Genital herpes simplex virus-2 (HSV-2) is the leading cause of genital ulcer disease worldwide.1 The HSV-2 increases the risk of human immunodeficiency virus (HIV) acquisition 2- to 3-fold, and nearly half of new HIV infections are attributable to previous infection with HSV-2 in high prevalence settings.2 Genital ulcers also increase the risk of HIV transmission.3 Neonatal herpes, which can be fatal or associated with permanent impairment, is another serious clinical consequence of HSV-2 which is estimated to affect 14,000 infants per year worldwide.4
The vast majority of new HSV-2 infections are transmitted from partners who have no history of genital ulcers.5 Even among persons with a known history of genital herpes, most transmissions occur in the absence of lesions.6 Few determinants of shedding rates have been described and the unpredictability of viral shedding is frustrating to patients.7 Though symptomatic infections are associated with a greater frequency of shedding, asymptomatic persons have been found to shed HSV-2 in the genital tract on approximately 10% of days.8 There is substantial individual variation in shedding frequency and quantity.9 A greater quantity of HSV-2 is detected in the presence of lesions compared to days without lesions. However, when no lesions are present, persons with symptomatic and asymptomatic HSV-2 infections appear to shed similar quantities of virus.8
The effect of female sex hormones on HSV-2 shedding and lesions is poorly understood. Previous studies suggest that vaginal immunity varies throughout the menstrual cycle in response to changes in endogenous sex hormones.10–12 The luteal phase of the cycle, which occurs after ovulation and is characterized by a specific hormonal profile including elevated serum progesterone and estradiol, is associated with increased inflammatory cytokines and decreased innate immune factors.10,13,14 Increased susceptibility to sexually transmitted infections has also been noted during this phase in animal models.12 Whether the frequency of HSV shedding or lesion occurrence varies throughout the menstrual cycle is not known. However some women anecdotally report more genital herpes outbreaks during menses and 1 study of the natural history of untreated genital herpes suggested such an association.15 Given the delay between known immune triggers (mucosal trauma or fever, for instance) and HSV-2 reactivation in neurons with latent infection, it is possible that a reduction in immunity during the luteal phase would lead to an increased risk of viral reactivation and the development of shedding or lesions during the subsequent weeks.
We aimed to determine whether there is an association between menstrual cycle phase, hormonal contraceptive use, and HSV-2 shedding or lesions in a large cohort of women. We hypothesized that HSV-2 shedding and lesions would be more frequent in the follicular phase of the cycle among women not using hormonal contraception and more frequent among hormonal contraceptive users.
MATERIALS AND METHODS
Participants
The HSV-2 seropositive women between ages 18 and 46 years were enrolled into prospective studies at the University of Washington Virology Research Clinic (Seattle, Washington) between February 1991 and September 2016. Women with and without a history of genital lesions were eligible for this analysis if they were seropositive for HSV-2, recorded menstrual dates using a daily diary and provided information regarding use of hormonal contraception. Women within a year of primary HSV-2 infection (laboratory documented or based on self-report) were excluded to remove potential confounding by time since acquisition. Individuals who were using antiviral medication at the time of the swabbing session were excluded from the analysis. This study was approved by the Institutional Review Board at the University of Washington.
Sample and Data Collection
Participants were instructed in the once-daily collection of mixed anogenital swabs as previously reported.8,16 Briefly, participants inserted a single Dacron swab into the vagina for 1 minute. The swab was then used to sample genital secretions from bilateral labia majora and minora, perineum, and perianal region, then placed into a 1-mL tube containing 1× PCR buffer. Women completed the same swabbing sequence in the presence and absence of menses. Participants completed a daily diary noting genital symptoms and lesions, sexual activity, and menstrual bleeding. Hormonal contraceptive use was determined based on participant self-report at the time of swabbing session initiation, typically indicated by a yes or no question; specific type of hormonal contraception was not recorded. For women with multiple swabbing sessions (distinct periods of time in which they collected daily swabs), we included only the first 2 sessions. Only the first session was included if contraceptive status changed over time.
Laboratory Studies
The HSV serostatus was determined by the University of Washington HSV Western Blot.17 To assess the presence of HSV-2 genital shedding, HSV DNA on genital swabs was detected with real-time fluorescence-based quantitative polymerase chain reaction (PCR) using primers to glycoprotein B, as previously described.18
Statistical Analysis
The HSV shedding frequency was defined as the number of days with genital swabs with HSV detected divided by the total number of days with genital swabs collected. Similarly, lesion frequency was defined as the number of days with reported genital lesions divided by the total number of days with diary data.
The menstrual cycle analysis included only women who indicated they were not using hormonal contraception. The HSV shedding and lesion data were included if they were within 14 days of recorded first day of menstrual bleeding. The menstrual cycle was divided into phases based on most proximate first day of menstrual bleeding. Follicular phase was defined as days 0 to 13 after the first day of bleeding, and luteal phase was defined as days 1 to 14 before bleeding. We further subdivided the follicular and luteal phases into four 7-day phases (early follicular: 0 to 6 days after first day of menses; late follicular: 7 to 13 days after first day of menses; early luteal: 8 to 14 days prior to first day of menses; late luteal: 1 to 7 days prior to first day of menses). Frequencies of HSV shedding were calculated by menstrual phase, and we assessed differences in shedding frequencies by menstrual phase using a Poisson mixed model, including a random intercept for individual to account for the correlation in repeated measures within individuals. In the analysis of genital lesions, we relied on a nonparametric approach due to sparse data. We used the Wilcoxon signed-rank test for paired data to compare lesion frequencies at different phases of the menstrual cycle. Due to concerns about misclassification of menstrual phase for days further from first day of menses, a sensitivity analysis was conducted using only measures taken within 10 days of the start of menses.
The HSV shedding frequency was compared in women using versus not using hormonal contraception with a Poisson mixed model. Lesion frequencies were compared between groups using the Wilcoxon rank-sum test. Assuming that most women on hormonal contraception in this population use cyclic oral contraceptives and initiate a hormone-free interval (placebo pills) just prior to menses, we used similar analyses to determine whether HSV shedding or lesion frequencies were associated with menses (cycle days 0 to 6) in hormonal contraceptive users.
For both the menstrual cycle analysis and the hormonal contraception analysis, the mean duration of HSV-2 shedding episodes and the quantity of HSV-2 DNA were compared using linear mixed effects models. A 2-sided P value less than 0.05 was considered to be statistically significant.
RESULTS
We identified 244 eligible women who collected 12,059 daily genital swabs and recorded a lesion diary on 13,237 days. The median age was 32 years (range, 19–46). The median time since HSV-2 infection was 6 years (range, 1–28 years), and women had a median of 4 genital herpes recurrences per year (range, 0–28).
Menstrual Cycle Phase
Among the 189 women who reported not using hormonal contraception, HSV DNA was detected on 1030 of 4936 days (20.9%) in the follicular phase of the cycle compared to 726 of 4073 days (17.8%) in the luteal phase of the cycle (rate ratio [RR], 1.19; 95% confidence interval [CI], 1.03–1.37; P = 0.02). Figure 1A demonstrates shedding rates among participants when menstrual cycle was further divided into early and late follicular, and early and late luteal. Though shedding frequency appeared higher in the early and late follicular phases, none of the individual comparisons were statistically significant.
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Figure 1: Bar plots of overall HSV-2 shedding frequency (A) and lesion frequency (B) at different phases of the menstrual cycle.
Genital lesions were reported on 687 (12.8%) of 5372 days in the follicular phase versus 478 (10.7%) of 4480 days in the luteal phase, though this difference was not statistically significant (P = 0.07) (Fig. 1B). Median duration of shedding episodes was 3 days for the follicular phase (range, 1–32 days) versus 2 days for the luteal phase (range, 1–17 days), and this difference was not statistically significant (P = 0.61). On days HSV-2 DNA was detected, the quantity of virus shed was the same (4.7 log10 copies/mL in the follicular and luteal phase; P = 0.61).
These findings were reproduced in the planned sensitivity analysis limited to data within 10 days before or after the first day of menses. HSV detection was higher in the follicular phase (21.4%) versus the luteal phase (17.3%) (RR, 1.25; 95% CI, 1.06–1.48, P = 0.01). Lesion frequency was also higher in the follicular phase (13.1% vs 10.2%, P = 0.05).
Hormonal Contraception
Sixty-eight (28%) of the 244 participants reported using hormonal contraception during the study period (Table 1). Women who did and did not use hormonal contraception were similar in age, number of genital herpes recurrences per year, and time since HSV-2 infection. HSV-2 DNA was detected on 675 of 3691 daily swabs (18.3%) for those using hormonal contraception and 2036 (19%) of 10715 swabs for women not using hormonal contraception (RR, 1.16; 95% CI, 0.75–1.79; P = 0.50) (Table 2). Genital lesions were present on 320 of 3677 days (8.7%) for those using hormonal contraception and 1272 of 11447 days (11.1%) for women not using hormonal contraception (P = 0.66). Scatter plots of individual shedding and lesion frequencies are shown in Figure 2. The duration of HSV-2 shedding episodes and the quantity of HSV-2 on the days that HSV was detected were similar for women using hormonal contraception versus not using hormonal contraception (P = 0.61 and P = 0.32, respectively) (Table 2).
TABLE 1: Characteristics of Study Participants Using Versus Not Using Hormonal Contraception
TABLE 2: HSV-2 Shedding and Lesions by Hormonal Contraceptive Use
Figure 2: Scatter plots of individual HSV-2 shedding frequencies (A) and genital lesion frequencies (B) comparing women using and not using hormonal contraception.
Among women who reported using hormonal contraception, HSV-2 shedding and lesion frequency were not found to differ during cycle days 0 to 6 compared with days 7 to 14 after first day of menses and 1 to 14 days before menses. HSV-2 DNA was detected on 127 of 809 swabs (15.7%) collected within the 7 days after the first recorded day of menstrual bleeding, and 436 of 2241 swabs (19.5%) collected during the remainder of a 28-day cycle (RR, 1.21; 95% CI, 0.91–1.59, P = 0.19). Lesions were present on 63 of 871 days (7.2%) during this first week, compared to 238 of 2413 days (9.9%) in the remainder of the cycle (P = 0.11).
DISCUSSION
This study represents the most comprehensive analysis to date of the association between HSV-2 shedding and lesions and menstrual cycle phase and hormonal contraceptive use. Among women with established HSV-2 infection who are not using hormonal contraception, the follicular phase of the cycle is associated with a higher frequency of HSV-2 shedding compared to the luteal phase. Based on our analysis limited to data within 10 days prior to or after the start of menses, HSV-2 DNA detection is approximately 25% more likely to occur during the follicular phase. A similar trend was found in the frequency of genital lesions by phases of the menstrual cycle. Hormonal contraception use was not significantly associated with genital HSV-2 DNA detection or lesions.
The biological mechanisms underlying the difference in HSV-2 shedding frequency observed at different phases of the menstrual cycle are unknown. HSV-2 resides in the sacral ganglia, and during reactivation, travels down the axon to skin and mucosal surfaces.19 A strong immune response within epithelial tissues is required to respond to frequent short bursts of genital shedding to attenuate viral replication and prevent the development of genital lesions. Prior studies have demonstrated that markers of innate and adaptive immunity and immune cell populations in the upper and lower genital tract vary during the menstrual cycle, and HSV-2 shedding may be impacted by such variations in immunity.10,12,14 Though studies are inconsistent, the luteal phase of the cycle appears to be associated with a decrease in innate immune factors and an increase in inflammatory cytokines,14,20 which potentially may lead to increased HSV-2 DNA detection in the subsequent week. It is interesting that the genital shedding of HIV and cytomegalovirus have been found in some studies to vary throughout the menstrual cycle.21,22
In this study, we did not detect a statistically significant association between HSV-2 shedding and lesions and hormonal contraception. The few available studies that have explored an association between HSV-2 shedding and hormonal contraceptive use have had inconsistent findings. An early study of HSV shedding among 27 women collecting daily swabs for viral culture found that contraceptive use and menstrual cycle were not associated with genital HSV-2 shedding.23 However, use of viral culture is about one fourth as sensitive as PCR, and in this study viral shedding was rare, limiting the power of the study.24 Cherpes et al collected vaginal swabs every 4 months for a year from 330 HSV-2 seropositive women which included both hormonal contraceptive users and nonusers.25 Overall, 9% of swabs had HSV-2 detected. The authors found that users of oral contraceptive pills or depot medroxyprogesterone acetate (DMPA) were approximately twice as likely to shed HSV-2. In the present study, participants collected daily samples providing a more precise measurement of HSV-2 shedding, a greater proportion of swabs were positive for HSV-2, and our analysis excluded women with HSV-2 seroconversion within the past year.
Most studies of hormonal contraception and HSV-2 acquisition have focused on injectable progestin contraceptives such as DMPA. This contraceptive method causes a variety of changes in innate and adaptive immunity and the vaginal microbiome.26 In a mouse model, both DMPA and levonorgestrel increased genital mucosal permeability and susceptibility to HSV-2.27 Among HSV-2 seronegative women, DMPA may increase the risk of HSV-2 acquisition. In a prospective cohort study of 682 HSV-2–negative women in Uganda women who consistently used DMPA had a 2-fold higher risk of HSV-2 acquisition.28 A recent study of sex workers in Canada found a 4-fold increased risk of HSV-2 acquisition among DMPA users.29 These observations may be related to decreased serum estradiol levels with DMPA and hypoestrogenic effects, which occur less commonly with other methods of hormonal contraception.
This study has several important limitations. First, menstrual cycle phase was estimated by the first day of bleeding reported in participant menstrual diaries. For most women, the shedding session encompassed 1 to 2 menstrual cycles. Cycle phase was not confirmed with serum progesterone testing, and whether study participants were ovulatory or had generally regular menstrual cycles was unknown; some participants may have had abnormal bleeding unrelated to menstruation. Length of cycle phases is highly variable among women, with the follicular (or proliferative) phase ranging from 10 to 23 days, and decreasing as women age.30 The luteal (or secretory) phase is less variable between women and within individuals, but can range from 7 to 19 days. We found a trend but not a significant difference in lesion rate during the follicular phase of the menstrual cycle. As noted in other natural history studies, lesions occurred on approximately half of the days with shedding;8 the lower frequency of lesions results in more limited power to detect differences. We lacked detail regarding specific type of hormonal contraception used, compliance, and specific formulation and dose for oral contraceptive users. Most hormonal contraceptive users in this study likely used the combined oral contraceptive pill on a cyclic regimen, as combined oral contraceptives represent approximately 75% of hormonal contraceptive use in the United States.31 However, this study encompasses 25 years of data, and trends in contraceptive use as well as available formulations of oral contraceptive pills have changed over time. Future studies need to investigate patterns of HSV-2 shedding and lesions among women using more precisely defined methods of hormonal contraception.
Our findings enable clinicians to reassure women with genital herpes that the use of hormonal contraception is unlikely to increase the frequency of herpes outbreaks or the risk of transmission to a susceptible partner. However, among women not using hormonal contraception, recurrences and shedding may be more frequent during the follicular phase of the cycle (including during menstruation). As recommended in the 2015 Centers for Disease Control and Prevention STD Treatment Guidelines, patients with HSV-2 infection should be encouraged to disclose their HSV status to sexual partners, to use condoms, and to use daily suppressive antiviral therapy to reduce the risk of HSV-2 transmission.32
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