Rebecca M. Brotman

Vaginal microbiome of reproductive-age women

The means by which vaginal microbiomes help prevent urogenital diseases in women and maintain health are poorly understood. To gain insight into this, the vaginal bacterial communities of 396 asymptomatic North American women who represented four ethnic groups (white, black, Hispanic, and Asian) were sampled and the species composition characterized by pyrosequencing of barcoded 16S rRNA genes. The communities clustered into five groups: four were dominated by Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii, whereas the fifth had lower proportions of lactic acid bacteria and higher proportions of strictly anaerobic organisms, indicating that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities. The proportions of each community group varied among the four ethnic groups, and these differences were statistically significant [χ2(10) = 36.8, P < 0.0001]. Moreover, the vaginal pH of women in different ethnic groups also differed and was higher in Hispanic (pH 5.0 ± 0.59) and black (pH 4.7 ± 1.04) women as compared with Asian (pH 4.4 ± 0.59) and white (pH 4.2 ± 0.3) women. Phylotypes with correlated relative abundances were found in all communities, and these patterns were associated with either high or low Nugent scores, which are used as a factor for the diagnosis of bacterial vaginosis. The inherent differences within and between women in different ethnic groups strongly argues for a more refined definition of the kinds of bacterial communities normally found in healthy women and the need to appreciate differences between individuals so they can be taken into account in risk assessment and disease diagnosis.

Temporal Dynamics of the Human Vaginal Microbiota

The vaginal microbiome is dynamic, varying over time in composition and function with implications for women’s health. What’s Up with Vaginal Microbes? The ability to properly identify women at risk of acquiring sexually transmitted infectious diseases or who might suffer from adverse obstetric sequelae is a critical first step in reducing their incidence and the unnecessary use of antibiotics. Currently, patients undergo a clinical examination of the vagina that includes measuring the pH and evaluating the amount and type of discharge and the presence of odor. These criteria are thought to be surrogates for the presence of an “abnormal” vaginal microbiota. Although these kinds of tests, done only once, could be used to diagnose conditions such as bacterial vaginosis, it is debatable whether they are accurate predictors of risk because little is known about how the composition and function of the vaginal microbiome changes over time. Previous studies have established that in healthy asymptomatic women, five types of vaginal microbiota exist that differ in the kinds of microbes they contain. It was thought that each type carries its own risks and particular response to environmental disturbances, such as sexual activity or hygiene practices. In an exciting new study, Gajer and colleagues now describe changes in the identity and abundance of bacteria in the vaginal communities of 32 women by analyzing vaginal samples obtained twice weekly over a 16-week period. The kinds of bacteria present in the samples were identified by classifying thousands of 16S rRNA gene sequences in each sample using high-throughput next-generation sequencing. The authors further characterized vaginal community function by determining the metabolites produced throughout the 16-week period. Gajer and colleagues found that there were five longitudinal patterns of change in vaginal microbial community composition. Moreover, in some women, the vaginal microbial community composition changed markedly and rapidly over time, whereas in others it was relatively stable. Using statistical modeling, the authors showed that the menstrual cycle influenced the stability of the vaginal communities. In many cases, the metabolite profiles indicated that vaginal community function was maintained despite changes in bacterial composition. Intervals of increased susceptibility to disease may occur because the vaginal microbiota varies over time. The authors envision that better knowledge of the causes and consequences of these changes to the host will lead to the development of new strategies to manage vaginal microbiomes in ways that promote health and minimize the use of antibiotics. Elucidating the factors that impinge on the stability of bacterial communities in the vagina may help in predicting the risk of diseases that affect women’s health. Here, we describe the temporal dynamics of the composition of vaginal bacterial communities in 32 reproductive-age women over a 16-week period. The analysis revealed the dynamics of five major classes of bacterial communities and showed that some communities change markedly over short time periods, whereas others are relatively stable. Modeling community stability using new quantitative measures indicates that deviation from stability correlates with time in the menstrual cycle, bacterial community composition, and sexual activity. The women studied are healthy; thus, it appears that neither variation in community composition per se nor higher levels of observed diversity (co-dominance) are necessarily indicative of dysbiosis.

Bacterial Vaginosis Assessed by Gram Stain and Diminished Colonization Resistance to Incident Gonococcal, Chlamydial, and Trichomonal Genital Infection

BACKGROUND We sought to assess the relationship between bacterial vaginosis (BV) assessed by Gram stain and incident trichomonal, gonococcal, and/or chlamydial genital infection. METHODS This longitudinal study included 3620 nonpregnant women aged 15-44 years who presented for routine care at 12 clinics in Birmingham, Alabama. Participants were assessed quarterly for 1 year. Vaginal smears were categorized by the Nugent Gram stain score (0-3, normal; 4-6, intermediate state; 7-10, BV). Pooled logistic regression was used to estimate the hazard ratios for the comparison of trichomonal, gonococcal, and chlamydial infection incidence in participants by Nugent score at the prior visit. Participants were censored at their first visit with a positive test result for trichomonal, gonococcal, and/or chlamydial infection. RESULTS Of the 10,606 eligible visits, 37.96% were classified by BV and 13.3% by positive detection of trichomonal, gonococcal, and/or chlamydial infection. An intermediate state or BV at the prior visit was associated with a 1.5-2-fold increased risk for incident trichomonal, gonococcal, and/or chlamydial infection (adjusted hazard ratio [AHR] for intermediate state, 1.41 [95% confidence interval {CI}, 1.12-1.76]; AHR for BV, 1.73 [95% CI, 1.42-2.11]; P= .058 for trend). Estimates were similar for trichomonal-only, gonococcal-only, and chlamydial-only infection outcomes. CONCLUSION BV microbiota as gauged by Gram stain is associated with a significantly elevated risk for acquisition of trichomonal, gonococcal, and/or chlamydial genital infection.

A Longitudinal Study of Vaginal Douching and Bacterial Vaginosis—A Marginal Structural Modeling Analysis

The etiology of bacterial vaginosis is unknown, and there are no long-term therapies for preventing this frequently recurring condition. Vaginal douching has been reported to be associated with bacterial vaginosis in observational studies. However, this association may be due to confounding by indication--that is, confounding by women douching in response to vaginal symptoms associated with bacterial vaginosis. The authors used marginal structural modeling to estimate the causal effect of douching on bacterial vaginosis risk while controlling for this confounding effect. In 1999-2002, nonpregnant women (n = 3,620) were recruited into a prospective study when they visited one of 12 public health clinics in Birmingham, Alabama, for routine care. Participants were assessed quarterly for 1 year. Bacterial vaginosis was based on a Nugents Gram stain score of 7 or higher. Thirty-two percent of participants douched in every study interval, and 43.0% never douched. Of the 12,349 study visits, 40.2% were classified as involving bacterial vaginosis. The relative risk for regular douching as compared with no douching was 1.21 (95% confidence interval: 1.08, 1.38). These findings indicate that douching confers increased risk of disruption of vaginal flora. In the absence of a large randomized trial, these findings provide the best evidence to date for a risk of bacterial vaginosis associated with douching.

Vaginal microbiome and sexually transmitted infections: an epidemiologic perspective

Vaginal bacterial communities are thought to help prevent sexually transmitted infections. Bacterial vaginosis (BV) is a common clinical syndrome in which the protective lactic acid-producing bacteria (mainly species of the Lactobacillus genus) are supplanted by a diverse array of anaerobic bacteria. Epidemiologically, BV has been shown to be an independent risk factor for adverse outcomes including preterm birth, development of pelvic inflammatory disease, and acquisition of sexually transmitted infections. Longitudinal studies of the vaginal microbiome using molecular techniques such as 16S ribosomal DNA analysis may lead to interventions that shift the vaginal microbiota toward more protective states.

Association between the vaginal microbiota, menopause status, and signs of vulvovaginal atrophy.

ObjectiveThe vaginal microbiota helps protect the female genital tract from disease. We sought to describe the composition of the vaginal microbiota in premenopausal, perimenopausal, and postmenopausal women and to explore the association between the microbiota and vulvovaginal atrophy (VVA). MethodsEighty-seven women (aged 35-60 y) were classified as premenopausal (n = 30), perimenopausal (n = 29), or postmenopausal (n = 28) according to Stages of Reproductive Aging Workshop guidelines. Midvaginal bacterial community composition was characterized by 16S ribosomal RNA gene analysis. ResultsBacterial communities clustered into six community state types (CSTs), of which four were dominated by Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, or Lactobacillus jensenii, and two (CST IV-A and CST IV-B) had low relative abundance of Lactobacillus. CST IV-A was characterized by Streptococcus and Prevotella, whereas CST IV-B was characterized by Atopobium. There were significant associations between menopause stage and CST (P = 0.004) and between VVA and CST (P = 0.002). Perimenopausal women were more likely to be classified as CST IV-A or L. gasseri CST, whereas postmenopausal women were often classified as CST IV-A. CSTs dominated by L. crispatus and L. iners were more prevalent in premenopausal women. Nineteen participants had signs of mild or moderate VVA. Compared with women with no VVA, the vaginal microbiota of women with mild or moderate atrophy had 25-fold greater odds of being classified as CST IV-A versus L. crispatus CST (adjusted odds ratio, 25.89; 95% credible interval, 2.98-406.79). ConclusionsA distinct bacterial community state (CST IV-A) with a low relative abundance of Lactobacillus is associated with VVA. Future studies recruiting a larger number of women are needed to replicate the findings. This study provides an impetus for future longitudinal studies designed to manage, modulate, and restore vaginal microbiota homeostasis, which would provide stronger evidence for a causal relationship with VVA and ultimately improve the treatment and prevention of atrophic vaginitis in menopause.

Interplay Between the Temporal Dynamics of the Vaginal Microbiota and Human Papillomavirus Detection

BACKGROUND We sought to describe the temporal relationship between vaginal microbiota and human papillomavirus (HPV) detection. METHODS Thirty-two reproductive-age women self-collected midvaginal swabs twice weekly for 16 weeks (937 samples). Vaginal bacterial communities were characterized by pyrosequencing of barcoded 16S rRNA genes and clustered into 6 community state types (CSTs). Each swab was tested for 37 HPV types. The effects of CSTs on the rate of transition between HPV-negative and HPV-positive states were assessed using continuous-time Markov models. RESULTS Participants had an average of 29 samples, with HPV point prevalence between 58%-77%. CST was associated with changes in HPV status (P<.001). Lactobacillus gasseri-dominated CSTs had the fastest HPV remission rate, and a low Lactobacillus community with high proportions of the genera Atopobium (CST IV-B) had the slowest rate compared to L. crispatus-dominated CSTs (adjusted transition rate ratio [aTRR], 4.43, 95% confidence interval [CI], 1.11-17.7; aTRR, 0.33, 95% CI, .12-1.19, respectively). The rate ratio of incident HPV for low Lactobacillus CST IV-A was 1.86 (95% CI, .52-6.74). CONCLUSIONS Vaginal microbiota dominated by L. gasseri was associated with increased clearance of detectable HPV. Frequent longitudinal sampling is necessary for evaluation of the association between HPV detection and dynamic microbiota.

Recent Advances in Understanding the Microbiology of the Female Reproductive Tract and the Causes of Premature Birth

Data derived from molecular microbiological investigations of the human vagina have led to the discovery of resident bacterial communities that exhibit marked differences in terms of species composition. All undergo dynamic changes that are likely due to intrinsic host and behavioral factors. Similar types of bacteria have been found in both amniotic fluid and the vagina, suggesting a potential route of colonization. Given that not all of the species involved in intrauterine infections are readily cultivated, it is important that culture-independent methods of analysis must be used to understand the etiology of these infections. Further research is needed to establish whether an ascending pathway from the vagina to the amniotic cavity enables the development of intrauterine infections.

Comparison of Self-Collected and Physician-Collected Vaginal Swabs for Microbiome Analysis

ABSTRACT To our knowledge, no data are available on whether the microbial species composition and abundance sampled with self-collected vaginal swabs are comparable to those of swabs collected by clinicians. Twenty healthy women were recruited to the study during a routine gynecological visit. Eligible women were between 18 and 40 years old with regular menstrual cycles. Participants self-collected a vaginal swab using a standardized protocol and then were examined by a physician, who collected an additional five swabs from the lateral wall of the mid-vagina. In this study, the self-collected and three physician-obtained swabs were analyzed and compared using terminal restriction fragment length polymorphism and sequence analyses of the 16S rRNA genes. Vaginal microbial community comparative statistical analyses of both T-RFLP and 16S rRNA gene sequence datasets revealed that self-collected vaginal swabs sampled the same microbial diversity as physician collected swabs of the mid-vagina. These findings enable large-scale, field-based studies of the vaginal microbiome.

The effect of vaginal douching cessation on bacterial vaginosis: a pilot study

OBJECTIVE The objective of the study was to evaluate the risk for bacterial vaginosis (BV) in a douching cessation trial. STUDY DESIGN Thirty-nine reproductive-age women who reported use of douche products were enrolled into a 20-week study consisting of a 4 week douching observation (phase I) followed by 12-weeks of douching cessation (phase II). In phase III, participants then chose to resume douching or continue cessation for the remaining 4 weeks. Self-collected vaginal samples were obtained twice weekly in the first 16 weeks, and 1 sample was collected during week 20 (1107 samples total). BV was diagnosed by Nugent score of 7 or greater. Conditional logistic regression was used to evaluate douching cessation on the risk of BV. RESULTS The adjusted odds ratio (aOR) for BV in the douching cessation phase, as compared with the douching-observation phase was 0.76 (95% confidence interval [CI], 0.33 to 1.76). Among women who reported their primary reason for douching was to cleanse after menstruation, BV was significantly reduced in douching cessation (aOR:0.23; 95% CI, 0.12 to 0.44). CONCLUSION Vaginal douching cessation may reduce the risk for BV in a subset of women.