Bacterial vaginosis is a syndrome characterized by a shift in the bacterial composition of the vaginal microbiota from a Lactobacillus species–predominant ecosystem to a mixed, diverse, primarily anaerobic community.1 Bacterial vaginosis is most prevalent in women of reproductive age2 and has clearly been linked to sexual activity,3 although the mechanism for this link is unclear. Several possibilities have been proposed including transmission of a specific bacterial pathogen,4 transmission of a phage that attacks the protective lactobacilli,5 or use of vaginal lubricants that disrupt the normal ecosystem.6 Initial studies suggested that women who had not had penile–vaginal intercourse were unlikely to have bacterial vaginosis7,8; however, more recent studies have shown that nonpenetrative sexual contact such as oral sex, use of sex toys, or mutual masturbation9 can increase the risk of altered vaginal microbiota.
Bacterial vaginosis has a high rate of recurrence after treatment10 and is linked to adverse reproductive health outcomes such as preterm delivery, pelvic inflammatory disease, and acquisition of sexually transmitted infections11; thus, prevention is important. Understanding how sexual behavior may transmit or facilitate development of bacterial vaginosis may be an important part of developing a prevention strategy. A cross-sectional study of young Australian women showed that many of the bacterial vaginosis–associated bacterial species such as Megasphaera, Leptotrichia, and the clostridia-like bacterial vaginosis–associated bacterium-1, -2, and -3 are less prevalent in sexually naïve women.12 Little is known about the normal vaginal microbiota in adolescents and sexually inexperienced women and whether initiation of sexual activity induces a change from a healthy Lactobacillus species–dominant microbiota toward one that might increase the risk of developing bacterial vaginosis. We undertook this study to examine whether initiation of sexual intercourse in a virginal population was associated with shifts in the vaginal microbiota that might indicate how sex predisposes women to develop bacterial vaginosis.
PATIENTS AND METHODS
This analysis used samples and data collected from 18- to 22-year-old women who were virgins enrolled in a longitudinal study of human papillomavirus (HPV) acquisition in Seattle, Washington, between December 2000 and March 2007.13 Both the parent study and this analysis were approved by the University of Washington institutional review board, and all participants provided written informed consent. Participants were eligible for the parent study if they reported never having penile–vaginal intercourse or a first episode of penile–vaginal intercourse in the last 3 months. All participants were college students at the University of Washington in Seattle. Only participants who had never had penile–vaginal intercourse at enrollment (167 of 250 enrolled participants) were eligible for inclusion in this analysis. Two visits were selected: one visit before and one after sexual debut (if the latter occurred during study follow-up) or two visits within 2 years of each other if the participant remained a virgin.
Study participants were seen every 4 months and completed a questionnaire about general health, contraception, vaginal medication use in the 7 days before the visit, sexual activity, and genital symptoms. Participants completed online diaries about sexual activity and condom use in the month before each study visit. A pelvic examination was performed by a clinician at each visit, in which the appearance of the external genitalia, vagina, and cervix was examined. Testing for HPV was performed at all visits as previously described.13 Bacterial vaginosis was evaluated using Amsel’s clinical criteria at all visits.14 Any ulcerative lesions were cultured for herpes virus, and tests were run for gonorrhea and Chlamydia infection at each visit by culture of cervical swabs.
Self-collected vaginal swabs were placed in 1 mL of specimen transport medium and stored at −80°C until DNA extraction was performed using the Qiagen DNA Blood mini kit. Previous studies have established that self-collected swabs provide similar results to provider-collected swabs, both in detection of bacterial presence and bacterial quantity.15 Extracted DNA was tested in a quantitative polymerase chain reaction (PCR) assay using primers targeting the human 18S rRNA gene to validate that successful DNA extraction occurred. An internal amplification control PCR using exogenous DNA from a jellyfish gene was used to test for presence of PCR inhibitors.16 Samples were then subjected to taxon-directed 16S rRNA gene quantitative PCR assays for the detection and quantification of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, Leptotrichia-Sneathia species (combined assay), Megasphaera species, and bacterial vaginosis–associated bacterium-1, -2, and -3, which have been described elsewhere.17,18 The assays use a TaqMan format and are run on an ABI 7500 Thermocycler or Eppendorf Mastercycler ep Realplex thermal cycler. Negative reactions were assigned a value at the lower limit of detection for the assay.
Demographic characteristics were compared between women who remained virgins and those who became sexually active using Student’s t test, χ2 test, or Mann-Whitney. Differences in prevalence of individual species at the first and second visits were assessed using McNemar’s test for paired data. Log-transformed quantities of bacteria were compared using t test or paired t test. For analyses of acquisition of bacterial vaginosis–associated bacterial colonization at the follow-up visit, women colonized with any of these species at baseline were excluded. Bacterial vaginosis–associated species were defined as A vaginae, Leptotrichia-Sneathia species, Megasphaera species, and bacterial vaginosis–associated bacterium-1, -2, and -3. G vaginalis was considered a commensal organism, because it can be found in a significant proportion of women both with and without bacterial vaginosis.19 Fisher’s exact test was used to assess the association between initiation of penile–vaginal sexual intercourse and the presence of individual bacterial species at the follow-up visit. Other potential confounding factors were also evaluated for association with colonization, including race, use of hormonal contraception, and menstrual phase using Fisher’s exact test. Menstrual phase was defined as proliferative if a visit was within 14 days of the last menstrual period, luteal if the visit was more than 14 days but less than 35 days since the last menstrual period, and oligomenorrheic if the last menstrual period was more than 35 days before the visit. Based on our available sample size for this analysis of 73 (after exclusion of 17 women with baseline colonization by bacterial vaginosis–associated species), we would have 80% power to detect a twofold difference in the proportion of women colonized by bacterial vaginosis–associated species with an α of .05.
The parent study enrolled 167 virginal participants, of whom 108 (65%) became sexually active with male partners and 59 (35%) did not. Of these enrolled virgins, 97 had available samples from at least two time points, including 71 women who became sexually active during the study and 26 who remained virgins. There were no significant differences in demographic characteristics at enrollment between these two groups of women (Tables 1 and 2 ): the majority were white and were not using any hormonal birth control methods at enrollment. Two women reported vaginal medication use in the 7 days before their study visit; both used vaginal antifungal creams for treatment of yeast. Between the initial and subsequent visit chosen for this subanalysis, women who initiated sexual intercourse became more likely to use birth control (primarily condoms and hormonal methods) (P<.01) and 88% reported only one new sexual partner (Table 2). Two women who reported remaining virgins also reported condom use but did not report any penile–vaginal sexual contact suggesting that condoms were used during nonpenile vaginal sexual activity. Among women who became sexually active, the mean number of penile–vaginal sex acts in the 4 weeks before the follow-up visit was 5.5±7.1 (range 0–31). Only 46 women reported using condoms, at a mean of 3.4±4.2 sex acts during that time, or a mean of 54% of sex acts. No participants were diagnosed with a sexually transmitted infection other than HPV, and no participants developed bacterial vaginosis. There was a significant increase in the number of women testing positive for HPV in the group who became sexually active (P=.03).
Very few participants had any of the bacterial vaginosis–associated species (Atopobium, Leptotrichia, Sneathia, Megasphaera, and bacterial vaginosis–associated bacterium-1, -2, or -3) detected either at the initial or follow-up visit (Table 3). Over half had at least one Lactobacillus species detected at both visits, and approximately 40% in each group had G vaginalis at the initial visit. Although prevalence of these species did not change in the women who remained virgins, there was a significant increase in the prevalence of G vaginalis in the group that became sexually active, from 28 of 71 (39%) at the initial visit to 40 of 71 (56%) at follow-up (P=.02) (Table 3). There were no significant differences in quantity of bacteria between women who remained virgins and those who became sexually active or between the initial visit and follow-up visit in either group (Fig. 1).
The majority of participants had similar patterns of colonization at both visits, no matter whether they remained virgins or became sexually active. Of participants who remained virgins throughout the study, 22 of 26 (85%) had at least one Lactobacillus species detected at both visits, and both of the two women colonized with A vaginae at enrollment were also colonized at follow-up. Of the 12 women colonized with G vaginalis at enrollment, 10 (83%) remained colonized at follow-up. Among women who became sexually active, 62 (87%) were colonized with at least one Lactobacillus species at both visits. If analysis is restricted to just L crispatus and L jensenii (both common H2O2-producers), 53 (75%) had persistent colonization. Colonization with bacterial vaginosis–associated species was more dynamic in this group. Of the six women colonized with either Leptotrichia or Sneathia species at enrollment, only one maintained that colonization at follow-up; of the four colonized with bacterial vaginosis–associated bacterium-2, only two were positive at follow-up, and, whereas two of eight women colonized with A vaginae lost colonization at follow-up, five additional women gained colonization. Presence of bacterial vaginosis–associated species at the initial or follow-up visits was not associated with presence of HPV at follow-up (P=.18 and P=.84, respectively).
After excluding women colonized with bacterial vaginosis–associated bacteria at baseline, only five women gained colonization with these species at follow-up. The likelihood of colonization by any bacterial vaginosis–associated species at the follow-up visit was not significantly associated with race, use of hormonal contraception, phase of the menstrual cycle, or initiation of sexual intercourse (Table 4). However, of the 80 women with no bacterial vaginosis–associated bacteria detected at baseline, the five who were colonized at follow-up were all women who had become sexually active (Table 5). Among the 53 women without baseline colonization who completed online diary information, the five who gained colonization with bacterial vaginosis–associated bacteria had a mean of 2±3.5 penile–vaginal sex acts in the 4 weeks before their clinic visit compared with 5.3±7 in 48 women who did not gain colonization with any of these species. Only 34 women reported condom use, two who gained colonization with bacterial vaginosis–associated species reported using condoms in a mean of 63% of acts, whereas the remaining 32 reported use in 55% of acts.
Our study offers a unique perspective on the effects of sexual intercourse on the vaginal microbiota by comparing samples from before and after initiation of sexual intercourse. In this cohort of sexually inexperienced women, we found that both bacterial vaginosis and vaginal colonization with bacterial vaginosis–associated bacterial species was rare and that the vaginal microbiota of women who were virgins appeared to be stable over the follow-up interval. Women who became sexually active during the study were more likely to gain colonization with G vaginalis, but there was no loss of colonization with Lactobacillus species after initiation of sexual intercourse nor increase in colonization with the fastidious bacterial vaginosis–associated species.
Although bacterial vaginosis is associated with sexual activity,2 the mechanism by which women lose vaginal Lactobacillus colonization and gain the polymicrobial community associated with bacterial vaginosis is unclear. Eschenbach et al20 studied the vaginal microbiota in sexually experienced women within 8–12 hours after one episode of intercourse with or without a condom and saw an increase in culture-based detection of Escherichia coli but did not evaluate the fastidious bacterial vaginosis–associated organisms described here. Fethers et al12 used similar quantitative PCR techniques to evaluate colonization in a cross-sectional study that combined sexually naïve and sexually experienced women. They found that G vaginalis, Sneathia species, and bacterial vaginosis–associated bacterium-3 were associated with more frequent penile–vaginal sex as well as with unprotected sex. Women with no sexual experience or who always used condoms were much less likely to be colonized with the bacterial vaginosis–associated species that were tested for in their study. Of note, their analysis did not assess changes in the microbiota over time. Our study confirms that vaginal colonization with bacterial vaginosis–associated bacterial species is rare in sexually inexperienced women and does not significantly increase with initiation of penile–vaginal sexual activity. Although we did see an increase in the proportion of women colonized with G vaginalis among those who became sexually active, this microbe is widely prevalent in women with and without bacterial vaginosis21 and for the purposes of this analysis is considered a commensal organism.
Our group has previously shown that the vaginal microbiota in sexually experienced women can be quite dynamic when sampled daily, attributable in part to the influence of menses.18 Other groups have shown significant variation in vaginal microbiota over time as well.22,23 Interestingly, in our data, women who remained virgins had very stable microbiota, whereas those who became sexually active began to show some variability in bacterial composition, although not to the extent reported in older women. This suggests that sexual activity as well as menses may play a role in disrupting or changing the vaginal microbial environment, although through what mechanism is not clear.
In a previous analysis from a similar cohort that excluded girls who remained virgins, presence of HPV was associated with diagnosis of bacterial vaginosis and more often preceded diagnosis of bacterial vaginosis than the opposite (bacterial vaginosis preceding HPV).24 In that analysis, the median time to first detection of HPV was 4 months, whereas median time to first bacterial vaginosis diagnosis was 12 months. Some have hypothesized that HPV infection, or the immune response, may predispose women to develop bacterial vaginosis. In this analysis, we had too few women with HPV at baseline to draw any firm conclusions but did not find any association between HPV and detection of bacterial vaginosis–associated bacteria.
Our study is limited in that we did not collect information on nonpenetrative sexual contact; therefore, we were unable to evaluate any associations with these species and nonpenetrative types of sexual activity, which have clearly been associated with an increased risk of bacterial vaginosis. Because our participants who became sexually active during the study had higher prevalence of bacterial vaginosis–associated bacteria at the first visit, we suspect that these women were already participating in nonpenetrative behaviors. This is consistent with a previous analysis of this cohort showing that women who initiated vaginal intercourse during the study had higher incidence of HPV infection before sexual debut, suggesting nonpenile exposure.25 Self-report of sexual activity could be incorrect, because some women may be uncomfortable disclosing sexual information to researchers. In addition, our sample size is small, as a result of the unique nature of the cohort, and thus affords limited power to detect any differences. Our participants are also a low-risk population for development of bacterial vaginosis: primarily white, well-educated, nonsmokers with few sexual partners. However, this allowed us to come closer to isolating the effect of sex alone on the vaginal microbiota. We tested for 11 individual bacterial species, both healthy commensals and well-described bacterial vaginosis–associated species, but this is not a comprehensive assessment of all potential species that may colonize the vagina.
Sexual activity is clearly linked to the development of bacterial vaginosis but likely through a more complex mechanism than some other sexually transmitted infections. Some have hypothesized that the change in vaginal pH resulting from semen is what drives the shift in microbiota that results in bacterial vaginosis. We did not have adequate numbers of women with reported information on condom use to draw any conclusions about the effect of semen independent of sexual activity. Interestingly, the women who did gain colonization with bacterial vaginosis–associated bacteria reported fewer penile–vaginal sex acts and a higher percentage of condom use with those acts.
Young, sexually inexperienced women are unlikely to have vaginal colonization with bacterial vaginosis–associated bacterial species, and the composition of the vaginal microbiota does not change rapidly after initiation of sexual activity. This suggests that the association between sex and the development of bacterial vaginosis is complex.
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© 2012 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
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