MRC Grant 0600048

Introduction: The human endometrium has a remarkable capacity for efficient cyclical repair following the inflammatory process of menstruation. Defective postmenstrual repair may contribute to the common complaint of heavy menstrual bleeding (HMB). The mechanisms and factors involved in endometrial repair are still to be fully elucidated. Endometrial function is governed by the ovarian hormones and pre-menstrually progesterone levels decline as the corpus luteum regresses. Consequently, the synthesis of prostaglandins (PG) is increased, namely PGE2 and the potent vasoconstrictor PGF2α. Subsequent vasoconstriction of endometrial spiral arterioles is believed to result in a transient hypoxic episode in the upper endometrial layer. Therefore, the aims of this thesis were to determine (i) the endometrial expression of putative repair factors across the menstrual cycle (ii) the regulation of these factors by hypoxia, PGE2 and PGF2α (ii) the role of hypoxia inducible factor (HIF)-1α in endometrial repair and (iii) differences in endometrium from women with objectively measured HMB (>80ml) and normal controls (<80ml). Methods/Results: Putative repair factors, with known angiogenic, mitogenic and proliferative functions, were identified in human endometrial samples by quantitative reverse transcription PCR and immunohistochemistry. Interleulin-8 (IL-8), vascular endothelial growth factor (VEGF), adrenomedullin (AM), connective tissue growth factor (CTGF) and endothelin-1 (ET-1) were all maximally expressed during the menstrual and/or proliferative phases of the cycle, consistent with the onset of endometrial repair. Endometrial cells and tissue explants treated with 100nM PGE2/F2α and/or hypoxia (0.5% O2) revealed up-regulation of IL-8, VEGF, AM and CTGF. An in vitro progesterone antagonism model revealed that progesterone withdrawal, hypoxia and prostaglandins are all necessary for significant increases in repair factor expression in endometrial tissue. HIF-1α was detected in human endometrium but exclusively in the late-secretory and menstrual phases. Using shorthairpin RNA against HIF-1α, it was determined that hypoxia up-regulated these factors 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 menstrual endometrium from women with objectively measured heavy and normal menstrual bleeding to provide an unbiased comparison of gene expression. 259 transcripts displayed significant changes between the two groups. Five candidate genes were validated using Q-RT-PCR. Bioinformatic analysis of the differentially expressed gene set identified bioprocesses that included positive regulation of biological and cellular processes, leukocyte differentiation, regulation of apoptosis and response to stress/hypoxia. The presence of HIF-1α protein was examined in menstrual endometrial tissue nuclear protein extracts by Western blot, revealing significantly decreased levels in women with HMB versus normal controls. Furthermore, the mRNA expression of known target genes of HIF-1α (VEGF, CXCR4) was also significantly decreased in these women. The functional impact of endometrial HIF-1α was assessed using an in vitro angiogenic assay. Silencing of HIF-1α in endometrial cells significantly reduced the angiogenic potential of culture supernatants when compared to untransfected cells or cells transfected with a scrambled sequence. Conclusions: Repair factors are significantly increased in the human endometrium following the onset of menstruation. Progesterone withdrawal, hypoxia via HIF-1α and prostaglandins appear necessary for the regulation of these factors at this time. Menstrual endometrium displays significant differences in gene expression and HIF- 1α protein levels between women with HMB and normal controls. The findings of this thesis contribute to the existing literature on both the physiological process of endometrial repair and the pathogenesis of HMB. Extension of this work may allow the identification of novel therapeutic targets for the treatment of this common, debilitating condition.