Longitudinal profiles of the vaginal microbiota of pre-, peri-, and postmenopausal women: preliminary insights from a secondary data analysis.

OBJECTIVE: Menopause is often accompanied by lowered Lactobacillus spp. relative abundance and increased abundance of diverse anaerobic/aerobic bacteria in the vaginal microbiota due in part to declines in estrogen. These microbiota are associated with urogenital symptoms and infections. In premenopause, vaginal microbiota can fluctuate rapidly, particularly with menstrual cycles and sexual activity; however, the longitudinal dynamics of vaginal microbiota are understudied in peri- and postmenopause. We described vaginal community stability across reproductive stages. METHODS: Pre- (n = 83), peri- (n = 8), and postmenopausal (n = 11) participants provided twice-weekly mid-vaginal samples (total, 1,556; average, 15 per participant) over 8 weeks in an observational study. Composition of the vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing, and a community state type (CST) was assigned to each sample. Clustering of longitudinal CST profiles, CST transition rates, duration of low-Lactobacillus/high bacterial diversity CSTs, and other metrics of bacterial community dynamics were assessed across reproductive stages. RESULTS: The proportion of participants with longitudinal CST profiles characterized by low-Lactobacillus CSTs was similar among pre- (38.6%), peri- (37.5%), and postmenopausal (36.4%) participants (P = 0.69). CST transition rates between consecutive samples were 21.1%, 16.7%, and 14.6% for pre-, peri-, and postmenopausal participants, respectively (P = 0.49). Low-Lactobacillus CST tended to persist for at least 4 weeks, irrespective of reproductive stage. CONCLUSIONS: Findings from this small yet frequently sampled cohort revealed vaginal bacterial fluctuations over 8 weeks that were similar across reproductive stages. Larger and longer-term studies based on these preliminary data could provide insights into the influence of microbiota dynamics on urogenital outcomes during menopause.

SpeciateIT and vSpeciateDB: Novel, fast and accurate per sequence 16S rRNA gene taxonomic classification of vaginal microbiota.

Clustering of sequences into operational taxonomic units (OTUs) and denoising methods are a mainstream stopgap to taxonomically classifying large numbers of 16S rRNA gene sequences. We developed speciateIT, a novel taxonomic classification tool which rapidly and accurately classifies individual amplicon sequences (https://github.com/Ravel-Laboratory/speciateIT). Environment-specific reference databases generally yield optimal taxonomic assignment. To this end, we also present vSpeciateDB, a custom reference database for the taxonomic classification of 16S rRNA gene amplicon sequences from vaginal microbiota. We show that speciateIT requires minimal computational resources relative to other algorithms and, when combined with vSpeciateDB, affords accurate species level classification in an environment-specific manner.

Untangling Associations of Microbiomes of Pregnancy and Preterm Birth.

This review illuminates the complex interplay between various maternal microbiomes and their influence on preterm birth (PTB), a driving and persistent contributor to neonatal morbidity and mortality. Here, we examine the dynamics of oral, gastrointestinal (gut), placental, and vaginal microbiomes, dissecting their roles in the pathogenesis of PTB. Importantly, focusing on the vaginal microbiome and PTB, the review highlights (1) a protective role of Lactobacillus species; (2) an increased risk with select anaerobes; and (3) the influence of social health determinants on the composition of vaginal microbial communities.

ZMapp reduces diffusion of Ebola viral particles in fresh human cervicovaginal mucus.

Vaginal transmission from semen of male Ebola virus (EBOV) survivors has been implicated as a potential origin of Ebola virus disease (EVD) outbreaks. While EBOV in semen must traverse cervicovaginal mucus (CVM) to reach target cells, the behaviour of EBOV in CVM is poorly understood. CVM contains substantial quantities of IgG, and arrays of IgG bound to a virion can develop multiple Fc-mucin bonds, immobilizing the IgG/virion complex in mucus. Here, we measured the real-time mobility of fluorescent Ebola virus-like-particles (VLP) in 50 CVM specimens from 17 women, with and without ZMapp, a cocktail of 3 monoclonal IgGs against EBOV. ZMapp-mediated effective trapping of Ebola VLPs in CVM from a subset of women across the menstrual cycle, primarily those with Lactobacillus crispatus dominant microbiota. Our work underscores the influence of the vaginal microbiome on IgG-mucin crosslinking against EBOV and identifies bottlenecks in the sexual transmission of EBOV.

Safety testing of Ovaprene: An investigational nonhormonal monthly vaginal contraceptive.

OBJECTIVES: Evaluate the safety of Ovaprene, an investigational nonhormonal vaginal contraceptive designed for monthly use. STUDY DESIGN: Open-label, multicenter study enrolling heterosexually-active women with previous permanent contraception who underwent assessments during five menstrual cycles: baseline postcoital test cycle, diaphragm postcoital test cycle, Ovaprene safety cycle, and two Ovaprene postcoital test cycles. Safety outcomes included treatment-emergent adverse events, systemic laboratory findings, pelvic examinations, colposcopies, Nugent scores, determination of community state types of vaginal microbiota, and anti-Escherichia coli activity and inflammatory markers in cervicovaginal fluids. RESULTS: We enrolled 38 participants. Of these, 33 used Ovaprene and completed 77 Ovaprene cycles. The most common product-related urogenital treatment-emergent adverse events were bacterial vaginosis and vaginal odor. The frequency of transitioning from Lactobacillus-dominated community state type to community state type IV (not Lactobacillus-dominated) was similar before Ovaprene use and afterwards. Mean Nugent scores were <4 at each visit without a discernible upward trend. Inflammatory markers showed wide variation but no upward trend, and E. coli inhibitory activity of cervical secretions did not change. We found no Staphylococcus aureus, the causative agent in toxic shock syndrome, on used Ovaprenes or in vaginal samples. No clinically important changes in systemic laboratory findings, pelvic examinations, or colposcopies occurred during Ovaprene use. CONCLUSIONS: Ovaprene use did not result in cervicovaginal irritation or adverse effects on resident vaginal microbiota and did not impact transitions from a Lactobacillus-dominated community state type to community state type IV. IMPLICATIONS: The finding that the use of Ovaprene, an investigational monthly user-controlled nonhormonal vaginal contraceptive, does not appear to result in adverse changes in vaginal health during short-term use supports further evaluation of the contraceptive potential of the device.

Inferring stability and persistence in the vaginal microbiome: A stochastic model of ecological dynamics.

The interplay of stochastic and ecological processes that govern the establishment and persistence of host-associated microbial communities is not well understood. Here we illustrate the conceptual and practical advantages of fitting stochastic population dynamics models to multi-species bacterial time series data. We show how the stability properties, fluctuation regimes and persistence probabilities of human vaginal microbial communities can be better understood by explicitly accommodating three sources of variability in ecological stochastic models of multi-species abundances: 1) stochastic biotic and abiotic forces, 2) ecological feedback and 3) sampling error. Rooting our modeling tool in stochastic population dynamics modeling theory was key to apply standardized measures of a community's reaction to environmental variation that ultimately depends on the nature and intensity of the intra-specific and inter-specific interaction strengths. Using estimates of model parameters, we developed a Risk Prediction Monitoring (RPM) tool that estimates temporal changes in persistence probabilities for any bacterial group of interest. This method mirrors approaches that are often used in conservation biology in which a measure of extinction risks is periodically updated with any change in a population or community. Additionally, we show how to use estimates of interaction strengths and persistence probabilities to formulate hypotheses regarding the molecular mechanisms and genetic composition that underpin different types of interactions. Instead of seeking a definition of "dysbiosis" we propose to translate concepts of theoretical ecology and conservation biology methods into practical approaches for the management of human-associated bacterial communities.

The DTC microbiome testing industry needs more regulation.

Tests lack analytical and clinical validity, requiring more federal oversight to prevent consumer harm.

Extracellular vesicles from vaginal Gardnerella vaginalis and Mobiluncus mulieris contain distinct proteomic cargo and induce inflammatory pathways.

Colonization of the vaginal space with bacteria such as Gardnerella vaginalis and Mobiluncus mulieris is associated with increased risk for STIs, bacterial vaginosis, and preterm birth, while Lactobacillus crispatus is associated with optimal reproductive health. Although host-microbe interactions are hypothesized to contribute to reproductive health and disease, the bacterial mediators that are critical to this response remain unclear. Bacterial extracellular vesicles (bEVs) are proposed to participate in host-microbe communication by providing protection of bacterial cargo, delivery to intracellular targets, and ultimately induction of immune responses from the host. We evaluated the proteome of bEVs produced in vitro from G. vaginalis, M. mulieris, and L. crispatus, identifying specific proteins of immunologic interest. We found that bEVs from each bacterial species internalize within cervical and vaginal epithelial cells, and that epithelial and immune cells express a multi-cytokine response when exposed to bEVs from G. vaginalis and M. mulieris but not L. crispatus. Further, we demonstrate that the inflammatory response induced by G. vaginalis and M. mulieris bEVs is TLR2-specific. Our results provide evidence that vaginal bacteria communicate with host cells through secreted bEVs, revealing a mechanism by which bacteria lead to adverse reproductive outcomes associated with inflammation. Elucidating host-microbe interactions in the cervicovaginal space will provide further insight into the mechanisms contributing to microbiome-mediated adverse outcomes and may reveal new therapeutic targets.

Sexual transmission of urogenital bacteria: whole metagenome sequencing evidence from a sexual network study.

Sexual transmission of the urogenital microbiota may contribute to adverse sexual and reproductive health outcomes. The extent of sexual transmission of the urogenital microbiota is unclear as prior studies largely investigated specific pathogens. We used epidemiologic data and whole metagenome sequencing to characterize urogenital microbiota strain concordance between participants of a sexual network study. Individuals who screened positive for genital Chlamydia trachomatis were enrolled and referred their sexual contacts from the prior 60-180 days. Snowball recruitment of sexual contacts continued for up to four waves. Vaginal swabs and penile urethral swabs were collected for whole metagenome sequencing. We evaluated bacterial strain concordance using inStrain and network analysis. We defined concordance as ≥99.99% average nucleotide identity over ≥50% shared coverage; we defined putative sexual transmission as concordance between sexual contacts with <5 single-nucleotide polymorphisms per megabase. Of 138 participants, 74 (54%) were female; 120 (87%) had genital chlamydia; and 43 (31%) were recruited contacts. We identified 115 strain-concordance events among 54 participants representing 25 bacterial species. Seven events (6%) were between sexual contacts including putative heterosexual transmission of Fannyhessea vaginae, Gardnerella leopoldii, Prevotella amnii, Sneathia sanguinegens, and Sneathia vaginalis (one strain each), and putative sexual transmission of Lactobacillus iners between female contacts. Most concordance events (108, 94%) were between non-contacts, including eight female participants connected through 18 Lactobacillus crispatus and 3 Lactobacillus jensenii concordant strains, and 14 female and 2 male participants densely interconnected through 52 Gardnerella swidsinskii concordance events.IMPORTANCEEpidemiologic evidence consistently indicates bacterial vaginosis (BV) is sexually associated and may be sexually transmitted, though sexual transmission remains subject to debate. This study is not capable of demonstrating BV sexual transmission; however, we do provide strain-level metagenomic evidence that strongly supports heterosexual transmission of BV-associated species. These findings strengthen the evidence base that supports ongoing investigations of concurrent male partner treatment for reducing BV recurrence. Our data suggest that measuring the impact of male partner treatment on F. vaginae, G. leopoldii, P. amnii, S. sanguinegens, and S. vaginalis may provide insight into why a regimen does or does not perform well. We also observed a high degree of strain concordance between non-sexual-contact female participants. We posit that this may reflect limited dispersal capacity of vaginal bacteria coupled with individuals' comembership in regional transmission networks where transmission may occur between parent and child at birth, cohabiting individuals, or sexual contacts.

ExpLOring the role of the intestinal MiCrobiome in InflammATory bowel disease-AssocIated SpONdylarthritis (LOCATION-IBD).

Background: Inflammatory Bowel Disease (IBD)-associated arthritis is a frequent and potentially debilitating complication of IBD, that can affect those with or without active intestinal disease, and is often difficult to treat. The microbiome is known to play a role in IBD development and has been shown to be associated with inflammatory arthritis without concomitant IBD, but its role in IBD-associated arthritis is still unexplored. Further, disease localization is associated with development of IBD-associated arthritis, and stool compositional profiles are predictive of disease localization, yet mucosal location-specific microbiomes have not been well characterized. To address this gap in understanding, we designed a study (LOCATION-IBD) to characterize the mucosa-associated intestinal microbiome and metabolome in IBD-associated arthritis. Methods: Adults with an established diagnosis of IBD undergoing clinical colonoscopy between May of 2021 and February of 2023 were invited to participate in this study; those interested in participation who met inclusion criteria were enrolled. Prior to enrollment, participants were stratified into those with or without IBD-associated arthritis. All participants were interviewed and had clinical and demographic data collected, and 97.8% completed clinical colonoscopy with biopsy collection. Results and conclusion: A total of 182 participants, 53 with confirmed IBD-associated arthritis, were enrolled in this study, resulting in 1151 biopsies obtained for microbiome and metabolome analysis (median 6, mean 6.3 per participant). Clinical and demographic data obtained from the study population will be analyzed with microbiome and metabolome data obtained from biopsies, with the goal of better understanding the mechanisms underpinning the host-microbiome relationship associated the development of IBD-associated arthritis.

Uterine fibroids and longitudinal profiles of the vaginal microbiota in a cohort presenting for transvaginal ultrasound.

Bacterial vaginosis, characterized in part by low levels of vaginal Lactobacillus species, has been associated with pro-inflammatory cytokines which could fuel uterine fibroid development. However, prior work on the associations between uterine fibroids and vaginal bacteria is sparse. Most studies have focused on assessment of individual taxa in a single sample. To address research gaps, we sought to compare short, longitudinal profiles of the vaginal microbiota in uterine fibroid cases versus controls with assessment for hormonal contraceptives (HCs), a possible confounder associated with both protection from fibroid development and increases in Lactobacillus-dominated vaginal microbiota. This is a secondary analysis of 83 reproductive-age cisgender women who presented for transvaginal ultrasound (TVUS) and self-collected mid-vaginal swabs daily for 1-2 weeks before TVUS (Range: 5-16 days, n = 697 samples). Sonography reports detailed uterine fibroid characteristics (N = 21 cases). Vaginal microbiota was assessed by 16S rRNA gene amplicon sequencing and longitudinal microbiota profiles were categorized by hierarchical clustering. We compared longitudinal profiles of the vaginal microbiota among fibroid cases and controls with exact logistic regression. Common indications for TVUS included pelvic mass (34%) and pelvic pain (39%). Fibroid cases tended to be older and report Black race. Cases less often reported HCs versus controls (32% vs. 58%). A larger proportion of cases had low-Lactobacillus longitudinal profiles (48%) than controls (34%). In unadjusted analysis, L. iners-dominated and low-Lactobacillus profiles had higher odds of fibroid case status compared to other Lactobacillus-dominated profiles, however these results were not statistically significant. No association between vaginal microbiota and fibroids was observed after adjusting for race, HC and menstruation. Results were consistent when number of fibroids were considered. There was not a statistically significant association between longitudinal profiles of vaginal microbiota and uterine fibroids after adjustment for common confounders; however, the study was limited by small sample size.

The role of neighborhood deprivation in the cervicovaginal microbiota.

BACKGROUND: Lactobacillus-deficient cervicovaginal microbiota is associated with spontaneous preterm birth and is more common among Black individuals. Persistent racial segregation in the United States has led to differential neighborhood exposures by race that can affect pregnancy outcomes. The extent to which neighborhood exposures may explain racial differences in the cervicovaginal microbiota is unknown. OBJECTIVE: This study aimed to determine whether neighborhood deprivation, defined as material community deprivation, is associated with a Lactobacillus-deficient cervicovaginal microbiota in a prospective cohort of pregnant individuals. Our hypothesis was that racial differences in neighborhood deprivation may explain the higher prevalence of Lactobacillus-deficient cervicovaginal microbiota in Black birthing people. STUDY DESIGN: This study analyzed data from Motherhood and Microbiome, a prospective pregnancy cohort enrolled from prenatal clinics in a single hospital system 2013-2016 in which a Lactobacillus-deficient cervicovaginal microbiota was previously shown to be associated with spontaneous preterm birth. This study geocoded addresses to obtain census tract neighborhood deprivation data from the Brokamp Nationwide Community Deprivation Index that uses weighted proportions of poverty, income, public assistance, lack of health insurance, and vacant housing. Generalized linear mixed models quantified associations of deprivation with the cervicovaginal microbiota accounting for geographic clustering by census tract and potential confounders. Because of different distributions of neighborhood deprivation and the cervicovaginal microbiota, race-stratified models were used. Mediation analyses quantified the extent to which deprivation may contribute to racial differences in the cervicovaginal microbiota. RESULTS: Higher neighborhood deprivation was associated with a Lactobacillus-deficient cervicovaginal microbiota. Per standard deviation increment of deprivation, participants had 28% higher adjusted odds (adjusted odds ratio, 1.28; 95% confidence interval, 1.04-1.58) of a Lactobacillus-deficient microbiota. Black participants had higher odds of a Lactobacillus-deficient microbiota than White participants (adjusted odds ratio, 4.00; 95% confidence interval, 2.05-8.26), and mediation analysis revealed that deprivation accounted for 22% (P=.046) of that disparity. CONCLUSION: Neighborhood deprivation was associated with Lactobacillus-deficient cervicovaginal microbiota and may partially explain Black-White disparities in the cervicovaginal microbiota. Mechanistic studies to explore how environmental exposures modify the cervicovaginal microbiota are warranted to identify novel opportunities for future interventional strategies to prevent preterm birth. As the findings demonstrate a potential biological effect from neighborhood conditions, policies that drive urban planning should be explored to improve pregnancy outcomes.

A Narrative Review on Spontaneous Clearance of Urogenital Chlamydia trachomatis: Host, Microbiome, and Pathogen-Related Factors.

ABSTRACT: Chlamydia trachomatis (CT) is the most commonly reported sexually transmitted infection in the United States. Untreated urogenital infection in women can result in adverse sequelae such as pelvic inflammatory disease and infertility. Despite national screening and treatment guidelines, rates continue to rise; because most infections are asymptomatic, the actual prevalence of CT infection is likely significantly higher than reported. Spontaneous clearance of CT in women (in the absence of antibiotic treatment) has been described in multiple epidemiologic studies. Given the serious consequences and high prevalence of CT infection, there is growing interest in understanding this phenomenon and factors that may promote CT clearance in women. Spontaneous CT clearance is likely the result of complex interactions between CT, the host immune system, and the vaginal microbiota (i.e., the communities of bacteria inhabiting the vagina), which has been implicated in CT acquisition. Herein, we briefly review current literature regarding the role of each of these factors in spontaneous CT clearance, identify knowledge gaps, and discuss future directions and possible implications for the development of novel interventions that may protect against CT infection, facilitate clearance, and prevent reproductive sequelae.

Gastroenterology Fellowship and Postdoctoral Training in Omics and Statistics-Part I: Why Is It Needed?

A multitude of federally and industry-funded efforts are underway to generate and collect human, animal, microbial, and other sources of data on an unprecedented scale; the results are commonly referred to as "big data." Often vaguely defined, big data refers to large and complex datasets consisting of myriad datatypes that can be integrated to address complex questions. Big data offers a wealth of information that can be accessed only by those who pose the right questions and have sufficient technical knowhow and analytical skills. The intersection comprised of the gut-brain axis, the intestinal microbiome and multi-ome, and several other interconnected organ systems poses particular challenges and opportunities for those engaged in gastrointestinal and liver research. Unfortunately, there is currently a shortage of clinicians, scientists, and physician-scientists with the training needed to use and analyze big data at the scale necessary for widespread implementation of precision medicine. Here, we review the importance of training in the use of big data, the perils of insufficient training, and potential solutions that exist or can be developed to address the dearth of individuals in GI and hepatology research with the necessary level of big data expertise.

Gastroenterology Fellowship and Postdoctoral Training in Omics and Statistics-Part II: How Can It Be Achieved?

Data are being generated, collected, and aggregated in massive quantities at exponentially increasing rates. This "big data," discussed in depth in the first section of this two-part series, is increasingly important to understand the nuances of the gastrointestinal tract and its complex interactions and networks involving a host of other organ systems and microbes. Creating and using these datasets correctly requires comprehensive training; however, current instruction in the integration, analysis, and interpretation of big data appears to lag far behind data acquisition. While opportunities exist for those interested in acquiring the requisite training, these appear to be underutilized, in part due to widespread ignorance of their existence. Here, to address these gaps in knowledge, we highlight existing big data learning opportunities and propose innovative approaches to attain such training. We offer suggestions at both the undergraduate and graduate medical education levels for prospective clinical and basic investigators. Lastly, we categorize training opportunities that can be selected to fit specific needs and timeframes.

Factors Associated With Extraintestinal Manifestations of Inflammatory Bowel Disease in SPARC-IBD.

BACKGROUND: Extraintestinal manifestations (EIMs) of inflammatory bowel diseases (IBDs) are a common and debilitating feature of disease, occurring in up to 40% of patients with IBD, yet predicting who may develop them is difficult. The goal of our study was to better characterize which patients may be at highest risk of developing not only 1 EIM, but also multiple EIMs, across both diseases. METHODS: A retrospective study of participants enrolled in the SPARC IBD (Study of Prospective Adult Research Cohort with IBD) registry was performed, and demographic and clinical data were analyzed. A total of 1211 patients with data available on EIMs were included, and differences among variables with vs without EIMs were assessed. RESULTS: A total of 329 participants with at least 1 EIM were identified, compared with 882 participants without any EIMs. Crohn's disease patients and women were more likely to have 2 or more EIMs (P = .005 and P ≤ .001, respectively). Participants with ocular manifestations were likeliest to have at least 2 EIMs (P ≤ .001). Even when diagnosis was controlled for, involvement of the right colon (P = .021) was predictive of IBD-associated arthritis across both diseases in a multivariate generalized linear model. CONCLUSIONS: This is the first comprehensive large-cohort assessment of how EIMs relate to one another at the individual vs systems levels. Further, our analysis is the first to recognize specific locations of colon involvement associated with EIMs of IBD, regardless of IBD type. These results are important in identifying patients at risk of developing future EIMs and may help with risk stratification when choosing treatments.

Although extraintestinal manifestations frequently complicate inflammatory bowel disease, predicting those at highest risk of developing them is difficult. We found female patients with Crohn's disease, ocular, and dermatologic manifestations are likeliest to develop multiple extraintestinal manifestations. Further, we found right-sided involvement is predictive of inflammatory bowel disease­associated arthritis.

Sub-communities of the vaginal microbiota in pregnant and non-pregnant women.

Diverse and non-Lactobacillus-dominated vaginal microbial communities are associated with adverse health outcomes such as preterm birth and the acquisition of sexually transmitted infections. Despite the importance of recognizing and understanding the key risk-associated features of these communities, their heterogeneous structure and properties remain ill-defined. Clustering approaches are commonly used to characterize vaginal communities, but they lack sensitivity and robustness in resolving substructures and revealing transitions between potential sub-communities. Here, we address this need with an approach based on mixed membership topic models. Using longitudinal data from cohorts of pregnant and non-pregnant study participants, we show that topic models more accurately describe sample composition, longitudinal changes, and better predict the loss of Lactobacillus dominance. We identify several non-Lactobacillus-dominated sub-communities common to both cohorts and independent of reproductive status. In non-pregnant individuals, we find that the menstrual cycle modulates transitions between and within sub-communities, as well as the concentrations of half of the cytokines and 18% of metabolites. Overall, our analyses based on mixed membership models reveal substructures of vaginal ecosystems which may have important clinical and biological associations.

Integrating compositional and functional content to describe vaginal microbiomes in health and disease.

BACKGROUND: A Lactobacillus-dominated vaginal microbiome provides the first line of defense against adverse genital tract health outcomes. However, there is limited understanding of the mechanisms by which the vaginal microbiome modulates protection, as prior work mostly described its composition through morphologic assessment and marker gene sequencing methods that do not capture functional information. To address this gap, we developed metagenomic community state types (mgCSTs) which use metagenomic sequences to describe and define vaginal microbiomes based on both composition and functional potential. RESULTS: MgCSTs are categories of microbiomes classified using taxonomy and the functional potential encoded in their metagenomes. MgCSTs reflect unique combinations of metagenomic subspecies (mgSs), which are assemblages of bacterial strains of the same species, within a microbiome. We demonstrate that mgCSTs are associated with demographics such as age and race, as well as vaginal pH and Gram stain assessment of vaginal smears. Importantly, these associations varied between mgCSTs predominated by the same bacterial species. A subset of mgCSTs, including three of the six predominated by Gardnerella vaginalis mgSs, as well as mgSs of L. iners, were associated with a greater likelihood of bacterial vaginosis diagnosed by Amsel clinical criteria. This L. iners mgSs, among other functional features, encoded enhanced genetic capabilities for epithelial cell attachment that could facilitate cytotoxin-mediated cell lysis. Finally, we report a mgSs and mgCST classifier for which source code is provided and may be adapted for use by the microbiome research community. CONCLUSIONS: MgCSTs are a novel and easily implemented approach to reduce the dimension of complex metagenomic datasets while maintaining their functional uniqueness. MgCSTs enable the investigation of multiple strains of the same species and the functional diversity in that species. Future investigations of functional diversity may be key to unraveling the pathways by which the vaginal microbiome modulates the protection of the genital tract. Importantly, our findings support the hypothesis that functional differences between vaginal microbiomes, including those that may look compositionally similar, are critical considerations in vaginal health. Ultimately, mgCSTs may lead to novel hypotheses concerning the role of the vaginal microbiome in promoting health and disease, and identify targets for novel prognostic, diagnostic, and therapeutic strategies to improve women's genital health. Video Abstract.