ABSTRACT: Transgender men (TGM) (persons who identify as male but assigned female sex at birth) often forgo gender- affirming surgery and retain their natal genitalia (i.e. vaginas). However, many TGM initiate masculinizing testosterone therapy. Much like estrogen-deficient cisgender women (CGW), TGM on testosterone experience decreased thickness of the vaginal epithelium and loss of menses. The vaginal microbiota of post-menopausal CGW may shift toward a vaginal dysbiosis due to loss of lactobacilli. Unlike post-menopausal CGW, TGM on testosterone also experience elevated testosterone levels. The impact of low estrogen/high testosterone on the vaginal microbiota of TGM is unknown and cannot be extrapolated from studies of CGW. Hormonal changes in conjunction with sexual behaviors due to increased sexual desire from use of testosterone could impact the vaginal microbiota of TGM, putting them at risk for bacterial vaginosis (BV) and, ultimately, HIV and other STIs. Data are limited regarding the composition of the vaginal microbiota after initiation of testosterone in TGM. A cross-sectional study comparing TGM on testosterone for ≥1 year to CGW found that the vaginal microbiota of 71% of TGM on testosterone was less likely to be lactobacillus-dominated and more likely to be enriched with >30 other bacterial species, many associated with BV. This study did not compare the vaginal microbiota of TGM pre- and post-testosterone initiation nor identify shifts in the microbiota that could precede vaginal dysbiosis or incident BV (iBV). We hypothesize that testosterone initiation will alter the composition of the vaginal microbiota in TGM. In the setting of increased sexual desire associated with testosterone and participation in sexual behaviors, iBV could be elicited by sexual transmission of BV-associated bacteria (BVAB). Our long-term goal is to identify ways to optimize the health benefits of testosterone in TGM while mitigating its potential risks. Aim 1. To investigate changes in the composition of the vaginal microbiota over time in TGM initiating testosterone treatment. We will obtain daily vaginal specimens and daily diaries from 40 TGM with baseline normal vaginal microbiota (no Amsel criteria, Nugent score 0-3), starting 7 days prior to testosterone initiation and 90 days after. 16S rRNA V4 sequencing will be performed on all 7 pre-testosterone specimens and weekly specimens thereafter to determine vaginal microbial community state type (CST) compositions over time. In TGM who experience a shift to vaginal dysbiosis (Nugent score ≥4 on at least 2 consecutive days), 16S sequencing will be performed on specimens starting 7 days pre-shift through 3 days post-shift for intensive sampling. Aim 2. To identify changes in the vaginal microbiota that precede iBV in TGM on testosterone. Vaginal specimens from 5 TGM in Aim 1 who develop iBV (i.e. Nugent score ≥7 for at least 2 consecutive days) (cases) and 5 controls who do not develop iBV will be identified. Daily vaginal specimens from 14 days prior to iBV, including the day of iBV, in cases and 14 days of vaginal specimens from controls (aligned with cases based on the day of testosterone initiation) will be characterized using shotgun metagenomics.

PROJECT NARRATIVE: Cross-sectional data comparing the vaginal microbiota of transgender men (TGM) on testosterone to cis-gender women (CGW) found that testosterone could have detrimental effects, including loss of healthy vaginal lactobacilli and an increased abundance of diverse bacteria, many associated with bacterial vaginosis (BV). The influence of testosterone on the vaginal microbiota paired with an increase in sexual desire from testosterone may put TGM at risk for vaginal dysbiosis and ultimately BV. The proposed studies in this R21 application will investigate changes in the composition of the vaginal microbiota in TGM after testosterone initiation.