AbstractThe softgel, vaginal 17β-estradiol (E2) insert (TX-004HR) significantly improved the maturation index of the vaginal epithelium, dyspareunia and vaginal dryness in menopausal women with vulvar and vaginal atrophy (VVA) and moderate to severe dyspareunia, without histological changes to the endometrium (Constantine G et al, Menopause 2017;24:409-416). The 4-μg and 10-μg E2 doses were FDA approved as Imvexxy® (TherapeuticsMD, Boca Raton, FL), for the treatment of moderate to severe dyspareunia, a symptom of VVA, due to menopause. The progesterone receptor (PR) is an estrogen-regulated gene with expression that is very sensitive to E2 exposure (Xiao CW and Goff AK, J Reprod Fertil.1999;115:101-109). Endometrial PR expression in the biopsies of women using the softgel vaginal 4-µg E2 insert was used as a marker of E2 exposure to determine whether sufficient E2 applied with the vaginal insert reaches the endometrium to upregulate PR expression. Our hypothesis posits that there would be insufficient E2 from the 4-µg E2 insert to stimulate an increase in endometrial PR expression.In this post hoc analysis of the REJOICE trial, endometrial biopsies from 25 women who had a normal baseline biopsy, an on-therapy biopsy after study day 70, and tissue readings from all pathologists were randomly selected from the 4-µg E2 vaginal insert and placebo groups. Endometrial tissue sections were stained to visualize PR expression using PgR1294 (Agilent; Santa Clara, CA). Cell staining was quantified using a trainable feature-recognition algorithm and mean expression levels between baseline and week 12 were analyzed by 2-sided t-tests.Acceptable PR expression results were available for 22 women in the 4-µg E2 group (three had insufficient tissue for analysis) and 25 women in the placebo group. For the 4-µg E2 group, mean ± SD (pmol/mg) PR expression was 0.455 ± 0.203 at baseline and 0.506 ± 0.226 at week 12. For the placebo group, mean PR expression was 0.579 ± 0.196 at baseline and 0.563 ± 0.213 at week 12. Mean PR expression levels at baseline and week 12 were not significantly different from each other within the 4-µg E2 (P=0.438) or placebo (P=0.783) group.No meaningful difference in endometrial PR expression was observed with the vaginal 4-µg E2 insert at week 12. These data support our hypothesis and the assertion that low-dose, local vaginal E2 exposure with the insert placed in the lower part of the vagina, does not pose an endometrial safety issue in postmenopausal women.