A Preliminary Study Of Endometrial Repair After Injury In Mice

The human endometrium demonstrates a remarkable regenerative capacity throughout the reproductive years of a woman’s life. Menstruation occurs in the absence of pregnancy and follows withdrawal of progesterone and estrogen as the corpus luteum regresses. This withdrawal of steroid hormones, progesterone in particular, leads to a cascade of molecular and cellular interactions that result in shedding of the upper two-thirds of the endometrium (the functional layer) and bleeding. Repair of the endometrium occurs in a cyclical fashion following the tissue destruction of menstruation. Aberrations in this tightly controlled sequence of injury and repair will contribute to menstrual disorders, such as heavy menstrual bleeding (HMB; menorrhagia).Elucidation of the processes involved in endometrial regeneration is essential for the development of novel medical treatment strategies and prevention of unnecessary surgical intervention. Endometrial injury has been the focus of much interest and the subject of recent reviews. However, much less is known regarding the local mechanisms and hormonal control of endometrial repair and regeneration. An increased understanding of endometrial repair will also serve as a paradigm for similar processes in tissues at other sites in the body. This study focuses on the local mechanisms involved in endometrial repair and regeneration, its hormonal control and other areas requiring investigation.Firstly, we investigate the effect of estradiol on endometrial restoration after menstrual-like changes. Pseudopregnant mice were ovariectomized following oil-induced decidualization and then were allocated into two groups randomly. In estradiol group,5ng of17β-E2in arachis oil was injected s.c. at1200, while equivalent arachis oil in solvent group. Morphology, hormone levels and wet weight of uterin were evaluated24h and48H after ovariectomization. The sections of uterin were examined for extent of breakdown and/or repair and analyzed using a scoring system from1-5as previously described. For analyzing the morphological differences between the two groups, both a Kruskal-Wallis test and a repeated measures analysis assuming mouse as a random effect were performed. At48H after ovariectomization, the morphological observation showed that restorations of both the stromal and epithelial components proceed faster in estradiol group. A significant effect of estradiol treatment has been observed in this model during the repair process. These data suggests that the level of estradiol may influence endometrial regeneration after menstrual-like changes. Secondly, we investigate the mechanism of endometrial regeneration using the physiological damage model and mechanical wounding model. Histological observation and reticular fiber staining results show that endometrial repair in the mechanical wounding model is slower than physiological damage model. Some pathologic change, such as stromal edema, could be observed even72H after mechanical injury. Meanwhile, the endometrium after physiological damage macroscopically appeared to be the same as the intact one. Antibodies specific for BrdU then were used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA during endometrial repair. In physiological damage model almost all epithelia were marked by BrdU, however only sporadic positive cells were observed in stroma. In mechanical wounding model, these BrdU positive cells contain epithelia, endothelia, and plenty of stromal cells. Furthermore, we found that the maximum expression of VEGF and the peak values of vascular density appear earlier in the physiological damage mode. So we thought that making preparations for the "expected damage" in physiological damage model makes endometrial regeneration progress faster.Methods of transfection have been developed and advanced during the last fifty years, which can be performed on living organism (in vivo) or cultured mammalian cells (in vitro). Transduction of the female genital tract is a major goal in reproductive biology and medicine. Different approaches have been used to deliver foreign genes into endometrium cells in vitro. On live animals, different experiments showed that the types and percentage of in-vivo transfected cells is very variable. Charnock-Jones et al. first realized uterine epithelium transduction with DNA-liposome mixture injection. From then on, some modified protocols had been reported, most of which were advantaged in the transfection of luminal and glandular epithelia. In addition to epithe;a, endometrial stromal cells have a well-characterized role in female reproduction, such as decidual response, menstruation, and embryonic development. However, efficient gene delivery targeting stromal cells by single injection of LV had not yet been achieved. We thought that it is probably the result of epithelial barrier which is a lining for uterine cavity and protects underlying tissue from viral aggression. This protection would act against the infection of viral vectors and make endometrium-specific gene transfer difficult. Therefore we hypothesized that manipulation in due time, such as menstrual phase, in which functional layer of endometrium sheds and the protective barrier breaks drastically, could act as an elegant solution of this problem. So we incorporated the mouse menstruation model into transgenic procedures. Our results, for the first time, demonstrated the feasibility of high efficient lentivirus-mediated transduction targeting endometrial stromal cells. This new method of in vivo transduction may provide an efficient and easy-to-use tool for studying gene functions in pregnancy or other aspects of reproductive process. On the other hand, it could provide a sound basis for future clinical trials of gene therapy applied to infertility and other pathological disorders.