The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair.

The current dogma surrounding endometrial regeneration after menses includes a critical need for estrogen-primed proliferation. Although some evidence suggests that estrogen may not be required for the initial reepithelialization of the uterine surface, it is widely believed that it is essential for successful stromal renewal. This study aimed to identify proliferating cell types during endometrial repair and to examine whether estrogen is required for successful repair using a previously developed mouse model. In the model, decidualization is artificially induced, and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and by 48 h has usually undergone complete repair. Although the mice are ovariectomized, restoration of both the stromal and epithelial components proceeds rapidly after breakdown and results in what appears to be a normal endometrium. However, potential estrogenic influences from extraovarian sources (particularly the diet and fat) remain. In this study, complete removal of extraovarian estrogen was achieved by maintenance of animals on a soy-free diet and administration of aromatase inhibitor letrozole. No significant differences in uterine weight or estrogen-responsive genes lactoferrin and progesterone receptor were observed compared with control ovariectomized but otherwise untreated mice, whereas significantly higher measurements were obtained from an estrogen-added group. Importantly, no significant difference in the rate of endometrial repair was observed in the complete absence of estrogen, demonstrating that estrogen is not essential for complete endometrial restoration in this model.

Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.

Breakthrough bleeding (BTB), a major side effect of long-acting progestogen (p)-only contraceptives in women, is the main reason for discontinuation of their use. To understand the mechanisms of BTB, a mouse model of endometrial breakdown and repair was adapted to evaluate the effects of long-term progestogens on the endometrium. Appropriately prepared mice received either an etonogestrel (ENG)-or levonorgestrel (LNG)-releasing subdermal implant. Forty eight hours after decidualization was induced in one uterine horn the majority of tissues were highly decidualized, designated 0 day (0d). Uteri were collected subsequently at 5-day intervals (to 45d) and both decidualized and non-decidualized horns were analysed for morphological changes, leukocyte infiltration and matrix metalloproteinase expression (MMP). In decidualized horns, large blood vessels (BV) developed and disturbance of tissue integrity was observed at 5d with substantial stromal breakdown by 10d, progressing until 25d when re-epithelialization was initiated. By 45d, the tissue was restored to its pre-decidualized state but with considerable tortuosity of the luminal epithelium. Tissue remodelling was not apparent in the non-decidualized horns before 35d, when hyperproliferation of the luminal epithelium resulted in tortuosity. Changes in morphology were similar with the two progestogens, but occurred more rapidly with LNG. Apart from macrophages, few leukocytes were present in non-decidualized horns but large infiltrates of neutrophils and uterine natural killer cells (uNK) were associated with tissue breakdown in decidualized tissue, many of these cells were MMP9-positive. MMP7 was primarily associated with tissue repair. Therefore, this model mimics some of the changes observed in endometria of women using p-only contraceptives and provides an opportunity for functional studies.