Comparison of shipping versus immediate freezer storage of vaginal samples for vaginal microbiota assessment.

ObjectivesWe evaluated how storing vaginal samples at room temperature in stabilising solutions versus immediate freezing affects 16S rRNA gene amplicon sequencing-based microbiota studies, aiming to simplify home and field collection. METHODS: Twenty participants self-collected six mid-vaginal swabs that were stored in two nucleic acid preservatives (three in modified Solution C2 (Qiagen) and three in Amies/RNALater (Sigma)) in January-February 2016. From each set, two were immediately frozen (-80°C) and one was shipped to the University of Idaho (Moscow, Idaho) with return shipping to the Institute for Genome Sciences (Baltimore, Maryland). Amplicon sequencing of the 16S rRNA gene was used to characterise the vaginal microbiota, VALENCIA was used to assign community state types (CSTs), and quantitative PCR (qPCR) of 16S rRNA genes was used to estimate bacterial abundance. Cohen's Kappa statistic was used to assess within-participant agreement. Bayesian difference of means models assessed within-participant comparisons between shipped and immediately frozen samples. RESULTS: There were 115 samples available for analysis. Average duration of transit for shipped samples was 8 days (SD: 1.60, range: 6-11). Within-participant comparisons of CSTs between shipped and immediately frozen samples revealed complete concordance (kappa: 1.0) for both preservative solutions. No significant differences comparing shipped and immediately frozen samples were found with taxon-level comparisons or bacterial abundances based on pan-bacterial qPCR. CONCLUSIONS: Short-term room temperature shipping of vaginal swabs placed in stabilising solutions did not affect vaginal microbiota composition. Home collection with mail-in of vaginal samples may be a reasonable approach for research and clinical purposes to assess the vaginal microbiota.

Nonoptimal Vaginal Microbiota After Azithromycin Treatment for Chlamydia trachomatis Infection.

We characterized the composition and structure of the vaginal microbiota in a cohort of 149 women with genital Chlamydia trachomatis infection at baseline who were followed quarterly for 9 months after antibiotic treatment. At time of diagnosis, the vaginal microbiota was dominated by Lactobacillus iners or a diverse array of bacterial vaginosis-associated bacteria including Gardnerella vaginalis. Interestingly, L. iners-dominated communities were most common after azithromycin treatment (1 g monodose), consistent with the observed relative resistance of L. iners to azithromycin. Lactobacillus iners-dominated communities have been associated with increased risk of C. trachomatis infection, suggesting that the impact of antibiotic treatment on the vaginal microbiota could favor reinfections. These results provide support for the dual need to account for the potential perturbing effect(s) of antibiotic treatment on the vaginal microbiota, and to develop strategies to protect and restore optimal vaginal microbiota.

Impact of Standard Bacterial Vaginosis Treatment on the Genital Microbiota, Immune Milieu, and Ex Vivo Human Immunodeficiency Virus Susceptibility.

BACKGROUND: Genital immunology is a key determinant of human immunodeficiency virus (HIV) susceptibility. Both factors are modulated by bacterial vaginosis (BV) and, to some extent, by Lactobacillus iners, the genital Lactobacillus spp. that predominates in African, Caribbean, and other Black (ACB) women. We conducted a clinical trial to assess the impact of oral metronidazole treatment on the genital immune parameters of HIV acquisition risks in Kenyan women with BV. METHODS: The primary endpoint was ex vivo cervical CD4+ T-cell HIV susceptibility after 1 month; secondary endpoints included genital cytokine/chemokine levels, cervical immune cell populations, and the composition of the cervico-vaginal microbiota by 16S ribosomal RNA gene amplicon sequencing. RESULTS: BV resolved (Nugent score ≤ 3) at 1 month in 20/45 participants, and cervical CD4+ T-cell HIV entry was moderately reduced in all participants, regardless of treatment outcome. Resolution of BV and reduced abundances of BV-associated gram-negative taxa correlated with reduced genital interleukin (IL)-1α/ß. However, BV resolution and the concomitant colonization by Lactobacillus iners substantially increased several genital chemokines associated with HIV acquisition, including interferon-γ inducible protein (IP)-10, macrophage inflammatory protein (MIP)-3α, and monokine induced by gamma interferon (MIG). In an independent cohort of ACB women, most of whom were BV-free, vaginal chemokines were again closely linked with L. iners abundance, though not other Lactobacillus spp. CONCLUSIONS: BV treatment reduced genital CD4+ T-cell HIV susceptibility and IL-1 levels, but dramatically increased the genital chemokines that may enhance HIV susceptibility; the latter effect was related to the restoration of an Lactobacillus iners-dominated microbiota. Further studies are needed before treatment of asymptomatic BV can be recommended for HIV prevention in ACB communities.

Comparative phase I randomized open-label pilot clinical trial of Gynophilus® (Lcr regenerans®) immediate release capsules versus slow release muco-adhesive tablets.

Gynophilus® (Lcr regenerans®) is a live biotherapeutic product (LBP) that contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35®, which is indicated to restore vaginal health. The aim of the study was to compare the safety, ease of use, and compliance of two formulations (immediate release: IR capsule and slow release: SR muco-adhesive tablets) as well as the colonization of Lcr35® in healthy women. This phase I study (Comprigel) is a parallel, randomized, 4-arm, and open-label clinical trial evaluating an IR daily capsule formulation vs. a SR tablet administered every 3, 4, or 5 days for 21 days. Self-collected vaginal swabs were used to quantify Lcr35® and characterize the composition and structure of the vaginal microbiota. Both LBPs were well-tolerated, and no severe adverse effects were reported. All groups had Lcr35® vaginal concentrations over 107 colony forming unit per milliliter of vaginal secretion on each day in the study. The new Gynophilus® slow release tablets administered either every 3, 4, or 5 days provided vaginal concentrations that were not significantly different from those of classic Gynophilus® (capsule) once-a-day regimen. The LBPs and the different regimens did not adversely influence the abundance of native Lactobacillus spp. and indeed tended to favor their growth and reduce colonization by non-Lactobacillus spp. This study illustrates that the SR muco-adhesive LBP tablet (Gynophilus® SR) administered every 3 or 4 days as a safe, well-tolerated, and efficacious alternative to a more demanding IR daily capsule and could protect women's healthy vaginal microbiome by promoting endogenous Lactobacillus spp.

A retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women.

BACKGROUND: We know very little about the microbiota inhabiting the upper female reproductive tract and how it impacts on fertility. AIMS: This pilot study aimed to examine the vaginal, cervical and endometrial microbiota for women with a history of infertility compared to women with a history of fertility. MATERIALS AND METHODS: Using a retrospective case-control study design, women were recruited for collection of vaginal, cervical and endometrial samples. The microbiota composition was analysed by 16S ribosomal RNA (rRNA) gene amplification and endometrial expression of selected human genes by quantitative reverse transcription polymerase chain reaction. RESULTS: Sixty-five specimens from the reproductive tract of 31 women were successfully analysed using 16S rRNA gene amplicon sequencing (16 controls and 15 cases). The dominant microbial community members were consistent in the vagina and cervix, and generally consistent with the endometrium although the relative proportions varied. We detected three major microbiota clusters that did not group by tissue location or case-control status. There was a trend that infertile women more often had Ureaplasma in the vagina and Gardnerella in the cervix. Testing for the expression of selected genes in the endometrium did not show evidence of correlation with case-control status, or with microbial community composition, although Tenascin-C expression correlated with a history of miscarriage. CONCLUSIONS: There is a need for further exploration of the endometrial microbiota, and how the microbiota members or profile interplays with fertility or assisted reproductive technologies.

Group B Streptococcus and the Vaginal Microbiota.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) is an important neonatal pathogen and emerging cause of disease in adults. The major risk factor for neonatal disease is maternal vaginal colonization. However, little is known about the relationship between GBS and vaginal microbiota. Methods: Vaginal lavage samples from nonpregnant women were tested for GBS, and amplicon-based sequencing targeting the 16S ribosomal RNA V3-V4 region was performed. Results: Four hundred twenty-eight of 432 samples met the high-quality read threshold. There was no relationship between GBS carriage and demographic characteristics, α-diversity, or overall vaginal microbiota community state type (CST). Within the non-Lactobacillus-dominant CST IV, GBS positive status was significantly more prevalent in CST IV-A than CST IV-B. Significant clustering by GBS status was noted on principal coordinates analysis, and 18 individual taxa were found to be significantly associated with GBS carriage by linear discriminant analysis. After adjusting for race/ethnicity, 4 taxa were positively associated with GBS, and 6 were negatively associated. Conclusions: Vaginal microbiota CST and α-diversity are not related to GBS status. However, specific microbial taxa are associated with colonization of this important human pathogen, highlighting a potential role for the microbiota in promotion or inhibition of GBS colonization.

Modeling the integration of bacterial rRNA fragments into the human cancer genome.

BACKGROUND: Cancer is a disease driven by the accumulation of genomic alterations, including the integration of exogenous DNA into the human somatic genome. We previously identified in silico evidence of DNA fragments from a Pseudomonas-like bacteria integrating into the 5'-UTR of four proto-oncogenes in stomach cancer sequencing data. The functional and biological consequences of these bacterial DNA integrations remain unknown. RESULTS: Modeling of these integrations suggests that the previously identified sequences cover most of the sequence flanking the junction between the bacterial and human DNA. Further examination of these reads reveals that these integrations are rich in guanine nucleotides and the integrated bacterial DNA may have complex transcript secondary structures. CONCLUSIONS: The models presented here lay the foundation for future experiments to test if bacterial DNA integrations alter the transcription of the human genes.

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.

Association between cigarette smoking and the vaginal microbiota: a pilot study.

BACKGROUND: Smoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV), a condition defined in part by decimation of Lactobacillus spp. The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose women to BV. BPDE increases bacteriophage induction in Lactobacillus spp. and is found in the vaginal secretions of smokers. We compared the vaginal microbiota between smokers and non-smokers and followed microbiota changes in a smoking cessation pilot study. METHODS: In 2010-2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (Phase A) and 9 smokers were enrolled and followed for a 12-week smoking cessation program (Phase B). Phase B included weekly behavioral counseling and nicotine patches to encourage smoking cessation. In both phases, participants self-collected mid-vaginal swabs (daily, Phase B) and completed behavioral surveys. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes (V1-V3 regions). Vaginal smears were assigned Nugent Gram stain scores. Smoking status was evaluated (weekly, Phase B) using the semi-quantitative NicAlert® saliva cotinine test and carbon monoxide (CO) exhalation. RESULTS: In phase A, there was a significant trend for increasing saliva cotinine and CO exhalation with elevated Nugent scores (P value <0.005). Vaginal microbiota clustered into three community state types (CSTs); two dominated by Lactobacillus (L. iners, L. crispatus), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobes (termed CST-IV). Women who were observed in the low-Lactobacillus CST-IV state were 25-fold more likely to be smokers than those dominated by L. crispatus (aOR: 25.61, 95 % CI: 1.03-636.61). Four women completed Phase B. One of three who entered smoking cessation with high Nugent scores demonstrated a switch from CST-IV to a L.iners-dominated profile with a concomitant drop in Nugent scores which coincided with completion of nicotine patches. The other two women fluctuated between CST-IV and L. iners-dominated CSTs. The fourth woman had low Nugent scores with L. crispatus-dominated CSTs throughout. CONCLUSION: Smokers had a lower proportion of vaginal Lactobacillus spp. compared to non-smokers. Smoking cessation should be investigated as an adjunct to reducing recurrent BV. Larger studies are needed to confirm these findings.

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 [χ(2)(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.

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.

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.

Short-term feeding of defatted bovine colostrum mitigates inflammation in the gut via changes in metabolites and microbiota in a chicken animal model.

BACKGROUND: Nondrug supplement strategies to improve gut health have largely focused on the effects of individual compounds to improve one aspect of gut homeostasis. However, there is no comprehensive assessment of the reproducible effects of oral, short-term, low-level colostrum supplementation on gut inflammation status that are specific to the ileum. Herein, a chicken animal model highly responsive to even mild gut inflammatory stimuli was employed to compare the outcomes of feeding a standard diet (CON) to those of CON supplemented with a centrifuge-defatted bovine colostrum (BC) or a nonfat dried milk (NFDM) control on the efficiency of nutrient use, ileal morphology, gut nitro-oxidative inflammation status, metabolites, and the composition of the microbiota. RESULTS: A repeated design, iterative multiple regression model was developed to analyze how BC affected ileal digesta-associated anti-inflammatory metabolite abundance coincident with observed changes in the ileal microbiome, mitigation of epithelial inflammation, and ileal surface morphology. An improved whole body nutrient use efficiency in the BC group (v CON and NFDM) coincided with the observed increased ileum absorptive surface and reduced epithelial cell content of tyrosine-nitrated protein (NT, biomarker of nitro-oxidative inflammatory stress). Metabolome analysis revealed that anti-inflammatory metabolites were significantly greater in abundance in BC-fed animals. BC also had a beneficial BC impact on microbiota, particularly in promoting the presence of the bacterial types associated with eubiosis and the segmented filamentous bacteria, Candidatus Arthromitus. CONCLUSION: The data suggest that an anti-inflammatory environment in the ileum was more evident in BC than in the other feeding groups and associated with an increased content of statistically definable groups of anti-inflammatory metabolites that appear to functionally link the observed interactions between the host's improved gut health with an observed increase in whole body nutrient use efficiency, beneficial changes in the microbiome and immunometabolism.

High-resolution functional description of vaginal microbiomes in health and disease.

Background: A Lactobacillus-dominated vaginal microbiome provides the first line of defense against numerous 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 limitation, we developed metagenomic community state types (mgCSTs) which uses metagenomic sequences to describe and define vaginal microbiomes based on both composition and function. 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 mgSs, as well as a mgSs of L. iners, were associated with a greater likelihood of Amsel bacterial vaginosis diagnosis. 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 as an easily applied, standardized method for use by the microbiome research community. Conclusions: MgCSTs are a novel and easily implemented approach to reducing the dimension of complex metagenomic datasets, while maintaining their functional uniqueness. MgCSTs enable 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 protection to 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.

Relationships between the vaginal microbiota and genitourinary syndrome of menopause symptoms in postmenopausal women: the Study of Women's Health Across the Nation.

OBJECTIVE: To describe vaginal microbiota classified by community state types (CST) in a diverse cohort of postmenopausal women and evaluate relationships among genitourinary syndrome of menopause (GSM) symptoms (vaginal dryness, vulvovaginal irritation, sexual pain, dysuria, urinary urgency), CSTs, estrogen, vaginal maturation index (VMI), and vaginal pH. METHODS: In the Study of Women's Health Across the Nation, 1,320 women aged 60.4 to 72.5 years self-collected (2015-2017) vaginal samples analyzed for microbiota composition and structure (CSTs) using 16S rRNA gene amplicon sequencing, VMI, and pH. GSM symptoms were collected with self-administered questionnaires; interviewers elicited estrogen use and measured body mass index. Serum E2 and E1 were measured using high-performance liquid chromatography. We analyzed data using Pearson χ2 tests, analysis of variance, Kruskal-Wallis tests, and binomial logistic regression. RESULTS: The most frequently occurring CST was low Lactobacillus species IV-C (49.8%); 36.4% of women had CSTs dominated by Lactobacillus species. More than half of the women with vaginal atrophy biomarkers (VMI <50 and pH >5) had CST IV-C0, whereas women using estrogen or with higher E1 and E2 levels had a higher prevalence of Lactobacillus crispatus -dominated CST I ( P values < 0.001). Sexual pain was associated with atrophy biomarkers and independently associated with Streptococcus species-dominated CST IV-C1 (odds ratio, 2.26; 95% confidence intervals, 1.20-4.23). For all other GSM symptoms, we found no consistent associations with E1 or E2 levels, atrophy biomarkers, or any CST. CONCLUSIONS: Although close relationships exist among estrogen, CSTs, VMI, and pH, sexual pain was the only GSM symptom associated with the structure of vaginal microbiota and atrophy biomarkers.

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.

Influence of Hormonal Contraceptive Use and HIV on Cervicovaginal Cytokines and Microbiota in Malawi.

Important questions remain on how hormonal contraceptives alter the local immune environment and the microbiota in the female genital tract and how such effects may impact susceptibility to HIV infection. We leveraged samples from a previously conducted clinical trial of Malawian women with (n = 73) and without (n = 24) HIV infection randomized to depot medroxyprogesterone acetate (DMPA) or the levonogestrel implant in equal numbers within each group and determined the effects of these hormonal contraceptives (HCs) on the vaginal immune milieu and the composition of the vaginal microbiota. Longitudinal data for soluble immune mediators, measured by multiplex bead arrays and enzyme-linked immunosorbent assays (ELISAs), and vaginal microbiota, assessed by 16S rRNA gene amplicon, were collected prior to and over a period of 180 days post-HC initiation. DMPA and levonogestrel had only minimal effects on the vaginal immune milieu and microbiota. In women with HIV, with the caveat of a small sample size, there was an association between the median log10 change in the interleukin-12 (IL-12)/IL-10 ratio in vaginal fluid at day 180 post-HC compared to baseline when these women were classified as having a community state type (CST) IV vaginal microbiota and were randomized to DMPA. Long-lasting alterations in soluble immune markers or shifts in microbiota composition were not observed. Furthermore, women with HIV did not exhibit increased viral shedding in the genital tract after HC initiation. Consistent with the results of the ECHO (Evidence for Contraceptive Options and HIV Outcomes) trial, our data imply that the progestin-based HC DMPA and levonorgestrel are associated with minimal risk for women with HIV. (This study has been registered at ClinicalTrials.gov under registration no. NCT02103660). IMPORTANCE The results of the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial, the first large randomized controlled clinical trial comparing the HIV acquisition risk of women receiving DMPA, the levonorgestrel (LNG) implant, or the copper intrauterine device (IUD), did not reveal an increased risk of HIV acquisition for women on any of these three contraceptives. Our study results confirm that the two different progestin-based hormonal contraceptives DMPA and levonogestrel will not increase the risk for HIV infection. Furthermore, DMPA and levonogestrel have only minimal effects on the immune milieu and the microbiota in the vaginal tract, attesting to the safety of these hormonal contraceptives.

Lactobacillus-dominance and rapid stabilization of vaginal microbiota in combined oral contraceptive pill users examined through a longitudinal cohort study with frequent vaginal sampling over two years.

BACKGROUND: Bacterial vaginosis (BV), a condition in which vaginal Lactobacillus spp. are in low abundance, is associated with vulvovaginal symptoms, obstetric outcomes and urogenital infections. Recurrent BV is difficult to manage, and emerging data indicate a reduced risk of BV with the use of hormonal contraception (HC). Despite widespread use, little longitudinal data is available on whether, and in what timeframe, combined oral contraceptive pills (COCs) may act to affect vaginal microbiota stability and Lactobacillus dominance. METHODS: We compared the vaginal microbiota of reproductive-age cisgender women during intervals on combined estrogen and progestin COCs with non-use intervals in a 2-year observational study. Vaginal microbiota were characterized by 16S rRNA gene amplicon sequencing. FINDINGS: COC users were more likely to have Lactobacillus-dominated microbiota and more stable microbiota over time. Stability increased and then plateaued four weeks after COC initiation. The associations between COCs and Lactobacillus spp. dominance, and microbiota stability, were statistically significant for White, but not African American women; however sample size was limited for African American participants. Findings were similar for other forms of HC and when excluding samples collected during menses. INTERPRETATION: Our study provides a methodologic framework to evaluate observational longitudinal microbiota data with exposure crossovers. We found COCs are associated with vaginal microbiota stability and a Lactobacillus-dominated state. COCs appear to impact stability within a month of initiation. Our findings have clinical implications for how soon benefits can be expected in (at least White) patients initiating COCs, and support the need for larger prospective trials to verify our results in ethnically diverse populations. FUNDING: R01-AI089878.

Vaginal microbiome, antiretroviral concentrations, and HIV genital shedding in the setting of hormonal contraception initiation in Malawi.

OBJECTIVE: The aim of this study was to understand how vaginal microbiota composition affects antiretroviral concentrations in the setting of hormonal contraception initiation. METHODS: Cervicovaginal fluid (CVF) concentrations of tenofovir, lamivudine, and efavirenz from 73 Malawian women with HIV were compared before and after initiation of depot-medroxyprogesterone acetate (DMPA) or levonorgestrel implant. We evaluated antiretroviral concentrations and vaginal microbiota composition/structure in the context of contraception initiation and predicted genital shedding using multivariable repeated measurements models fit by generalized estimating equations. RESULTS: Mean lamivudine CVF concentrations decreased 37% 1 month after contraception initiation. Subgroup analyses revealed a 41% decrease in women 1 month after initiating levonorgestrel implant, but no significant difference was observed in DMPA group alone. Tenofovir, lamivudine, and efavirenz CVF concentrations were positively correlated with anaerobic bacteria associated with nonoptimal vaginal microbiota. Risk of genital HIV shedding was not significantly associated with tenofovir or lamivudine CVF concentrations [tenofovir relative risk (RR): 0.098, P = 0.75; lamivudine RR: 0.142, P = 0.54]. Lack of association between genital HIV shedding and efavirenz CVF concentrations did not change when adjusting for vaginal microbiota composition and lamivudine/tenofovir CVF concentrations (RR: 1.33, P = 0.531). CONCLUSION: No effect of hormone initiation on genital shedding provides confidence that women with HIV on either DMPA or levonorgestrel implant contraception will not have compromised ART efficacy. The unexpected positive correlation between antiretroviral CVF concentrations and certain bacterial taxa relative abundance requires further work to understand the mechanism and clinical relevance.

Fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study.

BACKGROUND: High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation. METHODS: Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, ß-glucuronidase and ß-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, ß-glucuronidase and ß-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05. RESULTS: In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57-0.70, P=0.03-0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal ß-glucuronidase (R=0.36, P=0.04). In contrast, fecal ß-glucuronidase correlated inversely with fecal total estrogens, both conjugated and deconjugated (R≤-0.47, P≤0.01). Premenopausal female estrogen levels, which were collected across menstrual cycles and thus highly variable, were completely unrelated to fecal microbiome and enzyme parameters (P≥0.6). CONCLUSIONS: Intestinal microbial richness and functions, including but not limited to ß-glucuronidase, influence levels of non-ovarian estrogens via enterohepatic circulation. Thus, the gut microbial community likely affects the risk for estrogen-related conditions in older adults. Understanding how Clostridia taxa relate to systemic estrogens may identify targets for interventions.