{"paper_id":"7ae65d2a-fd20-42e6-ba78-3e306ab87281","body_text":"Regulation of endometrial repair and its impact on heavy menstrual bleeding\nItem Status\nEmbargo End Date\nDate\nAuthors\nMaybin, Jacqueline Ann\nAbstract\nINTRODUCTION: The human endometrium has a remarkable capacity for efficient\ncyclical repair following the inflammatory process of menstruation. Defective postmenstrual\nrepair may contribute to the common complaint of heavy menstrual\nbleeding (HMB). The mechanisms and factors involved in endometrial repair are still\nto be fully elucidated. Endometrial function is governed by the ovarian hormones and\npre-menstrually progesterone levels decline as the corpus luteum regresses.\nConsequently, the synthesis of prostaglandins (PG) is increased, namely PGE2 and\nthe potent vasoconstrictor PGF2α. Subsequent vasoconstriction of endometrial spiral\narterioles is believed to result in a transient hypoxic episode in the upper endometrial\nlayer.\nTherefore, the aims of this thesis were to determine (i) the endometrial expression of\nputative repair factors across the menstrual cycle (ii) the regulation of these factors\nby hypoxia, PGE2 and PGF2α (ii) the role of hypoxia inducible factor (HIF)-1α in\nendometrial repair and (iii) differences in endometrium from women with objectively\nmeasured HMB (>80ml) and normal controls (<80ml).\nMETHODS/RESULTS:\nPutative repair factors, with known angiogenic, mitogenic and\nproliferative functions, were identified in human endometrial samples by quantitative\nreverse transcription PCR and immunohistochemistry. Interleulin-8 (IL-8), vascular\nendothelial growth factor (VEGF), adrenomedullin (AM), connective tissue growth\nfactor (CTGF) and endothelin-1 (ET-1) were all maximally expressed during the\nmenstrual and/or proliferative phases of the cycle, consistent with the onset of\nendometrial repair. Endometrial cells and tissue explants treated with 100nM\nPGE2/F2α and/or hypoxia (0.5% O2) revealed up-regulation of IL-8, VEGF, AM and\nCTGF. An in vitro progesterone antagonism model revealed that progesterone\nwithdrawal, hypoxia and prostaglandins are all necessary for significant increases in\nrepair factor expression in endometrial tissue. HIF-1α was detected in human\nendometrium but exclusively in the late-secretory and menstrual phases. Using shorthairpin\nRNA against HIF-1α, it was determined that hypoxia up-regulated these\nfactors via HIF-1α, whereas PGF2α acted in a HIF-1α independent manner to increase repair factor expression. Finally, whole genome array analysis was performed on\nmenstrual endometrium from women with objectively measured heavy and normal\nmenstrual bleeding to provide an unbiased comparison of gene expression. 259\ntranscripts displayed significant changes between the two groups. Five candidate\ngenes were validated using Q-RT-PCR. Bioinformatic analysis of the differentially\nexpressed gene set identified bioprocesses that included positive regulation of\nbiological and cellular processes, leukocyte differentiation, regulation of apoptosis\nand response to stress/hypoxia. The presence of HIF-1α protein was examined in\nmenstrual endometrial tissue nuclear protein extracts by Western blot, revealing\nsignificantly decreased levels in women with HMB versus normal controls.\nFurthermore, the mRNA expression of known target genes of HIF-1α (VEGF,\nCXCR4) was also significantly decreased in these women. The functional impact of\nendometrial HIF-1α was assessed using an in vitro angiogenic assay. Silencing of\nHIF-1α in endometrial cells significantly reduced the angiogenic potential of culture\nsupernatants when compared to untransfected cells or cells transfected with a\nscrambled sequence.\nCONCLUSIONS: Repair factors are significantly increased in the human endometrium\nfollowing the onset of menstruation. Progesterone withdrawal, hypoxia via HIF-1α\nand prostaglandins appear necessary for the regulation of these factors at this time.\nMenstrual endometrium displays significant differences in gene expression and HIF-\n1α protein levels between women with HMB and normal controls. The findings of\nthis thesis contribute to the existing literature on both the physiological process of\nendometrial repair and the pathogenesis of HMB. Extension of this work may allow\nthe identification of novel therapeutic targets for the treatment of this common,\ndebilitating condition.\nThis item appears in the following Collection(s)","source_license":"CC0","license_restricted":false}