SUMMARY Transgender (trans) individuals are those who have a gender identity that does not match their birth-assigned sex. About 1.4 million adults in the United States identified as transgender in 2016. Trans people may choose to medically transition through gender-affirming hormones or surgeries. Trans people are at higher risk for HIV and other sexually transmitted infections (STI), but little is known of the effect of gender-affirming medical care on the genital microenvironment, including the microbiome and local immunology, which contribute to HIV and STI risk in cisgender individuals. The NIH has thus identified a major need for innovative research characterizing the biological and immunological impact of interventions used for gender reassignment. About ¼ of trans women (assigned male at birth but with a female gender identity) have undergone vaginoplasty (surgical creation of a neovagina, typically using penile and scrotal skin), and while these women frequently present with neovaginal dysbiosis, little is known about the underlying pathophysiology. The penile and vaginal microenvironment in cis men and women is a critical determinant of HIV and STI risk, yet our understanding of the composition and role of microbiota colonizing the neovagina is scarce and completely lacking for immune outcomes. In trans men, vaginectomy is rare (<2%) but an estimated 69% use gender-affirming testosterone therapy. Masculinizing hormone therapy has significant effects on the vaginal epithelium that result in reduced cellular proliferation and glycogen production. This is similar to vaginal atrophy observed during menopause in cis women, which affects the vaginal microbiota and has negative impacts on sexual health and quality of life. Little information is available on the relationship between vaginal microbiota and inflammation and the development of vaginal atrophy and HIV and STI risk in trans men. Given these unknowns, the short-term goals of this research are to describe the neovaginal and vaginal microbiomes in trans women and men, define microbes associated with local inflammation, and then test for associations between the genital microenvironment and medicines, genital exposures, and hormone therapy, to guide the development of novel interventions, and clinical and behavioral best-practices, which currently are lacking. We will achieve these goals by performing a thorough and rigorous longitudinal description of the vaginal and neovaginal microbiomes in trans men and women (Aim 1) and then defining the relationship between these microbiomes and the genital immunology, a determining factor of HIV and STI risk in cis individuals (Aim 2). Our study, TransBiota, will leverage a piloted and validated IRB-approved innovative home-based sampling strategy and an innovative statistical strategy that affords scientific rigor by correcting for experimental errors associated with microbiome analyses. Understanding the genital microenvironment and its role in...