Hormonal contraception alters vaginal microbiota and cytokines in South African adolescents in a randomized trial

Abstract

Young women in sub-Saharan Africa are disproportionally affected by HIV infection and unintended pregnancies. However, hormonal contraceptive (HC) use may influence HIV risk through changes in genital tract microbiota and inflammatory cytokines. To investigate this, 130 HIV negative adolescent females aged 15-19 years were enrolled into a substudy of UChoose, an open-label randomized crossover study (NCT02404038), comparing acceptability and contraceptive product preference as a proxy for HIV prevention delivery methods. Participants were randomized to injectable norethisterone enanthate (Net-En), combined oral contraceptives (COC) or etonorgesterol/ethinyl estradiol combined contraceptive vaginal ring (CCVR) for 16 weeks, then crossed over to another HC for 16 weeks. Cervicovaginal samples were collected at baseline, crossover and exit for characterization of the microbiota and measurement of cytokine levels; primary endpoints were cervical T cell activation, vaginal microbial diversity and cytokine concentrations. Adolescents randomized to COCs had lower vaginal microbial diversity and relative abundance of HIV risk-associated taxa compared to Net-En or CCVR. Cervicovaginal inflammatory cytokine concentrations were significantly higher in adolescents randomized to CCVR compared to COC and Net-En. This suggests that COC use may induce an optimal vaginal ecosystem by decreasing bacterial diversity and inflammatory taxa, while CCVR use is associated with genital inflammation.

Fig. 1. Study overview and randomization.

CONSORT diagram of the number of participants that completed each study visit and provided genital samples, and the distribution of participants on each of the hormonal contraceptive methods analyzed: combined oral contraceptives (COC), the Net-En injection, and the vaginally inserted combined contraceptive ring (CCVR) at crossover and exit. Note that at crossover, some participants could choose their next contraceptive method if assigned to CCVR as their first method. LTFU loss-to-follow-up.

Fig. 2. Vaginal community clusters in a South African adolescent cohort.

a Barplot depicting the relative abundance of the most abundant bacteria in the baseline samples identified by 16S rRNA microbiome profiling. Samples (n = 126) are grouped by community-state type (CST) established using soft k-means clustering with weighted UniFrac distances and ordered by the most abundant species in each CST (CST-I: L. crispatus, CST-III: L. iners, and CST-IV: G. vaginalis). Alpha diversity for each sample (Shannon Index) is depicted below the barplot. b–d Change in CST from baseline to crossover within study arms. Alluvial plot showing the change in CST distribution from baseline to crossover for matched participants randomized to one of the three study arms: b COC (n = 36), c Net-En (n = 32), and d CCVR (n = 34) in an intention-to-treat (ITT) analysis. Each line represents one adolescent and CST changes over time. The color of the line is based on the CST assigned at baseline. e–g Alpha diversity at baseline and crossover within study arms. Boxplot representing in alpha diversity (Shannon Index) of vaginal microbiota for matched participants at baseline and crossover randomized to one of the three study arms: e COC (n = 36), f Net-En (n = 32), and g CCVR (n = 34). h–j Alpha diversity within participants changing between hormonal contraceptive methods. Boxplots showing the alpha diverisity (Shannon Index) in h 62 vaginal samples from 29 participants changing from COC to Net-En or vice versa, i 52 vaginal samples from 26 participants changing from COC to CCVR or vice versa, and j 130 vaginal samples from 50 participants changing from Net-En to CCVR or vice versa. P values were generated using two-sided paired Wilcoxon signed-rank tests. Y axes are log10-transformed. Significance codes: *P < 0.05. Bounds of boxes show interquartile range (IQR) with the lower and upper hinges corresponding to the 25th and 75th percentiles, respectively, lines in the middle of the box indicate median, and top and bottom whiskers demonstrate value ranges within 1.5 × IQR from the hinge. Points beyond that are plotted individually. CCVR: combined contraceptive vaginal ring, COC: combined oral contraceptives. Source data are provided as a Source Data file.

Fig. 3. Cytokine concentrations from baseline to crossover according to hormonal contraceptive arm in an intent-to-treat analysis.

Boxplot showing the change in cytokine concentrations from baseline to crossover for matched participants within each of the three contraceptive arms: a adolescents randomized to COC (n = 36), b adolescents randomized to Net-En (n = 35), and c adolescents randomized to CCVR (n = 34). P values were generated using two-sided paired Wilcoxon signed-rank tests adjusted for multiple comparisons using the Benjamini–Hochberg (BH) method. Y axis log10-transformed. Significance codes: *P < 0.05, **P < 0.01, ***P < 0.001. Bounds of boxes show interquartile range (IQR) with the lower and upper hinges corresponding to the 25th and 75th percentiles, respectively, lines in the middle of the box indicate median, and top and bottom whiskers demonstrate the largest and lowest value within 1.5 × IQR from the hinge. Points beyond that are plotted individually. CCVR: combined contraceptive vaginal ring, COC: combined oral contraceptives. Source data are provided as a Source Data file.

Fig. 4. Effect of hormonal contraception on bacterial taxa and cervicovaginal cytokines implicated in HIV risk.

a A multilevel sparse partial least-squares discriminant analysis (sPLSDA) of cytokines and bacterial taxa associated with HIV grouped before (baseline) and after (crossover) use of COC, Net-En, or CCVR, respectively. b Barplot showing the loadings of components 1 and 2 from the sPLSDA analysis. The absolute value is an indication of the importance of the bacteria or cytokine, while the sign indicates positive/negative correlations between the variables. CCVR: combined contraceptive vaginal ring, COC: combined oral contraceptives. Source data are provided as a Source Data file.