PROJECT SUMMARY Studies have shown that cisgender women with type 2 diabetes (T2D) are at greater risk for recurrent urogenital infections, including urinary tract infections and vulvovaginal candidiasis (VVC), but it is unknown whether individuals with T2D have disproportionately higher rates of bacterial vaginosis (BV). BV is a common form of vaginitis with a 30% prevalence among North American women. BV is characterized by a Lactobacillus-deficient vaginal microbiota and is associated with increased risk for sexually transmitted infections, including HIV, as well as vulvovaginal symptoms that significantly affect quality of life. Sodium glucose cotransporter-2 (SGLT2) inhibitors, a favorable oral antidiabetic medication, function by inhibiting reabsorption of glucose in the kidneys, causing increased excretion of glucose in urine. This mechanism has been associated with increased incidence of VVC, but the influence of SGLT2 inhibitors on the genitourinary microbiota has not been investigated with molecular methods. In this F31, I propose a molecular epidemiologic project which assesses the vaginal microbiota associated with T2D and after SGLT2 inhibitor use. I hypothesize that T2D and SGLT2 inhibitors are associated with molecular-BV and a vaginal microenvironment low in lactic acid. The production of lactic acid by lactobacilli is an important function because it provides protection by acidifying the vaginal microenvironment (to pH <4.5). The specific aims are: 1) To assess the vaginal microenvironment in non-pregnant individuals with T2D compared to ethnicity/race-matched individuals without T2D. Cisgender women aged 45 years and greater will be recruited to a cross-sectional study at the University of Maryland Center for Diabetes and Endocrinology and a general health clinic at the Baltimore Midtown campus. Self-collected vaginal samples will be assessed for bacterial composition utilizing 16S rRNA gene amplicon sequencing, pan-bacterial quantitative PCR and lactic acid isomer assays. Questionnaires will collect important covariate information. 2) To compare the genitourinary microbiota of women before and after treatment with SGLT2 inhibitors, utilizing a repository of urine samples collected from an ongoing study in the Old Order Amish (R01-DK118942, PI: Taylor, co-sponsor). The single cross-over study design allows for assessment of changes in the genitourinary microbiota following SGLT2 inhibitors and eliminates confounding on time-independent factors because it allows each participant to serve as their own control. Aim 2 is based on repository urine samples, making it highly feasible; recently published studies suggest urine samples demonstrate high concordance with vaginal microbiota. The studies proposed in this F31 will provide the first characterization of the vaginal microbiota in the setting of T2D and assess the impact of SGLT2 inhibitors on the genitourinary microbiota. These findings will contribute to BV screening guidelines ...