(253) Chemotherapy and Ovariectomy Impact Epithelial Cell Structure in the Vagina and Induce an Enrichment of Basal Cells with Stem-like Activity

Julia L. Balough^1,2,3; Meena Sukhwani^2,3; Pamela A. Moalli^2,3; Kyle E. Orwig^1,2,3

1. Molecular Genetics and Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh PA, United States.
2.    Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, United States.
3.   Magee-Women’s Research Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, United States.

Abstract Text:

Vaginal function is integral to a woman’s sexual function and quality of life. The vaginal epithelium remodels by expanding and shrinking during the course of the menstrual cycle (human) or estrous cycle (mice). With ovariectomy, basal epithelial cells are maintained while differentiated epithelial cells are lost but can be recovered if exogenous estrogen is added which suggests a population of vaginal cells exist that self-renew and differentiate given the environment. We hypothesize that chemotherapy, which accelerates ovarian aging by destroying estrogen-producing follicles, will lead to vaginal dysfunction and atrophy similar to ovariectomy. C57BL/6 mice (6 weeks old; n=60) were divided between four treatment groups (n=15/group): ovariectomy (OVX)+chemotherapy (Chemo); OVX only; Chemo only; Control (no chemo or OVX). After sham or OVX surgery, mice were injected with busulfan (24 mg/kg) and cyclophosphamide (200 mg/kg) or vehicle (DMSO+water). Reproductive endpoints were measured at 7, 30, and 90 days post-injection. OVX, regardless of Chemo, induced irregular cycling with diestrus contributing to 74-100% of the estrous stages observed 7, 30 and 90 days after injection. Control animals maintained regular cycling with diestrus contributing to 4-25% of the cycle. Chemo-only animals maintained regular cycling with no difference to control animals at 7 and 30 days post-injection but were irregular with diestrus contributing to 70% of the stages at day 90. This phenotype aligns with loss of estrogen as Chemo significantly reduced the number of ovarian follicles 7 (1.57±0.29) and 30 days (0.04±0.07) with no follicles present at 90 days (0.00) after injection compared to control (5.13±1.64 follicles/mm²). Similarly, the weight of the reproductive tract per body weight was significantly reduced after OVX (2.93±1.14) compared to ovary-intact (6.98±0.58 mg/g) animals at all timepoints. However, Chemo significantly decreased the reproductive tract weight at 30 days (5.05±0.68) post-injection compared to control animals (7.48±1.66) that was not present at day 7 (6.96±1.56) and 90 (7.06±1.38 mg/g). When all samples were collected in diestrus, the thickness of the vaginal epithelium was significantly thinned after Chemo at day 7 (61.6±19.9) and 30 (47.8±9.23) but was significantly thicker at day 90 (96.7±15.3) compared to control (76.0±23.4 μm). OVX animals, regardless of Chemo, had significantly thinned epithelium (24.2±9.62) at all timepoints compared to ovary-intact animals (72.3±18.9 μm). Using immunohistochemistry, we observed the density of differentiating epithelial cells, marked by Cadherin-13, was significantly reduced by OVX at all timepoints (0.03±0.04) and Chemo at day 30 (0.19±0.07) compared to control (0.28±0.11 cells/mm). In addition, there was increased cell proliferation, marked by Ki67, in the basal epithelium after Chemo at day 7 (0.06±0.03) that was lost at day 30 (0.03±0.01) and 90 (0.03±0.02) compared to control (0.04±0.03 cells/mm). Finally, we observed an increase in Nerve Growth Factor Receptor, a marker of epithelial stem cells, in the basal epithelium with OVX and Chemo (0.11±0.05) that was maintained at all timepoints compared to control (0.06±0.05 cells/mm). Taken together we found OVX and Chemo do not induce equivalent effects on reproductive tract weight, vaginal epithelium thickness and proliferation suggesting estrogen may not be the only factor impacting differentiation of the vaginal epithelium. OVX and Chemo induce irregular cycling and an enrichment of a stem-like basal cells. Work is ongoing to characterize the serum and transcriptome of the basal epithelial cells under these conditions. This work is supported by the Magee-Womens Research Institute and the Richard King Mellon Foundation.