation of various immune responses including chemokine production. We have previously reported that P2RY14 is exclusively localized in human endometrial luminal and glandular epithelial cells (EEC) and that P2RY14 in EEC may behave as a novel danger sensor for UDP-glucose released by damaged or dying cells, thereby activating P2RY14 to stimulate IL-8 production and the recruitment of neutrophils and other immune cells into the damaged endometrium. Although the expression levels of P2RY14 in EEC are constant during the menstrual cycle, the exclusive localization of P2RY14 in EEC and a possible EEC damage induced by an implanted embryo prompted us to investigate whether UDP-glucose and its receptor P2RY14 in EEC are involved in embryo implantation. Material and Methods: We employed an in vitro implantation model in which spheroids prepared from a JAR choriocarcinoma cell line and Ishikawa cells, a human endometrial epithelial cell line, were co-incubated to allow for implantation of JAR spheroids onto the monolayer of Ishikawa cells. To distinguish between Ishikawa cells (EEC model) and JAR spheroids (embryo model) latters were pre-coated by a lipophilic-dye, followed to perform and analyzed by immunofluorescence and confocal microscopy. To determine the role of UDP-glucose and P2RY14 in implantation, Ishikawa cells were pretreated with and without P2RY14 siRNA or different concentrations of UDP-glucose and subjected to the in vitro implantation assay followed by counting the number of JAR spheroids adhered to Ishikawa cells. Furthermore, we examined the expression of P2RY14 in the co-culture of Ishikawa cells and JAR spheroids using confocal microscopy. Results: In vitro implantation assay revealed that JAR spheroids attached and adhered to Ishikawa cells in a UDP-glucose-dose dependent manner. Pretreatment of Ishikawa cells with P2RY14 siRNA resulted in a significant decrease in the number of adhered JAR spheroids as compared to control siRNA. Ishikawa cells closely adjacent to an implanted JAR spheroid exhibited the most prominent expression of P2RY14 as compared to the other areas more distant from the implanted site, as determined by confocal microscopy. Conclusions: The results of this study suggest that UDP-glucose and P2RY14 may participate in the process of embryo implantation. Several studies have implicated apoptosis of EEC in embryonic implantation. Hence, we here propose a possi