PROJECT SUMMARY/ABSTRACT Over one million people in the United States identify as transgender or gender nonconforming (TGNC), meaning their personal identity and gender do not correspond with their assigned sex at birth. Transmasculine individuals were assigned female at birth and often seek treatment with injectable testosterone for gender affirming hormone therapy. Transmasculine individuals are at high risk for sexually transmitted infections (STIs); at least a third have intercourse with cisgender men. We hypothesize that initiation of testosterone leads to vaginal atrophy and is associated with vaginitis symptoms, inflammation, and increases in vaginal bacteria associated with STI acquisition. We propose to enroll a cohort of 30 transmasculine individuals prior to gender-affirming hormone therapy. We will collect vaginal swabs and vaginal biopsies at baseline and after 3 months of injectable testosterone use. Using daily electronic dairies, we will track vaginal symptoms including dyspareunia, dryness, and discharge, sexual behaviors, and bleeding patterns. Our first aim is to assess vaginal inflammation and atrophy after testosterone initiation and identify associations with vaginitis symptoms and sexual behaviors. To assess inflammation, we will quantify cytokine (IL-1α, IL-1β, IL-10) and chemokine (MIP-1β, IP-10/CXCL10, IL- 8) concentrations in vaginal swabs, as these markers have been associated with HIV acquisition in prospective cohort studies. We will assess vaginal atrophy utilizing histological analysis of vaginal biopsies, specifically measuring epithelial thickness and glycogen content. We will correlate these outcomes with vaginitis symptoms and sexual behaviors recorded in daily diaries. As an exploratory outcome, we will assess gene expression in biopsies using RNA sequencing. The second aim is to assess changes in the vaginal microbiome. We will perform broad-range 16S rRNA gene PCR with high throughput sequencing to assess vaginal bacterial census, richness and community diversity on vaginal swabs. This project will address fundamental gaps in our understanding of how testosterone affects vaginal health and immunity, and identify key outcomes and confounders critical to designing future clinical trials assessing hormone therapy, HIV pre-exposure prophylaxis and vaginal drugs in this population. This work will be the first step towards identifying safer gender-affirming hormone regimens that will improve mucosal immunity, reduce vaginal symptoms, and optimize sexual health. We have substantial community engagement and experience recruiting transmasculine individuals into research studies and assessing reproductive health outcomes. Our proposal combines synergistic expertise in key areas including hormonal medications, gender affirming therapy, vaginal immunity, and the microbiome.

PROJECT NARRATIVE Transgender and gender nonconforming individuals using testosterone for gender affirming therapy are at high risk for sexually transmitted infections (STIs), yet the effects of testosterone on vaginal immunity are unknown. We will enroll a cohort of 30 transmasculine individuals prior to testosterone initiation and follow them for 3 months to assess changes in vaginal cytokines and chemokines, vaginal histology, and the microbiome. We aim to assess how testosterone affects vaginal health, and identify key outcomes and confounders critical to designing future clinical trials assessing hormone therapy, HIV pre-exposure prophylaxis, and vaginal drugs in this population.