Selective expression of granulysin in discrete menstrual phases

From the University of Washington and the Hladik lab, Vaccine and Infectious Disease Division

The cervicovaginal tract (CVT) is the first point of entry for both pathogens and sperm, making this tissue site unique in its ability to protect against infection while also allowing for reproduction. This paradox requires the CVT to be a tightly controlled immune environment, and changes to this regulation can lead to either infertility or the acquisition of sexually transmitted infections (STIs). Endogenous sex hormone cycling during the menstrual cycle, as well as hormonal contraceptives, drastically affect mucosal immune responses. “Sex hormones vary during the menstrual cycle, and these variations affect immunity in the female reproductive tract and susceptibility to STIs. Previous studies have suggested that immunity is suppressed during the luteal phase of the menstrual cycle, when the sex hormone progesterone is high,” said Sean Hughes, a researcher from the Hladik lab (Vaccine and Infectious Disease Division) and the lead author of a recent publication in the American Journal of Reproductive Immunology. Nevertheless, the immune effects of the female sex hormones estradiol and progesterone remain far from completely understood, “and even less is known about the effect of progestin-containing contraceptive vaginal rings (CVR; such as the NuvaRing), so we compared immunity during CVR use to both phases of the menstrual cycle,” Hughes explained. Vaginal delivery of novel contraceptives and microbicides is an enticing strategy. However, the baseline effects of sex hormones on CVT immunity must be defined before safely introducing hormone or microbicide products without the risk of decreased immunity to STI.

To characterize the baseline effects of sex hormones on the CVT’s immune regulation, Hughes and colleagues from the University of Washington compared gene expression and secreted cervicovaginal immune mediators at two phases of the menstrual cycle and in the presence of hormonal contraception. The authors analyzed vaginal cytobrush samples collected during the follicular phase, the luteal phase, and after one month of CVR from 17 adult women enrolled in a study of the NuvaRing, a contraceptive device that contains ethinyl estradiol and etonogestrel, forms of estrogen and progestin, respectively. They assessed immune-related gene expression by microarray at each of the time points. The findings suggest that immune-related genes are expressed most highly during CVR use, followed by the follicular and then luteal phases. This continuum is in line with previous findings and the “window of vulnerability” hypothesis, which suggests that CVT immune responses are dampened during the luteal phase. This tradeoff may increase the likelihood of conception while it also may increase the risk of STI acquisition.

Graphical abstract summarizing GNLY and immune mediator expression at different phases of the estrus cycle.
Graphical abstract summarizing GNLY and immune mediator expression at different phases of the estrus cycle. Figure provided by Dr. Hladik and Claire Levy.

A second aim of this study was to compare immune-related gene expression across distinct anatomical compartments of the CVT. Although the current study included only samples from the cervicovaginal compartment, previous studies have collected gene expression data from other female reproductive tract tissues such as fallopian tubes, endometrium, endocervix, and ectocervix. Hughes and colleagues re-analyzed these existing data sets to contextualize their findings. “Many studies have been published comparing gene expression during the phases of the menstrual cycle in individual anatomic sites throughout the female reproductive tract. To place our study in a wider context, we combined data from our and seven other studies. This reanalysis gave us a picture of gene expression throughout all the major sites of the female reproductive tract,” Hughes said. They found that gene expression patterns varied by location: “in the upper reproductive tract (fallopian tubes and uterus), immune-related gene expression tended to be higher during the luteal phase. The opposite was true in the cervix, where immune-related gene expression was higher during the follicular phase. In all sites, gene expression related to cell proliferation was higher during the follicular phase,” Hughes said. “The anatomic differences we observed in gene expression during the menstrual cycle show that immunity is not uniform throughout the female reproductive tract and that anatomic context is important,” Hughes said.

However, not all their results were expected. “Surprisingly, we found that despite containing a progesterone-like compound”—which is known to have immunosuppressive properties— “the NuvaRing was associated with higher immune-related gene and protein expression than the luteal phase or even the follicular phase,” Hughes explained. Furthermore, an “intriguing and unexpected finding was that the antimicrobial protein granulysin (GNLY) was highly expressed in the vagina during the follicular phase of the menstrual cycle and nearly absent during the luteal phase,” Hughes said. Although GNLY has been previously implicated in maintaining fetal-maternal tolerance in pregnancy, it’s not yet clear how GNLY affects CVT immunity, but the authors “have submitted a grant application to study the role of GNLY in cervicovaginal immunity. The profound change in levels of GNLY between the follicular and luteal phases suggests that it plays an important cyclical role. We think GNLY may play a role in protecting against STIs without harming the natural vaginal microbiota,” Hughes said. Going forward, the authors are also exploring how GNLY could be used as a biomarker of the follicular phase in vaginal study samples.

Hughes SM, Pandey U, Johnston C, Marrazzo J, Hladik F, Micks E. Impact of the menstrual cycle and ethinyl estradiol/ etonogestrel contraceptive vaginal ring on granulysin and other mucosal immune mediators. Am J Reprod Immunol. 2021 Feb 28;e13412. doi: 10.1111/aji.13412. Online ahead of print.