Norethisterone Enanthate Increases Mouse Susceptibility to Genital Infection with Herpes Simplex Virus Type 2 and HIV Type 1

Depot-medroxyprogesterone acetate (DMPA) and norethisterone enanthate (NET-EN) are injectable progestins commonly used for contraception. For pregnancy prevention, women receive i.m. NET-EN or DMPA injections every 2 or 3 mo respectively. Although both provide effective contraception, there is concern that women using exogenous progestins are more likely to acquire HIV and other sexually transmitted infections. This public health dilemma has been long-standing and fueled by inconsistent results from the clinical studies used to examine this relationship (1–9). For the most part, however, these studies were not specifically designed to explore connections between HIV and progestin use, and methodological limitations likely contributed to variability in their results (10). These limitations included insufficient sample size, confounding of HIV incidence data by higher frequencies of unprotected sex in couples using hormonal contraception, imprecise knowledge of progestin use at time of HIV acquisition, and undefined estimates of HIV risk among women using different forms of progestins (11–13). For example, although one study found a 2-fold higher risk of male-to-female HIV transmission among women using injectable contraception, it did not delineate this risk between women using DMPA or NET-EN (14, 15). More recently, a randomized clinical trial that compared HIV acquisition in women using DMPA, levonorgestrel (LNG) implant, or copper intrauterine device reported that risk of HIV was not significantly increased in women randomized to any of these contraceptive choices (16). This study, however, did not include a group of women randomized to use no type of long-acting reversible contraceptive and could provide information only on the relative risk of HIV acquisition in women using DMPA, LNG implant, or a copper intrauterine device (16). It likewise did not include women randomized to initiate use of the injectable contraceptive NET-EN (16).

In addition to the obstacles that hinder clinical studies from defining precise relationships between HIV susceptibility and exogenous progestins, this public health controversy is fueled by the belief that putative connections lack biological plausibility (17). Addressing this long-standing concern using basic science and clinical research, the capacity of progestins to promote genital mucosal epithelial thinning and genital inflammation, alter the vaginal microbiota, suppress antivirus immunity, and enhance HIV replication have been identified as mechanisms with the potential to underlie this relationship (18–22). Alternatively, we showed that pharmacologically relevant serum levels of medroxyprogesterone acetate (MPA) in mice were associated with reduced genital expression of the cell–cell adhesion molecules desmoglein-1α (Dsg1a) and desmocollin-1 (Dsc1) (23). We likewise identified that DMPA-treated mice have increased genital mucosal permeability to low m.w. molecules and activated leukocytes, compromised genital mucosal barrier function, and enhanced susceptibility to genital HSV type 2 (HSV-2) and cell-associated HIV type 1 (HIV-1) infection (24). Guided by mouse model results, we discovered analogous changes in the expression of cell–cell adhesion molecules and mucosal barrier function in ectocervical tissue from women using DMPA or an LNG-releasing intrauterine system (LNG-IUS) (24, 25). Based on these earlier results, in this study, we used two mouse models to explore the effects of NET-EN on genital mucosal barrier function and susceptibility to genital infection with HSV-2 or cell-associated HIV-1.

All procedures were approved by the Ohio State University Institutional Animal Care and Use Committee and the Stanford University Administrative Panel on Laboratory Animal Care. As indicated, 6– to 8–wk-old C57BL/6J (wild-type) and NOD-scid IL-2Rg^null (NSG) female mice were purchased from Jackson Laboratory (Bar Harbor, ME). As indicated, mice were s.c. injected for five consecutive days with 100 μl of PBS alone or a PBS solution containing 1 mg of NET-EN (Sigma-Aldrich, Laramie, WY) (prepared by glass-to-glass homogenization and sonication), whereas other mice were s.c. administered a single dose of 1 mg of DMPA (Depo-Provera) or 2 mg of methylprednisolone (MePRDL) (Depo-Medrol) (both Pfizer, New York, NY). Where indicated, cells collected by vaginal lavage were stained with crystal violet to identify wild-type and NSG mice in the estrus stage of the estrous cycle (26). Also as indicated, mice were euthanized 24 h after the fifth NET-EN treatment, and the serum was sent to the Endocrine Technologies Support Core at the Oregon National Primate Research Center to quantify norethisterone (NET) levels by liquid chromatography–tandem triple quadrupole mass spectrometry (27).

Prior to storage at −80°C, excised vaginal tissue was placed in RNAlater (Qiagen, Hilden, Germany) for 24 h at 4°C. Using the manufacturer’s instructions, RNA was isolated from stored tissues using the RNeasy Lipid Tissue Kit and RNase-free DNase Set (Qiagen). Samples were resuspended in nuclease-free water to quantify RNA using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) (samples routinely displayed 260/280 and 260/230 ratios > 1.8). Relative gene expression was calculated using 100 ng of RNA in the TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems, Foster City, CA) and the ∆ cycle threshold method with the following primers: glucocorticoid-induced leucine zipper (Mm00726417_s1), glucocorticoid receptor (GR) (Mm00433832_m1), arrestin β 2 (Mm00520666-g1), desmocollin-1 (Mm00496525_m1), desmoglein-1α (Mm00809994_s1), kallikrein-related peptidase 7 (Mm01197332_g1), kallikrein-related peptidase 13 (Mm01197335_m1), kallikrein-related peptidase 14 (Mm00805093_m1), and pyruvate carboxylase (Mm00500992_m1) (the latter used to provide a housekeeping gene) (all primers are from Life Technologies, Pleasanton, CA). Quantitative real-time PCR (RT-qPCR) analyses were performed using the StepOne Real-Time PCR system (Applied Biosystems).

As indicated, 5- to 10-μm sections were obtained from formaldehyde-fixed and paraffin-embedded vaginal tissue and deparaffinized by the sequential immersion in 100% xylene, 100 and 96% ethanol, and sterile water. Ag retrieval was performed with sodium citrate buffer. Samples were incubated overnight at 4°C in PBT (i.e., PBS with 0.05% Tween 20 that contained 10% normal donkey serum; Abcam, Cambridge, MA) and for 1 h at room temperature with rabbit antidesmoglein-1 [clone EPR6766(B); Abcam] (1:200 dilution). Slides were washed and incubated 1 h at room temperature with Alexa Fluor 488–labeled donkey anti-rabbit IgG (Abcam) (1:500 dilution) and counter-stained with DAPI (23) for examination with a Confocal A1 Microscope (Nikon Instruments, Melville, NY) (all images were acquired using identical microscope settings). To quantify DSG1 protein levels, images were analyzed via ImageJ software as previously described (24, 28). All analyses were performed by investigators unaware of the treatment groups the mice had been assigned.

As previously described, mice in estrus- and progestin-treated mice were sedated and intravaginally (ivag) administered 10 μl of PBS containing 50 μg of Lucifer yellow CH lithium salt (molecular mass = 457.2 Da) and 62.5 μg of dextran Texas Red (molecular mass = 70 kDa) (both Life Technologies) to assess vaginal permeability to low–molecular mass molecules (23–25). As already described, human PBMC (hPBMC) were activated and labeled with 5 μM carboxyfluorescein succinimidyl ester (CellTrace CFSE; Life Technologies) to evaluate genital leukocyte permeability (25). Briefly, cells were resuspended in RPMI 1640 (10⁸ cells per milliliter), and 10 μl of this suspension was ivag administered to sedated animals. Mice were euthanized 15 h later for use in confocal microscopy studies that assessed hPBMC penetration into genital tissue. Using Image J software, hPBMC incursion into vaginal mucosa was defined by measuring the depth (micrometer) that CFSE-hPBMC penetrated between the epithelial apical and basal layers, whereas entry of cells into the vaginal lamina propria was defined by quantifying lamina propria fluorescence from CFSE-labeled hPBMC (pixels/10⁴ μm²).

Using methods described previously (24), untreated mice in estrus or mice 1 d after their fifth NET-EN treatment or 5 d after a single DMPA treatment were sedated for ivag inoculation with 10⁴ PFUs of HSV-2 333 in 10 μl of RPMI 1640 (HSV-2 was kindly provided by Dr. R. L. Hendricks). HSV-induced pathology was evaluated daily via a 5-point scale: 0, no pathology; 1, mild vulvar erythema; 2, moderate vulvar erythema; 3, severe vulvar erythema; 4, perineal ulceration; and 5, extension of perineal ulceration to surrounding areas (29). Mice were euthanized when pathology scores ≥3 were detected or if they displayed encephalopathic changes. In other studies, identical groups of mice were ivag infected with 3 × 10⁶ PFUs of HSV-1q–GFP in 10 μl of RPMI 1640 as previously described (24) and euthanized 24 h later to assess fluorescent virus dissemination in vaginal tissue by confocal microscopy.

Using methods already described (24), NSG mice were engrafted with hPBMC (10⁶ cells per mouse). Nine days after engraftment, mice remained untreated or were s.c. administered DMPA or NET-EN. Mice were sedated 14 d after hPBMC engraftment and ivag inoculated with 2 × 10⁶ HIV-1–_Ba-L–infected hPBMC in 10 μl of RPMI 1640 (untreated mice in estrus at the time of infection provided controls). Mice were monitored daily, and those remaining alive 14 d postinfection were euthanized. In separate studies, identically engrafted and progestin-treated mice were euthanized 10 d after ivag inoculation with 10⁶ HIV-1–_Ba-L–infected hPBMC in 10 μl of RPMI 1640. Plasma was obtained to measure HIV-1 RNA copies by Abbott RT-qPCR assay, and ex vivo splenocyte culture supernatants were used to detect infectious HIV-1 particles via TZM-BL luciferase assay (splenocyte culture supernatants from uninfected PBMC-engrafted mice and HIV-1–_Ba-L aliquots provided negative and positive controls, respectively) (30–36).

Statistical analyses were performed using Prism 7 (GraphPad, La Jolla, CA). Normality was assessed using the D’Agostino and Pearson omnibus test or Shapiro–Wilk test (when sample numbers were <8). Depending on data distribution, unpaired Student t tests or unpaired Mann–Whitney U tests were used to compare two groups. For comparing more than two groups, one-way ANOVA with Tukey or Dunnett post hoc test or Kruskal–Wallis test with Dunn post hoc test were used (depending again on data distribution). Area under the curve analyses were used to compare HSV-induced pathology between untreated mice infected in estrus and NET-EN– or DMPA-treated mice. Survival was evaluated with Kaplan–Meier curves and log-rank tests used to compare cumulative survival incidence after genital infection with HSV-2 or cell-associated HIV-1. Any p values ≤ 0.05 were deemed statistically significant.

Because DMPA is the progestin predominantly used in experimental models to promote genital pathogen susceptibility (37), we needed to first define an NET-EN dosing schedule that produces pharmacologically relevant serum levels in mice. Although initial work using RIAs indicated that peak serum NET levels in women after i.m. injection with 200 mg of NET-EN were 4–20 ng/ml (38, 39), more recent pharmacokinetic data generated with liquid chromatography–tandem mass spectrometry showed this dose generated peak serum NET concentrations that were closer to 35 ng/ml (40). Using established guidelines for converting drug doses between humans and laboratory animals (41), we created a dosing schedule for mice that was designed to achieve serum NET levels comparable to these clinical values. In these studies, we identified that daily administration of 1 mg of NET-EN for 5 d produced mean NET serum concentrations of 33 ng/ml, providing strong evidence that use of this dosing regimen in subsequent experiments would define genital tract changes induced by pharmacologically relevant serum concentrations of the drug (Fig. 1A).

Progestin binding to intracellular steroid receptors is known to initiate events that alter gene expression (42). Whereas most progestins, including LNG and NET-EN, demonstrate high progesterone receptor (PR) affinity and low GR affinity, MPA is characterized by high PR affinity but greater GR affinity relative to other progestins (43–45). However, previous research from our laboratory demonstrated that vaginal tissue from mice treated with DMPA do not exhibit the signature changes in glucocorticoid-responsive gene expression seen in mice administered the glucocorticoid MePRDL (24). Based on those results, we hypothesized that glucocorticoid-responsive gene expression is comparable in the vagina of NET-EN– and DMPA-treated mice but dissimilar from that found in mice treated with the pure GR agonist MePRDL. As posited, we found expression of multiple glucocorticoid-responsive genes, including glucocorticoid-induced leucine zipper (Tsc22d3), arrestin β 2 (Arrb2), and the GR (Nr3c1), was similar in vaginal tissue from mice in estrus and NET-EN– and DMPA-treated mice but significantly different from mice treated with MePRDL (Fig. 1B). These results were consistent with a prior report that NET-EN possesses negligible GR-binding affinity (45) and implied that changes in genital mucosal permeability or pathogen susceptibility elicited in NET-EN–treated mice would not be sequalae to NET-EN–mediated activation of the GR.

In earlier work, we identified that DMPA- and LNG-treated mice display significant increases in genital mucosal permeability compared with mice in the estrus stage of the estrous cycle (24). Extending these results in the current investigation, we showed that compared with estrus-stage mice, DMPA- and NET-EN–treated mice exhibit similar increases in genital mucosal permeability to low m.w. molecules (Fig. 2A). We also saw that entry of ivag administered activated human leukocytes into the vaginal epithelial tissue was facilitated by DMPA or NET-EN treatment (Fig. 2B, 2C). Although leukocyte incursion into more superficial layers of the vaginal epithelium was comparable in mice treated with DMPA or NET-EN, deeper penetration of these cells into the vaginal lamina propria was greater in DMPA- versus NET-EN–treated mice (Fig. 2B, 2D). Taken together, these studies established that treatment of mice with DMPA or NET-EN increases permeability of the genital mucosa, while also implying DMPA is more likely than NET-EN to facilitate leukocyte penetration into deeper layers of vaginal tissue.

In prior publication, we reported that DMPA- and LNG-mediated increases in genital mucosal permeability were associated with reduced genital expression of the cell–cell adhesion molecules Dsg1a and Dsc1 (24). Using similar methodology in the current study, we likewise found that compared with levels detected in estrus-stage mice, expression of the cell–cell adhesion molecule genes Dsg1a and Dsc1 was significantly reduced in vaginal tissue from mice treated with DMPA or NET-EN (Fig. 3A). Although DSG1 protein levels were also significantly decreased in vaginal tissue from DMPA- and NET-EN–treated mice versus mice in estrus (Fig. 3B), more profound decreases in DSG1 protein were induced by DMPA treatment (Fig. 3C). To explore a potential mechanism for this differential decrease in DSG1 protein, we used vaginal tissue from DMPA- and NET-EN–treated mice to quantify the expression of kallikrein-related peptidases, serine proteases shown to cleave DSG1 protein (46, 47). Consistent with the reduced levels of DSG1 protein in vaginal tissue from DMPA- versus NET-EN–treated mice (Fig. 3C), we uncovered that DMPA treatment was associated with significantly higher levels of kallikrein-related peptidase 7 (Klk7), kallikrein-related peptidase 13 (Klk13), and kallikrein-related peptidase 14 (Klk14) (Fig. 3D). Taken together, these findings identified that DMPA and NET-EN similarly inhibited vaginal Dsg1a and Dsc1 gene expression and also suggested that the reduced levels of DSG1 protein detected in DMPA-treated mice were secondary to DMPA-mediated increases in vaginal kallikrein-related peptidase expression.

Prior report showed that although vaginal epithelial thinning was comparable among mice in diestrus and DMPA-treated mice, only the latter group displayed significant increases in genital mucosal permeability and susceptibility to genital HSV-2 infection (24). Because our current investigation identified that DMPA- and NET-EN–treated mice display similar increases in genital mucosal permeability (Fig. 2), we hypothesized that NET-EN– and DMPA-treated mice are equally susceptible to genital HSV-2 infection. As posited, we detected widespread genital tissue dissemination of fluorescent HSV-1 in DMPA- and NET-EN–treated mice, but not in mice infected in estrus (Fig. 4A). Consistent with these results, ivag infection of estrus-stage wild-type mice with 10⁴ PFUs of HSV-2 caused no morbidity or mortality, whereas infection of DMPA- and NET-EN–treated mice uniformly produced genital pathology and lethal encephalopathic changes (Fig. 4B–D). Although HSV-2 infection induced 100% mortality in mice treated with DMPA or NET-EN, more severe genital pathology and reduced survival times were generated by DMPA treatment (Fig. 4B–D).

We concluded the current investigation by using hPBMC-engrafted NSG mice to compare the effects of DMPA or NET-EN treatment on susceptibility to ivag infection with cell-associated HIV-1–_Ba-L. In initial studies, hPBMC-engrafted NSG mice (i.e., humanized mice) were untreated or administered DMPA or NET-EN. Fourteen days after hPBMC engraftment, untreated mice in estrus and mice treated with progestin were ivag inoculated with 2 × 10⁶ HIV-1–infected hPBMC. Two weeks postinfection, we saw 100, 65, and 0% survival in untreated controls, NET-EN–treated mice, and DMPA-treated mice, respectively (Fig. 5A). In separate studies, identically engrafted and progestin-treated NSG mice were ivag infected with a smaller inoculum of 10⁶ HIV-1–infected hPBMC. Euthanizing all mice 10 d postinfection, systemic HIV-1 infection was detected in 100% of mice treated with DMPA or NET-EN but in no mice infected in estrus. Compared with NET-EN–treated mice, however, DMPA-treated mice demonstrated significant increases in both infectious HIV-1 in cultured splenocyte supernatants (Fig. 5B) and HIV-1 RNA copy numbers in plasma (Fig. 5C). Although these studies identified uniform susceptibility to ivag infection with cell-associated HIV-1–_Ba-L in DMPA- and NET-EN–treated humanized mice, they also implied that DMPA is more likely than NET-EN to promote systemic spread of the virus after genital inoculation.

The favorable impact of contraception on maternal health and life expectancy (48) does not mitigate the need to comprehensively define the effects of DMPA and other long-acting reversible contraceptives on antivirus immunity and HIV transmission efficiency. Among currently available options, the relationship between DMPA and HIV susceptibility is the one most often examined in clinical investigation, and meta-analytic assessments of all available higher quality studies identified a 1.4-fold greater risk of HIV acquisition in women using DMPA versus no hormonal contraception (10). Although a relatively large number of studies were available for this risk estimate calculation, the efficiency of HIV transmission among women using LNG-IUS versus no hormonal contraception is mostly unexplored (49). Likewise, although no clinical study identified NET-EN as a significant HIV risk factor, relatively few of these studies were deemed of sufficient quality to be informative (10, 50). Moreover, among the five clinical studies considered suitably informative for inclusion in a meta-analytic review, four identified an increased but statistically nonsignificant risk of HIV acquisition among women using NET-EN versus no hormonal contraception (10, 50–55).

Because the effects of NET-EN have also been underexplored in experimental models of genital infection, we used the current study to compare genital mucosal barrier function and genital pathogen susceptibility in estrus-stage, DMPA-treated, and NET-EN–treated mice. These studies newly identify that mice with pharmacologically relevant serum levels of NET display significantly reduced genital expression of the cell–cell adhesion molecule DSG1, increased genital mucosal permeability, and increased susceptibility to genital HSV-2 and cell-associated HIV-1 infection. As mucosal barrier function is compromised in the genital tract of both DMPA- and NET-EN–treated mice, our findings further imply that these progestins share a biological effect with the capacity to promote genital pathogen transmission. Conversely, direct comparison of DMPA- and NET-EN–treated mice in the current investigation identifies DMPA as more likely to reduce vaginal DSG1 protein levels, facilitate deeper penetration of genital tissue by activated leukocytes, and promote systemic dissemination of genitally inoculated cell-associated HIV-1. The current investigation also reveals that treatment with DMPA is more likely than NET-EN to enhance genital tissue expression of kallikrein-related peptidases. As these proteases cleave cell–cell adhesion molecules, their increased expression in DMPA-treated mice may contribute to the lower levels of DSG1 protein and greater compromise of genital mucosal barrier function in these animals. However, alternative explanation exists for the differential effects of MPA and NET in the female mouse genitals. Prior research suggests NET has lower affinity for the PR than MPA (43, 56, 57), a characteristic of the drug that has potential to reduce in vivo NET potency. Likewise, unlike MPA, a portion of NET is aromatized in vivo to ethinyl estradiol, a potent estrogen with high estrogen receptor affinity (58). Because susceptibility of DMPA-treated humanized mice to genital infection with cell-associated HIV-1 is eliminated by concomitant treatment with exogenous estrogen (25), it is possible that aromatic conversion of NET to a more estrogenic compound in vivo makes NET-EN less likely than DMPA to enhance genital pathogen susceptibility.

Although defining precise mechanisms that cause greater compromise of antivirus immunity in the genital tract of DMPA- versus NET-EN–treated mice will require further investigation, our studies do establish that NET-EN treatment promotes mouse susceptibility to genital infection with HSV-2 and cell-associated HIV-1. Additional investigation will likewise be needed to resolve if NET-EN causes similar effects in women. Despite these limitations, it seems likely that current findings will inform interpretation of clinical data and help prioritize future clinical research. As analogous changes in genital DSG1 protein levels, genital mucosal barrier function, and genital mucosal permeability are seen in DMPA- and LNG-treated mice and women using DMPA and LNG (23, 59), our findings also imply that similar changes may occur in the genital tract of women using NET-EN. Likewise, current evidence that treating mice with DMPA or NET-EN significantly increases susceptibility to genital HIV-1 infection intimates that even large-scale clinical studies may have a reduced capacity to resolve variable HIV transmission rates in women using DMPA, NET-EN, LNG-IUS, or other forms of unopposed progestin. Our work may also have relevance for data interpretation from studies where all participants are assigned to use some form of reversible contraception or when participants are randomized to conceptive choices that have uncertain effects on HIV transmission. Although identifying the forms of long-acting reversible contraception least likely to promote HIV transmission remains both an important and daunting obligation, our findings provide a strong indication that these efforts will continue to be informed by exploration of murine and other appropriate experimental models.

We thank the Endocrine Technologies Core (Oregon National Primate Research Center) for the serum progestin levels that were quantified. HIV-1–_Ba-L was obtained through the National Institutes of Health (NIH) AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH (from Dr. Suzanne Gartner, Dr. Mikulas Popovic, and Dr. Robert Gallo).

The authors have no financial conflicts of interest.