Impact of Hormonal Contraceptives on Cervical T-helper 17 Phenotype and Function in Adolescents: Results from a Randomized, Crossover Study Comparing Long-acting Injectable Norethisterone Oenanthate (NET-EN), Combined Oral Contraceptive Pills, and Combined Contraceptive Vaginal Rings

Abstract

Background: Adolescents in sub-Saharan Africa are at risk for human immunodeficiency virus (HIV) infection and unintended pregnancies. Observational studies suggest that injectable hormonal contraceptives (HCs) increase the HIV risk, although their effects on genital inflammation, particularly HIV-susceptible T-helper 17 (Th17) cells, are unknown. In a randomized crossover study, the effect of injectable norethisterone oenanthate (NET-EN), combined contraceptive vaginal rings (CCVR; NuvaRing), and combined oral contraceptive pills (COCPs) on cervical Th17 cells and cytokines were compared.

Methods: Adolescents (n = 130; 15-19 years) were randomly assigned 1:1:1 to NET-EN, CCVR, or COCPs for 16 weeks, then subsequently crossed over to another HC for 16 weeks. Estrogen, follicular stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured. Chemokine receptor 5 (CCR5), human leukocyte antigen (HLA) DR isotope, and cluster of differentiation 38 (CD38) expression by cervical cytobrush-derived CD4+ T cells was assessed by fluorescence-activated cell sorting. Th17 cells were defined as CCR6+ and CCR10-. Cervicovaginal Th17-related cytokines were measured by Luminex.

Results: CCVR use for the first 16 weeks was associated with reduced Th17 frequencies and lower FSH and LH concentrations, as compared to NET-EN and COCPs, with FSH concentrations and Th17 frequencies correlating significantly. However, Th17-related cytokine concentrations (interleukin [IL]-21, IL-1β, tumor necrosis factor-α, interferon-γ) and CCR5, HLA-DR, CD38, and Th17 frequencies were significantly higher in CCVR than NET-EN and COCP. At crossover, CCVR users changing to COCPs or NET-EN did not resolve activation or cytokines, although switching from COCP to CCVRs increased cytokine concentrations.

Conclusions: CCVR use altered endogenous hormone levels and associated cervical Th17 cell frequencies to a greater extent than use of NET-EN or COCPs, although Th17 cells were more activated and Th17-related cytokine concentrations were elevated. While CCVRs may impact the HIV risk by regulating Th17 numbers, increased activation and inflammation may balance any risk gains.

Keywords: CCVR; FSH; NET-EN; Th17; cytokines.

Figure 1.

Cervical Th17 cells were identified by expression of CCR6 and CCR10 (CCR6+ CCR10-). A, Cervical CD4, CD3, and T-cell frequencies were defined as CCR6+ CCR10- (Th17) or CCR6- CCR10- (enriched for Th1/2 cells). The expression of CCR5 is shown (B) alone and in combination with (C) HLA-DR, (D) CD38, or (E) HLA-DR and CD38 by Th17 cells (CCR6+ CCR10-; black bars) and Th1/2-like T cells (CCR6- CCR10-; clear bars). Mann-Whitney U tests were applied to compare the markers between the groups, and P values ≤ .05 were considered significant. Abbreviation: Th17, T-helper 17.

Figure 2.

Phenotype of Th17 cells before (baseline) and after (16 weeks) being on (A) NET-EN, (B) COCPs, and (C) CCVR. The frequencies of Th17 cells and expression of CCR5, CD38, and HLA-DR on these cells were assessed before and after 16 weeks of adolescents being on the randomized HC. A Wilcoxon matched pairs signed rank test was applied and a P value ≤ .05 was considered significant. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; HC, hormonal contraceptive; NET-EN, injectable norethisterone oenanthate; Th17, T-helper 17.

Figure 3.

Comparison of genital concentrations of Th17-related cytokines at baseline and crossover (16 weeks). A, Concentrations of Th17-related cytokines before and after using NET-EN for 16 weeks. B, Concentrations of Th17-related cytokines before and after using COCPs for 16 weeks. C, Concentrations of Th17-related cytokines before and after using CCVR for 16 weeks. Cytokine concentrations were compared using a Mann-Whitney U test, and only significant P values are displayed (P ≤ .05). Cytokines highlighted by the gray-shaded box remained significant after adjusting for multiple comparisons. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; IFN-γ, interferon-gamma; IL, interleukin; NET-EN, injectable norethisterone oenanthate; sCD40L, soluble CD40 ligand; Th17, T-helper 17; TNF-α, tumor necrosis factor-alpha.

Figure 4.

Unsupervised hierarchical clustering of cervicovaginal cytokine concentrations in adolescents randomized to use NET-EN (n = 34; blue blocks), COCPs (n = 37; green blocks), or CCVR (n = 34; pink blocks) for 16 weeks. Adolescents using the CCVR (pink blocks) were enriched in the upregulated cervicovaginal cytokine cluster, compared to those using either NET-EN (blue blocks) or COCPs (green blocks). Cytokine concentrations are indicated using a color scale that ranges from green (low) through black to red (high). The dendrogram above the heat map illustrates the degrees of relatedness between genital cytokine profiles evident within the various adolescents. The dendrogram on the left-hand side of the heat map indicates relationships between the expression profiles of the analyzed cytokines across all of the women assessed in this study. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; HC, hormonal contraceptive; IFN-γ, interferon-gamma; IL, interleukin; NET-EN, injectable norethisterone oenanthate; sCD40L, soluble CD40 ligand; TNF-α, tumor necrosis factor-alpha.

Figure 5.

Longitudinal changes in phenotype and activation of cervical Th17 cells in adolescents randomized to use CCVR first and then either crossing to (A) NET-EN (n = 14) or (B) COCPs (n = 4), and those randomized to use (C) NET-EN (n = 8) or (D) COCPs (n = 12) first before crossing over to CCVR. Each line connects a single participant across all the 3 visits. Box and whiskers show the median, IQR, and range. The Mann-Whitney U test was applied. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; IQR, interquartile range; NET-EN, injectable norethisterone oenanthate; Th17, T-helper 17.

Figure 6.

Longitudinal changes in Th17-related cytokine concentrations in adolescents initially randomized to the CCVR and subsequently crossed over to (A) NET-EN (n = 21) or (B) COCPs (n = 4). The samples were compared using a Mann-Whitney U test. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; IFN-γ, interferon-gamma; IL, interleukin; NET-EN, injectable norethisterone oenanthate; Th17, T-helper 17; TNF-α, tumor necrosis factor-alpha.

Figure 7.

Longitudinal changes in Th17-related cytokine concentrations in adolescents initially randomized to (A) NET-EN (n = 24) or (B) COCPs (n = 20) and subsequently crossed over to CCVR. The samples were compared using a Mann-Whitney U test. Abbreviations: CCVR, combined contraceptive vaginal ring; COCPs, combined oral contraceptive pills; IFN-γ, interferon-gamma; IL, interleukin; NET-EN, injectable norethisterone oenanthate; Th17, T-helper 17; TNF-α, tumor necrosis factor-alpha.