Levonorgestrel and Female Genital Tract Immunity: Time for a Closer Look

As so few publications have explored the immunomodulatory effects of the exogenous progestin levonorgestrel (LNG), it was interesting to read the results from the investigation by Eastman et al that evaluated the course of genital Chlamydia trachomatis infection in olive baboons administered a human-use LNG-releasing intrauterine system (LNG-IUS) prior to infection [1]. Although this study failed to identify significant differences in the incidence of pelvic inflammatory disease in untreated versus LNG-treated baboons, it reinforced findings from an earlier publication involving these same animals that reported LNG-IUS administration was associated with delayed Chlamydia clearance [2]. This important finding supports results from an observational study that found impaired genital clearance of high-risk human papillomavirus in women using a LNG-IUS [3] and from our work in a murine model of intranasal C. trachomatis infection (in which susceptibility is not affected by sex steroids) that saw LNG treatment hinder dendritic cell activation and function, formation of Chlamydia-specific T-cell immunity, and pulmonary chlamydial clearance [4]. Although the studies by Eastman et al did not demonstrate a mechanism for the impaired chlamydial clearance seen in baboons in which LNG-IUS was inserted prior to infection, our mouse model findings indicate that the formation of protective Chlamydia-specific T-cell responses in baboons may have been similarly impaired by LNG treatment.

Surprisingly, Eastman et al reported no significant differences in susceptibility to Chlamydia infection in untreated controls versus LNG-treated baboons [1]. Numerous murine and nonhuman primate genital infection models use the exogenous progestin depot-medroxyprogesterone acetate (DMPA) to achieve uniform susceptibility to infection [5], and we found that in vivo treatment of mice with LNG or DMPA dramatically increased genital mucosal permeability and made mice uniformly susceptible to genital herpes simplex virus type 2 infection [6]. We further identified analogous diminution of genital mucosal barrier function in women initiating use of DMPA or LNG-IUS [6, 7]. While comparable susceptibility to Chlamydia infection in untreated baboons and animals administered LNG-IUS in the study by Eastman et al implies IUS insertion itself was not a risk factor for infection, repetitive inoculation of 10⁷ inclusion-forming units (IFU) of C. trachomatis directly onto the ectocervix of study animals may have impaired the ability of this study to detect between-group differences in susceptibility to Chlamydia infection. Studies in mice show that C. trachomatis more effectively infects the cervical epithelium than the vaginal epithelium [8], while the Chlamydia inoculum of 10 million IFU used to infect baboons in the Eastman study are considerably greater than the 10²–10⁴C. trachomatis bacterial load associated with human sexual transmission [9].

To understand the LNG-mediated effects on susceptibility to C. trachomatis infection with biologically relevant infectious inoculums, we used 10⁴ IFU of C. trachomatis serovar D elementary bodies (VR-885; ATCC, Manassas, VA) in 10 μL of sucrose-phosphate-glutamate buffer to intravaginally infect 8-week-old wild-type Balb/cJ mice (Jackson Laboratory, Bar Harbor, ME) that were in the estrus stage of the estrous cycle or intravaginally administered 1.25μg of LNG suspended in 10μL of phosphate-buffered saline daily for 8 days (from 5 days before until 3 days after infection). Using methods described elsewhere [10] and approved by The Ohio State University Institutional Animal Care and Use Committee, cervicovaginal lavages collected at indicated days after infection to quantify levels of C. trachomatis DNA by quantitative reverse transcription polymerase chain reaction analysis showed that 100% of LNG-treated mice were infected with C. trachomatis, while all mice infected during estrus were resistant to infection (Figure 1). This finding adds to the sparse but accumulating evidence from experimental models indicating that LNG alters host susceptibility to genital infection and weakens both innate defense (ie, genital mucosal barrier function) and pathogen-specific cell-mediated immunity. These data further indicate that LNG and DMPA comparably inhibit host immunity, while implying these progestins similarly affect a woman’s susceptibility to genital infection. It thus seems essential to fully define LNG immunomodulatory effects in appropriate preclinical animal models and conduct clinical studies that explore changes in infection susceptibility mediated by LNG-IUS use and define LNG-mediated effects on the development of pathogen-specific immunity and rates of pathogen clearance.

Figure 1.

Levonorgestrel (LNG) increases mouse susceptibility to genital Chlamydia trachomatis infection. Untreated mice in estrus and LNG-treated mice were intravaginally inoculated with 10⁴ inclusion-forming units of C. trachomatis serovar D elementary bodies. Cervicovaginal lavages (CVL) were obtained at indicated time points, and C. trachomatis DNA levels were measured by quantitative reverse transcription polymerase chain reaction analysis. The left panel shows C. trachomatis DNA levels, and the right panel depicts total C. trachomatis load calculated as area under the curve (AUC) of each animal. Displayed data were pooled from 2 independent experiments with 5 animals per group in each experiment. Values are median (range). ND, nondetectable.

Notes

Disclaimer.  The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.

Financial support. This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD072663), The Ohio State University College of Medicine, and Stanford University School of Medicine.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.