Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives

Abstract

Problem: Contraceptive hormones are systemically active, potent, and likely to invoke biological responses other than known fertility regulation impacts. We hypothesized that initiation of depot medroxyprogesterone acetate (DMPA) would increase genital HIV-target-cells and soluble immune mediators compared with baseline and initiation of other contraceptive methods.

Method of study: We collected cervical cytobrushes and cervicovaginal fluid from healthy Zimbabwean women aged 18-34 to assess immune cell populations, cytokines, and innate anti-HIV activity at baseline and after 30, 90, and 180 days use of DMPA (n = 38), norethisterone enanthate (n = 41), medroxyprogesterone acetate/estradiol cypionate (n = 36), levonorgestrel implant (n = 43), etonogestrel implant (n = 47), or copper intrauterine device (Cu-IUD) (n = 45). Cells were quantified by flow cytometry, cytokines were detected by multiplex assays, and innate anti-HIV activity was assessed by in vitro HIV challenge.

Results: Compared to baseline, the number of cervical HIV target cells (#CD4 cells P < .04 and #CD11c cells P < .04), the concentration of the inflammatory cytokine IL-1β (P < .01), and the innate in vitro anti-HIV activity (P < .001) significantly decreased following DMPA initiation. In Cu-IUD users, genital HIV target cells increased (#CD4 cells P < .001, #CD4CCR5 cells P = .02, #CD4CD69 cells P < .001, #CD8CD69 P = .01, and #CD11c cells P = .003) at day 30 and resolved by day 180. IFN-γ (P < .001), IL-1β (P < .001), IL-6 (P < .001), IL-8 (P < .001), IL-10 (P < .01), and RANTES (P < .001) were also significantly increased at day 30. Minimal alterations were observed following initiation of subdermal implantable contraceptives.

Conclusions: This head-to-head study compared six contraceptives and found increased HIV target cells and cervical inflammation temporally associated with Cu-IUD initiation. Use of hormonal contraception, including DMPA, did not increase cervical HIV target cells or inflammation. Clinical Trial Number: NCT02038335.

Keywords: CCR5; biomarkers; contraception; copper; medroxyprogesterone acetate; receptors.

Figure 1

Diagram of participant flow from eligibility assessment to final categorization

Figure 2

Systemic progestin concentrations after initiation and use of injectable and implantable contraceptives. Serum progestin concentrations for all evaluable participants at day 30, 90, and 180 following initiation and continuous use of (A) DMPA, (B) Net‐En, (C) MPA/estradiol cypionate, (D) levonorgestrel implant, and (E) etonogestrel implant. Mean serum concentrations with SD are indicated with red bars and black brackets, respectively. DMPA, depot medroxyprogesterone acetate; Net‐En, norethisterone enanthate; MPA, medroxyprogesterone acetate; LNG, levonorgestrel; ENG, etonogestrel

Figure 3

Flow cytometric gating strategy and baseline blood and cervical CD4 cells expressing CCR5 and CD69. Recovery of CD3⁺CD4⁺CCR5⁺ and CD3⁺CD4⁺CD69⁺ cells from (A) cervical cytobrush, (B) peripheral blood mononuclear cells (PBMC), (C) CD3⁺CD4⁺CCR5CD69⁺ double positives from cervical cytobrushes and PBMC (D) the proportion of CD3⁺CD4⁺CCR5⁺ and CD3⁺CD4⁺CD69⁺ from PBMC and cervical cytobrush demonstrating gating. Two senior laboratory scientists trained in advanced flow cytometry and masked to contraceptive group, independently reviewed and agreed upon gating parameters for each sample. The percent (D) and number (E) of CD3⁺CD4⁺CCR5⁺ and CD3⁺CD4⁺CD69⁺ cells in PBMC and cervical cytobrush samples collected from all evaluable participants at baseline, prior to initiation of any contraception. Mean values with SD are indicated with red bars and brackets, respectively

Figure 4

Impact of injectable contraceptives on genital immune cells and soluble mediators. Cervical immune cells (A, C, and E) collected by endocervical cytobrush and quantified by flow cytometry are expressed as % change in cell number from baseline to follow‐up at days 30 and 180 after initiation of depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (Net‐En), or medroxyprogesterone acetate and estradiol cypionate (MPA/EC) as indicated. Soluble mediators and innate anti‐HIV activity (B, D, and F) measured in cervicovaginal lavage expressed as the % change from baseline to follow‐up at days 30 and 180 after injectable contraceptive initiation. P‐values from Wilcoxon signed‐rank test comparing baseline (prior to contraceptive initiation) to follow‐up 180 d after initiation and continuous use of contraceptive as indicated; P‐values adjusted using the Holm‐Bonferroni multiple test procedure

Figure 5

Impact of implantable contraceptives on genital immune cells and soluble mediators. Cervical immune cells (A and C) collected by endocervical cytobrush and quantified by flow cytometry are expressed as % change in cell number from baseline to follow‐up at days 30 and 180 after initiation of contraception with levonorgestrel implant (LNG‐I) or etonogestrel implant (ENG‐I) as indicated. Soluble mediators and innate anti‐HIV activity (B and D) measured in cervicovaginal lavage expressed as the % change from baseline to follow‐up at days 30 and 180 after implantable contraceptive initiation. P‐values from Wilcoxon signed‐rank test comparing baseline (prior to contraceptive initiation) to follow‐up 180 d after initiation and continuous use of contraceptive as indicated; P‐values adjusted using the Holm‐Bonferroni multiple test procedure

Figure 6

Impact of copper intrauterine device on genital immune cells and soluble mediators. Cervical immune cells (A) collected by endocervical cytobrush and quantified by flow cytometry are expressed as % change in cell number from baseline to follow‐up at days 30 and 180 after initiation of contraception with copper intrauterine device (Cu‐IUD). Soluble mediators and innate anti‐HIV activity (B) measured in cervicovaginal lavage expressed as the % change from baseline to follow‐up at days 30 and 180 after intrauterine contraceptive initiation. P‐values from Wilcoxon signed‐rank test comparing baseline (prior to contraceptive initiation) to follow‐up 180 d after initiation and continuous use of contraceptive as indicated; P‐values adjusted using the Holm‐Bonferroni multiple test procedure