Use of contraceptive depot medroxyprogesterone acetate is associated with impaired cervicovaginal mucosal integrity

Abstract

Background: Injectable depot medroxyprogesterone acetate (DMPA) is one of the most popular contraception methods in areas of high HIV seroprevalence. Evidence is accumulating that use of DMPA might be associated with an increased risk of HIV-1 acquisition by women; however, mechanisms of this association are not completely understood. The goal of this study was to gain insight into mechanisms underlying the possible link between use of DMPA and risk of HIV-1 acquisition, exploring transcription profiling of ectocervical tissues.

Methods: Healthy women received either DMPA (n = 31) or combined oral contraceptive (COC), which has not been linked to an increased risk of HIV acquisition (n = 32). We conducted a comparative microarray-based whole-genome transcriptome profiling of human ectocervical tissues before and after 6 weeks of hormonal contraception use.

Results: The analysis identified that expression of 235 and 76 genes was significantly altered after DMPA and COC use, respectively. The most striking effect of DMPA, but not COC, was significantly altered expression (mostly downregulation) of many genes strategically involved in the maintenance of mucosal barrier function; the alterations, as indicated by Ingenuity Pathway Analysis (IPA), were most likely due to the DMPA-induced estrogen deficiency. Furthermore, IPA predicted that transcriptome alterations related to ectocervical immune responses were in general compatible with an immunosuppressive effect of DMPA, but, in some women, also with an inflammatory-like response.

Conclusion: Our results suggest that impairment of cervicovaginal mucosal integrity in response to DMPA administration is an important mechanism contributing to the potential increased risk of HIV-1 acquisition in DMPA users.

Trial registration: ClinicalTrials.gov NCT01421368.

Funding: This study was supported by the United States Agency for International Development (USAID) under Cooperative Agreement GPO-A-00-08-00005-00.

Keywords: AIDS/HIV; Expression profiling; Innate immunity; Reproductive Biology; Sex hormones.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Flow chart of the study.

Figure 2. Heatmaps of hierarchical clustering of the genes differentially expressed in the ectocervical epithelium of hormone contraception users.

(A) DMPA users. (B) COC users. Columns represent participants; rows represent genes. Gene expression levels are indicated by color: red denotes upregulation; blue denotes downregulation.

Figure 3. Representative images of immunohistochemical detection of KRT10 and DSG1 in the vaginal epithelium at baseline (n = 10 in DMPA cohort; n = 5 in COC cohort) and after contraception use (n = 16 in DMPA cohort; n = 5 in COC cohort).

See Supplemental Figure 5 for more details. BL, baseline. Scale bars: 100 μm.

Figure 4. Graphical Spearman correlation matrix of selected ectocervical genes differentially expressed in the DMPA users.

Spearman correlation r values were determined using GraphPad Prism 7. Colors are added for better visualization. See Supplemental Figure 2 for significance of Spearman correlation.

Figure 5. Functional analysis of genes differentially expressed in the ectocervical epithelium of HC users conducted using IPA.

(A) Top biological processes most significantly associated with the DEGs. (B) Activation state of biological functions in the ectocervical epithelium in HC users as characterized and sorted by Z scores. The overwhelming majority of computable functions was predicted to be suppressed (negative Z scores). Shown are the top computable biological functions.

Figure 6. Heatmap of hierarchical clustering based on the genes differentially expressed in the ectocervical epithelium of DMPA users, significant at parametric P value < 0.001 and [FC] > 2.

Boxes on top of the heatmap show groups (G1, G2, G3) designated for analysis of gene expression variability in the DMPA users.

Figure 7. Volcano plots of gene expression changes in 3 groups of the DMPA users.

Colored circles represent genes significant at false discovery rate set at 0.05. Red circles, upregulated genes; blue circles, downregulated genes.

Figure 8. Activation status of functions related to immune cell migrations in 3 groups of the DMPA users (IPA analysis).

(A) Networks showing relationships of DEGs with functions. (B) Heatmaps of the activation status of the biological functions in the groups.

Figure 9. Intact cervicovaginal epithelium.

Healthy cervicovaginal epithelium presents an effective barrier against HIV-1 transmission. The epithelium consists of several layers of continuously differentiating keratinocytes interconnected by cell junctional structures. The outermost epithelial layer, the stratum corneum (SC), is the first physical barrier for most microorganisms. Proteins such as RPTN, FLG, LCE3D, LOR, TGM3, and ALOX12B are essential in maintaining the SC. Corneocytes form the SC and are joined by corneodesmosomes, cell junctional structures whose intercellular part consists of DSG, DSC, and CDSN, proteins that are degraded by KLK peptidases in the uppermost part of the SC to promote cell separation and shedding. The multiple keratinocyte layers beneath the SC present the next level of physical protection due to strong cytoskeleton supported by KRTs and cell junctional structures, largely desmosomes, the transmembrane part of which is formed by DSGs and DSCs. Activities of KLK peptidases beneath the SC are suppressed by serine protease inhibitors including SPINKs and SERPINs. In addition to physical barriers, Lactobacillus spp., which are most often the dominant types of bacteria in healthy vaginal lumen, produce lactic acid — a factor that is involved in direct anti–HIV-1 activity. Vaginal colonization and lactic acid production by lactobacilli depend on available glycogen synthesized by keratinocyte GYS2. The expression pattern of epithelial barrier–related proteins, encoded by genes differentially expressed in DMPA users, is shown on the right. Only potential HIV-1 target immune cells are shown for simplicity.

Figure 10. Model of cervicovaginal mucosal changes in DMPA users plausibly linked to an enhanced HIV-1 susceptibility.

Use of DMPA results in altered expression of many genes involved in barrier functions of cervicovaginal mucosa. (A) Decrease in markers of differentiating keratinocytes (such as KRT10, KRT1, KRT6B) indicates a compromised epithelial differentiation. Downregulation of the molecules involved in the SC organization (FLG, RPTN, LCE3D, ALOX12B, TGM3, LOR, and CDSN) and cell junctional proteins in all layers (DSG1 and DSC2) leads to breaches in the epithelial barrier, which is exacerbated by an untimely activation of KLK peptidases due to a decreased production of peptidase inhibitors (specifically SPINK6 and SERPINB7). Decrease in GYS2 expression implicates a drop in the glycogen level and, therefore, lower abundance of Lactobacillus spp. (B) In more expanded responses, molecular changes in the cervicovaginal epithelium are intensified: alterations in the barrier-supporting genes dramatically increase in magnitude, which results in more spacious epithelial breaches and production and release of proinflammatory chemokines that attract more HIV target cells (such as CD4+ T cells, dendritic cells, macrophages) and damage-related neutrophils. In A and, on a larger scale, in B, the cervicovaginal epithelium of the DMPA users is characterized by loss of epithelial integrity, which allows HIV virions to penetrate and/or transverse the epithelium and reach HIV-1 target cells for productive infection or be transferred to the draining lymph node or circulating blood. See the legend to Figure 9 for more details.