Cell-Free Fat Extract Prevents Vaginal Atrophy in an Ovariectomized Model by Promoting Proliferation of Vaginal Keratinocytes and Neovascularization

Background: Most perimenopausal and postmenopausal women experience estrogen deficiency-induced vaginal atrophy. However, estrogen replacement therapy has contraindications and side effects, which makes it unsuitable for most women. Cell-free fat extract (CEFFE) has pro-proliferative and proangiogenic tissue regeneration activities. Objectives: The purpose of this study was to evaluate the effect of topical application of CEFFE in the vagina and the effect of CEFFE on vaginal keratinocytes. Methods: Ovariectomized mice were treated with CEFFE via vaginal topical application for 2 weeks. The vaginal mucosal cell layer number, mucosal thickness, and vaginal collagen volume were determined by histologic analyses. Vaginal mucosa proliferation and lamina propria angiogenesis were evaluated with anti-proliferating cell nuclear antigen and anti-CD31 staining, respectively. For in vitro analysis, VK2/E6E7 cells were administered, increasing the CEFFE concentration. Cell proliferation and cell-cycle distribution were analyzed by Cell Counting Kit 8 assay and flow cytometry, respectively. Mucosal migration was evaluated with a wound-healing assay. The expression of Ki-67 and estrogen-related proteins was detected by western blotting. Results: CEFFE-treated mice showed increased mucosal thickness and number of vaginal mucosal cell layers and reduced vaginal atrophy compared to ovariectomized mice. The number of proliferating cell nuclear antigen-positive cells and CD31-positive capillaries also increased. In addition, CEFFE promoted the proliferation and migration of VK2/E6E7 cells, upregulated the expression of Ki-67, and inhibited the expression of estrogen-related proteins and the PI3K/AKT pathway. Conclusions: CEFFE prevents estrogen deficiency-induced vaginal atrophy by promoting vaginal mucosal proliferation and increasing neovascularization, but not through the estrogen/estrogen receptor pathway, in an ovariectomized mouse model.