38The hormonal contraceptive Medroxyprogesterone Acetate (MPA) is associated with increased 39 risk of Human Immunodeficiency Virus (HIV), via incompletely understood mechanisms. 40 Increased diversity in the vaginal microbiota modulates genital inflammation and is associated 41 with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal 42 microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually 43 transmitted infections (STIs), with Nugent Scores <7 (N=58 of 370 screened), MPA correlated 44 with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene 45 sequencing. MPA was also significantly associated with low vaginal glycogen and α-amylase, 46 factors implicated in vaginal colonization by lactobacilli, bacteria believed to protect against STIs. 47Introduction: 56 Meta-analyses suggest the injectable progestin-based contraceptive Depot-Medroxyprogesterone 57 Acetate (DMPA) increases heterosexual acquisition of Human Immunodeficiency Virus (HIV-1) 58 1.4-fold (1), while a prospective study reported women using injectable progestins were at 3.5 59 times increased risk of HIV acquisition compared to women not using long-term hormonal 60 contraceptives (2). These statistics are particularly troubling because DMPA is a popular 61 contraceptive in Africa (3), where HIV-1 prevalence is greatest. Studies have suggested several 62 biological mechanisms, including hypo-estrogenism, by which DMPA might enhance 63 susceptibility to HIV-1 and other sexually transmitted infections (STIs) (4). However, few (5-10) 64 examine the relationship between hormonal contraceptives and the vaginal microbiota (VMB), 65 even though diverse VMB, low in Lactobacillus species, confers a 4-fold increased risk of HIV-1 66 (11). Herein, we determined the effect of MPA (active component of DMPA) on several factors 67 within the vaginal microenvironment of Kenyan sex workers, including diversity of the VMB. 68 69 The VMB is a bacterial community lining vaginal epithelial cells (12). Unlike the diverse gut 70 microbiota, the VMB is generally low in diversity, and five community state types, including four 71 dominated by a Lactobacillus species have been described (12). Two factors thought to maintain 72 vaginal lactobacilli are glycogen, stored in epithelial cells and available as free glycogen in vaginal 73 fluid (13-15), and α-amylase, an enzyme that catabolizes glycogen for use by lactobacilli and other 74 bacteria (16) as energy. Similar to the gut, the VMB modifies immunity. The VMB alters 75 inflammation in the female genital tract (17), and is implicated in reproductive health and disease. 76 Several factors including ethnicity (12), STIs (18), bacterial vaginosis (BV) (19, 20), and sex work 77 (21) have been reported to impact VMB composition. This study was designed to examine the 78 4 effect of hormonal contraceptives on the VMB of healthy, asymptomatic Kenyan sex workers to 79 determine if DMPA is associated with changes...