Project Summary The progestin-only contraceptive Depo-Provera 150mg delivered intramuscularly (150-IM), the most widely used injectable contraceptive worldwide, has been associated with increased HIV acquisition in many observational studies. The absence of causal evidence from randomized clinical trials has resulted in uncertainty about whether the observed association is due to methodological limitations or a real biological effect. Due to this uncertainty, and the risks associated with unplanned pregnancies, the World Health Organization advises that women at high risk of HIV can continue to use progestin injectables with counseling. Data on the effects of other long-acting contraceptives on HIV acquisition, such as implants and intrauterine devices (IUDs) are even more scarce; and there are no data on Sayana® Press (DMPA-SQ 104mg, hereafter 104-SQ) and other lower dose DMPA formulations in development. This study will leverage three ongoing randomized trials to conduct timely, innovative, and cost efficient research to evaluate the impact of multiple contraceptives – including 150-IM, the levonorgestrel (LNG) implant, the copper IUD, 104- SQ, and novel low dose DMPA formulations - on the female genital tract (FGT) microbial and immune environment. The aims of this research are to: 1) analyze the vaginal microbiome of women by 16S rRNA gene survey and metagenomic sequencing for high-resolution analysis of bacteria, viruses, fungi, and parasites before and after initiation of 150-IM, 104-SQ, LNG implant or Cu-IUD; 2) evaluate levels of vaginal cytokines and antimicrobial proteins before and after initiation of 150-IM, 104-SQ, LNG implant or Cu-IUD; 3) evaluate changes in the frequency and activation of CCR5+, CCR6+CCR10- (Th17-like) and α4β7 CD4+ T cells in the cervix among women using 150-IM, 104-SQ, LNG implant or Cu-IUD; 4) evaluate changes in the vaginal microbiome, cytokines and antimicrobial proteins with use of lower MPA doses (45, 75 and 105mg) compared to 150-IM, and the effect of systemic and genital endogenous and exogenous hormone levels on these outcomes; and 5) conduct a discovery metaproteomics analysis to evaluate alterations in vaginal human and microbial proteins following initiation of 150-IM, 104-SQ, LNG implant or Cu-IUD. This research will clarify the complex associations between contraceptive exposure, endogenous and exogenous hormones and the FGT microbiome, markers of immunity and inflammation, recruitment of HIV target cells and activation of those cells and markers of epithelial barrier repair. These data will inform contraceptive use and policy as well as provide targets and safety endpoints for the development of future contraceptives. Moreover, these data will provide the first evidence of FGT microbiome and immune changes that occur following initiation of other MPA based contraceptives, including 104-SQ and novel low dose MPA formulations which will be critically needed if the ECHO trial identifies increased acqu...