Background The effects of hormonal contraception and pregnancy on the vaginal microbiome (by molecular methods), acquisition and persistence of sexually transmitted infections (STIs), and genitourinary mucosal immunology are still largely unknown.

Methods HIV-negative, non-pregnant female sex workers (n = 397) in Kigali, Rwanda, were followed for two years. Demographic, behavioural, clinical, STI and pregnancy data were collected at regular intervals. The vaginal microbiome was cross-sectionally determined using a phylogenetic microarray (n = 174). Women with STIs were purposefully oversampled in this subsample. Inflammatory cytokines were measured in cervicovaginal fluid using Luminex and ELISA methodology (n = 343). Hormonal exposure was defined as use of hormonal contraception (oral or injectable) or a positive urine pregnancy test. Women in the exposure groups were compared to non-pregnant women who did not use hormonal contraception. Adjustments were made for demographic data and sexual risk taking.

Results At baseline, 12% of the women used hormonal injectables, and 6% oral contraceptives (OC); 7.7% was pregnant. OC use was associated with higher HPV prevalence (aOR 3.09; 95% CI 1.42–7.72), higher Chlamydia trachomatis incidence (aOR 7.13; 95% CI 1.40–36.30), and lower syphilis prevalence (0% vs 7.2% in controls) and incidence (0% vs 1.2%). Hormonal injectables were associated with higher HSV-2 prevalence (aOR 2.08; 95% CI 1.23–3.50). Pregnancy was weakly associated with higher Trichomonas vaginalis(aOR 1.67; 95% CI 0.97–2.88) and vaginal yeast (aOR 1.95; 95% CI 0.99–3.82) incidence. Six vaginal microbiome clusters were identified. No associations between hormonal exposure status and vaginal microbiome clusters were found; however, pregnant women had lower Gardnerella vaginalis levels. Pregnant women had higher IL-8 levels in cervicovaginal fluids than non-exposed women.

Conclusions Both hormonal contraception and pregnancy were associated with higher STI incidence. Overall, vaginal inflammation and microbiome composition were similar among groups, but pregnant women had lower Gardnerella and higher IL-8 levels.