Investigations into inflammation and apoptosis in the ‘perimenstrual’ human endometrium and a mouse model of menstruation
Armstrong, Gregory Martin
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Menstruation is triggered by a fall in circulating progesterone (P4), and to a lesser extent, oestradiol (E2) concentrations, and characterised by classical inflammatory features in the endometrium: breakdown of the basal lamina, tissue oedema and an influx of migratory leucocytes. During and following menstruation, endometrial inflammation is resolved and the endometrium is repaired. The successful resolution of acute inflammation in other tissues involves apoptosis and the phagocytic clearance of apoptotic cells. Human endometrial tissues were collected with informed patient consent and local research ethics committee approval. C57Bl/6 mice underwent an induced menstruation protocol (via sequential E2 and P4 exposure followed by P4 withdrawal), both with and without experimental inhibition of apoptosis (using the pan-caspase inhibitor, Q-VD-OPh). Coordinated apoptosis and neutrophil recruitment were hypothesised to be components of the menstrual event and to precede menstrual shedding in the human endometrium. Immunoreactivity histoscoring for cleaved caspase-3 (CC3) revealed extensive apoptosis in the normal human endometrium early in the ‘perimenstrual’ period, and careful stereological delineation of neutrophil (elastase+) recruitment showed a significant influx coincident with menstrual tissue breakdown. Apoptosis and neutrophil recruitment were hypothesised to follow similar courses in the endometria of mice undergoing an induced menstruation protocol, recapitulating human menstrual events. Immunoreactivity histoscoring for CC3 and stereological investigation into neutrophil (Ly6G+) recruitment in mouse endometrial tissues revealed almost identical extents and timings of apoptosis and neutrophil recruitment in women. Whole genome array evidence of differential apoptosis-related gene transcription in the endometria of women with heavy menstrual bleeding (HMB) compared to those of women with normal menstrual bleeding (NMB) led to the hypothesis that apoptosis may be dysregulated in women with HMB and that perhaps this may delay timely repair of the endometrium and lead to prolonged bleeding in consequence. Candidate differentially-regulated gene transcripts identified by the whole genome array were validated by means of RT-qPCR, although immunoreactivity histoscoring for CC3 did not reveal any differences in apoptosis or its localisation between women with NMB and HMB at the menstrual cycle time-points examined. Building on evidence of apoptotic transcriptional dysregulation in the endometria of women with HMB, it was hypothesised that experimental inhibition of apoptosis (via Q-VD-OPh) in a mouse model of induced menstruation could delay endometrial repair and delay resolution of endometrial inflammation. Some evidence of delayed early repair was obtained, alongside the discoveries of delayed inflammatory gene transcription and increased decidual proliferation (BrdU+) in apoptosis-inhibited mice. Apoptosis precedes the classical inflammatory features of menstruation in the human and mouse endometrium, with inhibition of apoptosis in the latter altering repair and the inflammatory micro-environment. An apoptosis-inhibited mouse model of menstruation may therefore represent a viable model for the further study of heavy menstrual bleeding.