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Background: The study aimed to determine the effect of menopausal status and hormone therapy on the introitus and labia majora at the levels of histology and gene expression., Methods: Three cohorts of 10 women each (pre-menopause, post-menopause and post-menopause + hormone therapy) were selected based on the presentation of clinical atrophy and vaginal pH. Biopsies were obtained from the introitus (fourchette) and labia majora and processed for histology and gene expression analyses with microarrays. Other data collected included self-assessed symptoms, serum estradiol, testosterone, serum hormone binding globulin and the pH of the vagina and labia majora., Results: The introitus appears exquisitely sensitive to hormone status. Dramatic changes were observed in histology including a thinning of the epithelium in post-menopausal subjects with vaginal atrophy. Furthermore, there was differential expression of many genes that may contribute to tissue remodeling in the atrophic introitus. Levels of expression of genes associated with wound healing, angiogenesis, cell migration/locomotion, dermal structure, apoptosis, inflammation, epithelial cell differentiation, fatty acid, carbohydrate and steroid metabolism were significantly different in the cohort exhibiting atrophy of the introitus. While changes were also observed at the labia, that site was considerably less sensitive to hormone status. The gene expression changes observed at the introitus in this study were very similar to those reported previously in the atrophic vagina providing further evidence that these changes are associated with atrophy., Conclusions: The histological and gene expression changes occurring within the introitus after menopause may contribute to the constellation of symptoms that constitute the genitourinary syndrome of menopause., Competing Interests: MA Freedman and KB Sharma are consultants for The Procter & Gamble Company and received funding from Procter & Gamble for this study. RL Binder, MA Farage, Y Wang, C Combs, D Moore, JP Tiesman, CC Bascom, RJ Isfort and R Warren are current or former employees of Procter & Gamble., (Copyright 2019, Binder et al.)