PROJECT SUMMARY Gender affirming hormone therapy (GAHT) is used by transgender (TG) people to alleviate gender dysphoria. GAHT for male to female TG subjects (transwomen) consists of a gonadotropin-releasing hormone agonist (GnRHa) to block testosterone production and cross-sex hormone treatment (CSHT) with estrogen. GAHT for female to male TG subjects (transmen) is based on testosterone CSHT without a GnRHa. CSHT is often started at 12-16 years of age, before the pubertal surge in bone mass and the completion of skeletal maturation. In addition, male and female adolescents with gender dysphoria are sometime treated temporarily with a GnRHa without CSHT to suppress puberty. The effects of puberty blockade followed by CSHT and those of CSHT without prior puberty blockade on skeletal maturation are mostly unknown. The gut microbiome is pivotal regulator of skeleton postnatal maturation, bone health, and bone responsiveness to GnRHa and sex steroids. Moreover, the composition of the microbiome is regulated by sex steroids. Thus, modifications to the gut microbiome composition may mediate the effects of GnRHa and CSHT on the skeleton. Supporting this hypothesis, our preliminary metagenomic analysis revealed that CSHT induced differences in the composition of the gut microbiome. Preliminary studies also showed that CSHT impacts gut permeability, which can lead to further changes in the gut microbiome composition. We further show that CSHT-induced modifications to the composition of the gut microbiome alters indices of bone volume and structure, and the frequency of intestinal and bone marrow (BM) T regulatory cells (Tregs), which is a T cell lineage expanded by estrogen and testosterone. Tregs are essential in the regulation of bone formation and bone resorption. Based on our preliminary data and on published reports, we hypothesize that GnRHa treatment and CSHT affect skeletal maturation, and that these effects are mediated, in part, by modifications in gut microbiome composition and changes in gut permeability. Aim 1a will investigate the extent to which microbiome depletion by antibiotic treatment alters the skeletal effects of the GnRHa Leuprolide with and without subsequent CSHT in young male and female mice. Aim 1b will utilize fecal material transfers (FMTs) to directly determine the extent to which stool microbiome contributes to the skeletal effects of GnRHa treatment with and without subsequent CSHT in young male and female mice. Aim 2a will determine the contribution of microbiome dependent expansion and migration of gut Tregs to the skeletal effects of GnRHa treatment with and without subsequent CSHT. Aim 2b will determine the contribution of increased gut permeability to the skeletal effects of GnRHa treatment with and without subsequent CSHT, and Aim 2c will investigate if colonization of GF mice with bacteria mediating the skeletal effects of Leuprolide with and without subsequent CSHT restores effects that are absent in GF mice. This project will determine the effects of GnRHa treatment with and without ensuing CSHT on skeletal maturation, and whether such effects are mediated by modifications to the microbiome.

PROJECT NARRATIVE This project will determine the contribution of the gut microbiome to the effects of gender affirming hormone therapy (GAHT) in mice. Our study will provide information on whether GAHT exerts effects on skeletal maturation, and the mechanisms whereby the gut microbiome mediates the skeletal effects of GAHT. The project is important because the skeletal effects of GAHT in humans is a critical medical concern in transgender medicine.