HIV infection does not occur after every virus exposure. Rather, the transmission probability per exposure in women is 1 in 200 to 2000.1 The virus levels in the transmitting person are a key factor for transmission risk, but host factors also play a role. Among them, the physical properties of the vaginal epithelium and innate and adaptive mucosal immune factors impact susceptibility to vaginal infection. These factors can be influenced by estrogen and progesterone levels during pregnancy or contraceptive use2-5 and presumably also during the menstrual cycle. In the first, follicular phase of the menstrual cycle, high estrogen levels create an environment of heightened immunity against invading pathogens (reviewed in Ref. 6). During the second, luteal phase of the cycle with high progesterone levels, immunity is reduced, allowing potential implantation of an allogeneic embryo. We hypothesized that these milieu alterations in the follicular and luteal phases of the menstrual cycle would impact macaque susceptibility to SHIV infection. It is well established that the administration of synthetic progesterone enhances simian immunodeficiency virus (SIV) transmission in macaques, probably through the thinning of the vaginal epithelial lining, depression of certain immune responses, an increase in vaginal pH, altered microflora, and reduced cervical mucus.7-9 Susceptibility fluctuations to HIV or SIV during the menstrual cycle have been postulated before.6 Sodora et al7 found that, in the follicular phase, only 1 of 7 rhesus macaques exposed to SIV became infected, whereas 3 of 6 became infected in the luteal phase. The difference was not statistically significant due to the small number of study animals. Thus, further examination of menstrual cycle impact on the susceptibility to SIV or SHIV infection is needed.
To address the influence of the undisturbed menstrual cycle on susceptibility to SHIV infection, we use adult female pigtail macaques, which have continuous lunar menstrual cycles as do humans. We infected them with SHIVSF162P3, a CCR5-using virus strain that can be mucosally transmitted.10 Cycle lengths in pigtail macaques are an average of 32.8 days, with ovulation occurring between days 17 and 19.11 Our hypothesis was that animals would be more vulnerable to infection during the luteal phase when progesterone levels are high. Initial viremia would then be detected more often in the follicular phase of the menstrual cycle after a 7- to 14-day viral eclipse phase following viral transmission.
MATERIALS AND METHODS
Nineteen adult, female, pigtail macaques were cycling normally based on visual inspection for menstrual blood and progesterone measurements. We performed a retrospective analysis of macaques previously enrolled in untreated or placebo-treated control arms of prevention trials, where they received repeat low-dose SHIV exposures without administration of hormones.12,13 Experiments were performed according to National Institutes of Health (NIH) guidelines on the care and use of laboratory animals, approved by the Institutional Animal Care and Use Committees of both the Centers for Disease Control and Prevention (CDC) and Yerkes National Primate Research Center (Atlanta, GA).
Virus Challenges, Hormone Analysis
Macaques were intravaginally challenged once (n = 4) or twice (n = 15) weekly for up to 14 weeks with SHIVSF162P3 (Fig. 1A) obtained from the NIH AIDS Research and Reference Reagent Program,10 followed by propagation on pigtail peripheral blood mononuclear cells.13 Stocks were titrated on fresh peripheral blood mononuclear cells to determine the tissue culture infective dose (TCID)-50 and titrated in vivo to cause infection after an average 2-6 vaginal challenges (data not shown). Fourteen animals were challenged with stock 1 (10 TCID50),13 5 with a more recent stock 2 (n = 1, 10 TCID50; n = 4, 50 TCID50). Viral load and progesterone were measured in blood collected immediately before each challenge. Plasma viral RNA was measured by reverse-transcriptase polymerase chain reaction12,13 with a detection limit of 50 copies per milliliter. Virus exposures were stopped at signs of initial viremia. For progesterone measurements, plasma was frozen at −80°C and shipped to the University of Wisconsin National Primate Research Center.14
A two-sided one sample test for proportion was conducted using SAS 9.2 (SAS institute, Cary, NC) to examine if initial viremia detection in the luteal phase was different from that in the follicular phase. Day 1 of the menstrual cycle was defined as the time point after the steepest progesterone decline.
We determined the time point of infection relative to the menstrual cycle. Figures 1B and C show representative progesterone data used to determine day 1 of the menstrual cycle for 2 macaques (remaining data are found in Supplemental Digital Content 1, http://links.lww.com/QAI/A173). Based on progesterone data, the median cycle length was 32 days among our pigtail macaques, determined from 18 complete cycles, and in accordance with published data.11 Using this median, animal cycles were divided into the follicular (days 1-16) and luteal phases (days 17 and up). The time point of initial viremia was found to be in the follicular phase for 18 macaques (95%), as compared with that in the luteal phase for only 1 macaque (5%; Fig. 2A). The difference was statistically significant (P value <0.0001). Thus, the number of infections detected in the follicular phase was 18 times as high as during the luteal phase. The mean occurrence of initial viremia was on day 6 of the cycle. There is a viral eclipse phase of approximately 7-14 days before nascent SHIVSF162P3 infection is detectable in this model (Fig. 1A, and Ref. 12). We thus calculated a window of highest susceptibility and of most frequent virus transmission late in the luteal phase, between days 24 and 31 of the menstrual cycle (Fig. 2B). This calculation was done by subtracting 7 - 14 days (eclipse phase) from day 6 (mean occurrence of initial viremia) of a 32-day cycle, which gave us an estimated susceptibility window between days 24 and 31. The median number of challenges required for infection was 4 for the detection of infection in the follicular phase, whereas it was 10 in the animal with initial viremia in the luteal phase. This observation was consistent with rapid infection when macaques started virus challenges in the luteal phase.
This study represents the first demonstration of a significant link between viral susceptibility and phase of menstrual cycle in macaques. Such a link has been suspected before6,7 but has not been demonstrated with statistical significance in macaques or in humans. We show that viremia is detected significantly more often in the follicular phase when compared with the luteal phase. Due to an eclipse phase of 7-14 days before nascent infection is detected in our experimental model, we can conclude that the susceptibility to vaginal SHIV infection is significantly elevated in the luteal phase of the menstrual cycle.6,15 Our findings are consistent with a postulated—but not demonstrated—susceptibility window to HIV infection for this phase of the cycle in humans,6 based on a reported depression of immunity for this hormonal period characterized by high progesterone levels. Reduced immunity is thought to prevent an immune attack directed at a potentially implanting fertilized egg that harbors foreign epitopes. The estimated window of SHIV susceptibility relative to the luteal phase is schematically represented in Figure 2B, together with a summary of different biologic changes associated with menstrual cycle phases.3,15,16
Our results are supported by earlier observations in a small number of rhesus macaques SIV challenged at different times of the menstrual cycle. The study also reported increased vulnerability to SIV infection in the luteal phase, although the authors could not ascertain this association with statistical significance.7 Rhesus macaques have seasonally varying cycles, with longer cycles in summer, and significant atrophy of vaginal epithelial lining during the luteal phase of their cycle.15 Untreated pigtail macaques, on the other hand, have continuous lunar cycles as do humans and are therefore relevant for human HIV infection.
In humans, although still being debated, HIV acquisition is reported to be more efficient in several high-progesterone situations including pregnancy and oral contraceptive usage (reviewed in Ref. 17), suggesting that progesterone engenders an environment conducive to viral entry and transmission in both humans and macaques. Our study thus adds to the growing body of research linking HIV-1 transmission and progesterone levels.
Study limitations include lack of daily progesterone measurements, which could have generated a more complete picture of the menstrual cycle. Also, nonavailability of frequent samples for estradiol measurements resulted in the inability to pinpoint the exact time of ovulation. Another limitation was that we used archived samples from completed studies in an effort to reduce animal numbers in research, and this precluded collection of mucosal tissues, the analyses of which could have shed light on local tissue changes, including the distribution of immune cells and the thickness of vaginal epithelium. In addition, we used an eclipse phase of 7-14 days to calculate when transmission occurred. The exact eclipse phase in each individual macaque was not known in our study, because it is not possible to determine which of the repeated challenges caused infection. However, of the 19 macaques, none showed evidence of viremia between days 1 and 6 after the first challenge, and 1, 3, and 3 macaques had measurable viremia on days 7, 10, and 14 after first challenge, respectively (Fig. 1A). We therefore used a wide range of 7-14 days for an estimated eclipse phase. This is consistent with a recent report of an 8.5-day median eclipse phase after challenge with a low rectal SIV dose, resulting in a 33% infection rate in exposed monkeys.18 Nonetheless, there remains the possibility that individual pigtail macaques in our study, challenged vaginally, had longer or shorter eclipse phases.
HIV prevention methods are needed for women at all reproductive stages, for example, for women with undisturbed menstrual cycles, for women on contraceptives, and for pregnant or menopausal women. Animal testing of female HIV prevention strategies could be improved by deliberate testing throughout undisturbed menstrual cycles and also in high progesterone conditions. Researchers have treated pigtail and rhesus macaques with Depo-Medroxyprogesterone acetate to obtain consistent rates of SIV infection, a practice that abrogates menstrual cycling and induces a prolonged state of high progesterone and high susceptibility.19,20 This may perturb specific susceptibility factors for SIV or SHIV infection, including mucosal antibodies, levels of mucus and mucins,21 and immune mediators such as defensins, elafin, and secretory leukocyte protease inhibitor (reviewed in Ref. 22). Depo-Medroxyprogesterone acetate-treated macaques may be an appropriate model for high progesterone conditions such as pregnancy or progesterone-based contraception but not for normally cycling women. We suggest that untreated pigtail macaques with their continuous lunar cycles similar to humans are a suitable animal model for the testing of HIV prevention strategies designed for women of reproductive age and not using hormonal contraception.
Our study raises awareness of significant and fundamental aspects of HIV transmission in women that are not yet fully explored, despite the fact that vaginal HIV transmission is responsible for more new cases of HIV infection than any other known route.1 A better understanding and further characterization of mucosal immune factors that are influenced by reproductive hormones could lead to the identification of natural resistance factors for HIV infection, with potential to develop novel prevention strategies.
The authors thank J. Mitchell, Dr F.J. Novembre, Dr E. Strobert, Dr K.S. Paul, and the animal care staff at both Yerkes National Primate Research Center and the CDC for animal procedures; Dr J.M. Smith for valuable scientific input; Dr T.E. Ziegler and D. Wittwer (University of Wisconsin National Primate Research Center) for progesterone measurements and advice on menstrual cycle analysis; Caryn Kim for administrative leadership. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH: SHIVSF162P3 (catalog#6526) from Drs Janet Harouse, Cecilia Cheng-Meyer, Ranajit Pal and the DAIDS, National Institute of Allergy and Infectious Diseases.
1. Hladik F, Hope TJ. HIV infection of the genital mucosa in women. Curr HIV/AIDS Rep
2. Gray RH, Li X, Kigozi G, et al. Increased risk of incident HIV during pregnancy in Rakai, Uganda: a prospective study. Lancet
3. Sheffield JS, Wendel GD Jr, McIntire DD, et al. The effect of progesterone
levels and pregnancy on HIV-1
coreceptor expression. Reprod Sci
4. Baeten JM, Lavreys L, Sagar M, et al. Effect of contraceptive methods on natural history of HIV: studies from the Mombasa cohort. J Acquir Immune Defic Syndr
5. Morrison CS, Chen PL, Kwok C, et al. Hormonal contraception and HIV acquisition: reanalysis using marginal structural modeling. AIDS
. 17;24:1778- 1781.
6. Wira CR, Fahey JV. A new strategy to understand how HIV infects women: identification of a window of vulnerability during the menstrual cycle
7. Sodora DL, Gettie A, Miller CJ, et al. Vaginal transmission of SIV: assessing infectivity and hormonal influences in macaques inoculated with cell-free and cell-associated viral stocks. AIDS Res Hum Retroviruses
8. Marx PA, Spira AI, Gettie A, et al. Progesterone
implants enhance SIV vaginal transmission and early virus load. Nat Med
9. Smith SM, Mefford M, Sodora D, et al. Topical estrogen protects against SIV vaginal transmission without evidence of systemic effect. AIDS
10. Harouse JM, Gettie A, Tan RC, et al. Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science
11. Blakley GB, Beamer TW, Dukelow WR. Characteristics of the menstrual cycle
in nonhuman primates. IV. Timed mating in Macaca nemestrina. Lab Anim
12. Parikh UM, Dobard C, Sharma S, et al. Complete protection from repeated vaginal simian-human immunodeficiency virus exposures in macaques by a topical gel containing tenofovir alone or with emtricitabine. J Virol
13. Otten RA, Adams DR, Kim CN, et al. Multiple vaginal exposures to low doses of R5 simian-human immunodeficiency virus: strategy to study HIV preclinical interventions in nonhuman primates. J Infect Dis
14. Saltzman W, Schultz-Darken NJ, Scheffler G, et al. Social and reproductive influences on plasma cortisol in female marmoset monkeys. Physiol Behav
15. Poonia B, Walter L, Dufour J, et al. Cyclic changes in the vaginal epithelium of normal rhesus macaques. J Endocrinol
16. Trunova N, Tsai L, Tung S, et al. Progestin-based contraceptive suppresses cellular immune responses in SHIV
-infected rhesus macaques. Virology
17. Hel Z, Stringer E, Mestecky J. Sex steroid hormones, hormonal contraception, and the immunobiology of human immunodeficiency virus-1 infection. Endocr Rev
18. Liu J, Keele BF, Li H, et al. Low dose mucosal SIV infection restricts early replication kinetics and transmitted virus variants in rhesus monkeys. J Virol
19. Jiang Y, Tian B, Saifuddin M, et al. RT-SHIV
, an infectious CCR5-tropic chimeric virus suitable for evaluating HIV reverse transcriptase inhibitors in macaque models. AIDS Res Ther
20. Veazey RS, Shattock RJ, Pope M, et al. Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1
gp120. Nat Med
21. Lai SK, Hida K, Shukair S, et al. Human immunodeficiency virus type 1 is trapped by acidic but not by neutralized human cervicovaginal mucus. J Virol
22. Ghosh M, Fahey JV, Shen Z, et al. Anti-HIV activity in cervical-vaginal secretions from HIV-positive and -negative women correlate with innate antimicrobial levels and IgG antibodies. PLoS One