Depot-medroxyprogesterone acetate reduces genital cell-cell adhesion molecule expression and increases genital herpes simplex virus type 2 infection susceptibility in a dose-dependent fashion

Abstract

Objective: Analyzing ectocervical biopsy tissue from women before and after they initiated use of depot-medroxyprogesterone acetate (DMPA), we previously reported this progestin reduces levels of the cell-cell adhesion molecule (CCAM) desmoglein-1 and increases genital mucosal permeability. We likewise saw treating mice with 1.0 mg of DMPA reduced vaginal CCAM expression and increased genital pathogen susceptibility. Herein, we used dose-response studies to delimit DMPA doses and serum MPA levels in mice associated with impaired genital mucosal barrier function and enhanced susceptibility to low-dose herpes simplex virus type 2 (HSV-2) infection.

Study design: We compared genital CCAM expression, genital mucosal permeability, and susceptibility to genital inoculation with 10³ plaque-forming units of HSV-2 among mice in estrus vs. after treatment with 0.01 mg, 0.1 mg, 0.3 mg, or 1.0 mg of DMPA.

Results: Compared to mice in estrus, DMPA treatment in a dose-dependent fashion significantly reduced desmoglein 1α (Dsg1a) and desmocollin-1 (Dsc1) gene expression, reduced DSG1 protein levels, and increased genital mucosal permeability to a low molecular weight molecule. While no mice infected with HSV-2 in estrus died, we respectively saw 50% and 100% mortality in mice administered 0.1 mg or 0.3 mg of DMPA. At time of infection, mean serum MPA levels in mice administered the 0.1 mg or 0.3 mg doses were 3.8 nM and 13.0 nM respectively (values comparable to trough and peak MPA serum levels in women using DMPA).

Conclusions: Mice with pharmacologically relevant serum MPA concentrations display significant changes in genital CCAM expression, genital mucosal barrier function, and HSV-2 susceptibility.

Keywords: DMPA; Desmoglein-1; Genital HSV-2 infection; Genital mucosal barrier function.

Figure 1.. DMPA decreases genital levels of CCAM and increases genital mucosal permeability in a dose-dependent fashion.

Mice in estrus and mice administered indicated DMPA doses 5 days previously were euthanized to process vaginal tissue for RNA isolation and immunofluorescence staining. A) Bar graphs show DMPA dose-dependent decrease in the relative expression of Dsg1a and Dsc1 mRNA. B) Images on left are representative of formaldehyde-fixed, paraffin-embedded vaginal tissue from mice in estrus and DMPA-treated mice that were stained with rabbit anti-DSG-1 and Alexa Fluor 488-conjugated anti-rabbit IgG (green) and counterstained with DAPI (blue) for confocal microscopy. Right-sided panel depicts the DMPA dose-dependent decrease in levels of DSG1 protein. C) Groups of mice treated as described in Figure 1B were anesthetized for intravaginal administration of a PBS solution containing Lucifer yellow (457 Da) (green) and 70 KDa dextran-Texas Red (red). Mice were euthanized after 45 minutes, and vaginal tissue processed and counterstained with DAPI (blue) for confocal microscopy. Images on left illustrate penetration of LY into vaginal mucosal epithelium. Right-sided panel shows the DMPA dose-dependent increase for penetration of this LMW fluorescent molecule into the vaginal epithelium. Data normality was defined by evaluation of the residuals. CCAM expression in DMPA treatment groups was compared to mice in estrus using one-way ANOVA and Dunnett’s post hoc tests. CCAM, cell-cell adhesion molecule; DAPI, 4,6-diamidino-2-phenylindole; DMPA, depot-medroxyprogesterone acetate; scale bar denotes 100 μm; Dsg1a, desmoglein-1α; Dsc1, desmocollin-1; and DSG1, desmoglein-1; *, **, ***, and **** denote p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

Figure 2.. DMPA-treated mice with MPA serum levels at the time of infection comparable to C_trough serum concentrations in women were more susceptibility to genital HSV-2 infection.

Mice in estrus and mice treated with 0.01 mg, 0.1 mg, 0.3 mg, or 1.0 mg of DMPA 5 days earlier were genitally inoculated with 10³ pfu of HSV-2. Immediately before infection, peripheral blood was collected to quantify serum concentrations of estradiol and MPA. A) Panel depicts positive linear correlation between DMPA dose and MPA serum levels (R² = 0.9643). B) Survival curve data shows that HSV-2 was uniformly lethal in mice administered ≥ 0.3 mg DMPA, whereas 50% and 0% of mice that received 0.1 mg or 0.01 mg of DMPA respectively succumbed to infection. Between-group comparisons were performed using the long-rank test. C) Panel depicts relationship between serum MPA levels at infection and HSV-2-induced mortality rates. Red and blue dotted vertical lines denote typical values for C_trough (red) and C_max (blue) serum MPA levels in women using DMPA. D) Though mortality differed significantly in mice administered DMPA doses of 0.1 mg (50% mortality) vs. 0.3 mg or 1.0 mg (both 100% mortality), serum estradiol levels in these groups were comparable. Data normality was determined by evaluation of the residuals. Serum estradiol levels were compared by one-way ANOVA and Tukey’s post hoc tests (all DMPA doses examined decreased serum estradiol levels compared to mice in proestrus and diestrus. DMPA, depot-medroxyprogesterone acetate; HSV-2, herpes simplex virus type 2; pfu, plaque forming unit; * and ** denote p < 0.05 and p < 0.01 respectively.