Regulation of endometrial regeneration; mechanisms contributing to repair and restoration of tissue integrity following menses

Abstract

The human endometrium is a dynamic, multi-cellular tissue that lines the inside of the uterine cavity. During a woman’s reproductive lifespan the endometrium is subjected to cyclical episodes of proliferation, angiogenesis, differentiation/decidualisation, shedding (menstruation), repair and regeneration in response to fluctuating levels of oestrogen and progesterone secreted by the ovaries. The endometrium displays unparalleled, tightly regulated, tissue remodelling resulting in a healed, scar-free tissue following menses or parturition. Mechanisms responsible for initiation of menses have been well documented: following progesterone withdrawal there is an increase in inflammatory mediators, focal hypoxia and induction and activation of matrix-degrading enzymes. In contrast, the molecular and cellular changes responsible for rapid, regulated, tissue repair at a time when oestrogen and progesterone are low are poorly understood. Histological studies using human menstrual phase endometrium have revealed that tissue destruction and shedding occur in close proximity to re-epithelialisation/repair. It has been proposed that re-epithelialisation involves proliferation of glandular epithelial cells in the remaining basal compartment; there is also evidence for a contribution from the underlying stroma. A role for androgens in the regulation of apoptosis of endometrial stromal cells has been proposed but the impact of androgens on tissue repair has not been investigated. Studies using human xenografts and primates have been used to model some aspects of the impact of progesterone withdrawal but simultaneous shedding (menses) and repair have not been modelled in mice; the species of choice for translational biomedical research. In the course of the studies described in this thesis, the following aims have been addressed: 1. To establish a model of menses in the mouse which mimics menses in women, namely; simultaneous breakdown and repair, overt menstruation, immune cell influx, tissue necrosis and re-epithelialisation. 2. To use this model to determine if the stromal cell compartment contributes to endometrial repair. 3. To examine the impact of androgens on the regulation of menses (shedding) and repair. An informative mouse model of endometrial breakdown that was characterised by overt menses, as well as rapid repair, was developed. Immunohistological evidence for extensive tissue remodelling including active angiogenesis, transient hypoxia, epithelial cell-specific proliferation and re-epithelialisation were obtained by examining uterine tissues recovered during an “early window of breakdown and repair” (4 to 24 hours after progesterone withdrawal). Novel data included identification of stromal cells that expressed epithelial cell markers, close to the luminal surface following endometrial shedding, suggesting a role for mesenchymal to epithelial transition (MET) in re-epithelialisation of the endometrium. In support of this idea, array and qRTPCR analyses revealed dynamic changes in expression of mRNAs encoded by genes known to be involved in MET during the window of breakdown and repair. Roles for hypoxia and tissue-resident macrophages in breakdown and tissue remodelling were identified. Treatment of mice with dihydrotestosterone to mimic concentrations of androgens circulated in women at the time of menses had an impact on the timing and duration of endometrial breakdown. Array analysis revealed altered expression of genes implicated in MET and angiogenesis/inflammation highlighting a potential, previously unrecognised role for androgens in regulation of tissue turnover during menstruation. In summary, using a newly refined mouse model new insights were obtained, implicating androgens and stromal MET in restoration of endometrial tissue homeostasis during menstruation. These findings may inform development of new treatments for disorders associated with aberrant repair such as heavy menstrual bleeding and endometriosis.