Cervicovaginal Microbiota Research Articles

12: Impact of high-risk cervicovaginal microbial communities and perceived stress on recurrent spontaneous preterm birth

While select cervicovaginal (CV) microbiota and psychosocial factors have been associated with spontaneous preterm birth (sPTB), their impact on the risk of recurrence remains unclear. It is also unknown whether psychosocial factors, such as maternal stress, amplify underlying biologic risk. This study sought to determine the effect of high-risk CV microbial communities and perceived stress on the risk of recurrent sPTB. This was a secondary analysis of a prospective pregnancy cohort, Motherhood and Microbiome. The Cohen’s Perceived Stress Scale (PSS-14) was administered and cervical swabs were obtained between 16-20 weeks. PSS-14 scores ≥30 reflected high perceived stress. We analyzed CV microbiota using 16S rRNA sequencing and classified microbial communities into community state types (CST). CST IV is a high-risk CV microbial community characterized by anaerobes and a lack of Lactobacillus. The final cohort included women with prior sPTB who had recurrent sPTB or term birth. Multivariable logistic regression modeled adjusted associations between CST IV and sPTB as well as high PSS-14 and sPTB. Among the 201 women, 33 (16.4%) had CST IV and 45 (22.5%) had recurrent sPTB. While proportions of recurrent sPTB v. term birth among women with CST IV were not different (48.8% v. 30.2%; p=0.082), models adjusted for African American race and marital status revealed a significant association between CST IV and recurrent sPTB (aOR 2.99; CI 1.13-7.96). High PSS-14 was a confounder on this association. After adjusting for high PSS-14, the odds of recurrent sPTB with CST IV were higher (aOR 3.83; CI 1.30-11.33). Among women with a prior sPTB, high-risk CV microbial communities confer increased risk of recurrent sPTB. Adjustment for perceived stress amplified associations between CST IV and recurrent sPTB. Identification of modifiable social or behavioral factors may unveil novel non-pharmacologic interventions to decrease recurrent sPTB among women with underlying biologic risk. (R01NR01478; ME)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Diverse Expression of Antimicrobial Activities Against Bacterial Vaginosis and Urinary Tract Infection Pathogens by Cervicovaginal Microbiota Strains of Lactobacillus gasseri and Lactobacillus crispatus.

We aimed to analyze the strain-by-strain expression of a large panel of antimicrobial activities counteracting the virulence mechanisms of bacterial vaginosis-associated Prevotella bivia CI-1 and Gardnerella vaginalis 594, pyelonephritis-associated Escherichia coli CFT073, and recurrent cystitis- and preterm labor-associated IH11128 E. coli by Lactobacillus gasseri and Lactobacillus crispatus clinical strains, and L. gasseri ATCC 9857 and KS 120.1, and L. crispatus CTV-05 strains isolated from the cervicovaginal microbiota of healthy women. All L. gasseri and L. crispatus strains exerted antimicrobial activity by secreted lactic acid, which killed the microbial pathogens by direct contact. Potent bactericidal activity was exerted by a very limited number of resident L. gasseri and L. crispatus strains showing the specific ability to a strain to produce and release antibiotic-like compounds. These compounds eradicated the microbial pathogens pre-associated with the surface of cervix epithelial cells, providing efficient protection of the cells against the deleterious effects triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Furthermore, these compounds crossed the cell membrane to kill the pre-internalized microbial pathogens. In addition, all L. gasseri and L. crispatus cells exhibited another non-strain specific activity which inhibited the association of microbial pathogens with cervix epithelial cells with varying efficiency, partially protecting the cells against lysis and detachment triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Our results provide evidence of strain-level specificity for certain antimicrobial properties among cervicovaginal L. gasseri and L. crispatus strains, indicating that the presence of a particular species in the vaginal microbiota is not sufficient to determine its benefit to the host. A full repertory of antimicrobial properties should be evaluated in choosing vaginal microbiota-associated Lactobacillus isolates for the development of live biotherapeutic strategies.

Cervicovaginal microbial communities deficient in Lactobacillus species are associated with second trimester short cervix

The cervix functions as a barrier to ascending pathogens in pregnancy. Short cervical length and lack of cervicovaginal Lactobacillus species are risk factors for spontaneous preterm birth; however, whether they interact to increase risk remains unknown. We sought to examine the relationship between cervicovaginal microbiota and short cervix as well as their combined impact on spontaneous preterm birth risk. This was a secondary analysis of a prospective nested, case-control pregnancy study. Cervical swabs were collected between 16 and 20 weeks of gestation. Cervical length was measured per standard clinical care during a clinically indicated ultrasound at approximately 20 weeks of gestation. Cervicovaginal microbiota were analyzed with 16S ribosomal RNA gene sequencing and classified into community state types among 67 cases of spontaneous preterm birth, 47 cases of medically indicated preterm birth, and 358 cases of term births. Logistic regression was used to model associations of community state type IV, a community characterized by a paucity of Lactobacillus species and a wide array of anaerobic bacteria, and short cervix (<25 mm) as well as to model the association of a combination of short cervix and community state type IV with the odds of spontaneous preterm birth. Among the 472 women in the data set, there were 38 with short cervix (8.1%) and 177 with community state type IV (37.5%). Short cervix was associated with spontaneous preterm birth (adjusted odds ratio, 15.59; 95% confidence interval, 6.77-35.92). Women with community state type IV had higher odds of short cervix (adjusted odds ratio, 2.17; 95% confidence interval, 1.04-4.53) as well as spontaneous preterm birth (adjusted odds ratio, 1.97; 95% confidence interval, 1.06-3.65). While the interaction of community state type IV and short cervix was not significant (P= .771), women with both short cervix and community state type IV (n= 20) had higher odds of spontaneous preterm birth compared with women with both normal cervical length and community state types I, II, III, or V (n= 277) (adjusted odds ratio, 21.8; 95% confidence interval, 6.78-70.2). Community state type IV, characterized by a diverse set of strict and facultative anaerobes and a paucity of Lactobacillus species, is associated with increased odds of short cervix. Women with both community state type IV and short cervix have higher odds of spontaneous preterm birth than women with either factor alone. Determining the cascade of events leading to premature cervical shortening, including dysbiosis, may be critical in preventing spontaneous preterm birth.

The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection.

The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(-) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota's role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

The vaginal microbiota, human papillomavirus and cervical dysplasia: a systematic review and network meta-analysis.

Persistent infection with high-risk human papillomavirus can lead to cervical dysplasia and cancer. Recent studies have suggested associations between the composition of the vaginal microbiota, infection with human papillomavirus (HPV) and progression to cervical dysplasia and cancer. To assess how specific cervico-vaginal microbiota compositions are associated with HPV infection, cervical dysplasia and cancer, we conducted a systematic review and network meta-analysis (registered in PROSPERO: CRD42018112862). PubMed, Web of science, Embase and Cochrane database. All original studies describing at least two community state types of bacteria (CST), based on molecular techniques enabling identification of bacteria, and reporting the association with HPV infection, cervical dysplasia and/or cervical cancer. For the meta-analysis, a network map was constructed to provide an overview of the network relationships and to assess how many studies provided direct evidence for the different vaginal microbiota compositions and HPV, cervical dysplasia or cancer. Thereafter, the consistency of the model was assessed, and forest plots were constructed to pool and summarise the available evidence, presenting odds ratios and 95% confidence intervals. Vaginal microbiota dominated by non-Lactobacilli species or Lactobacillus iners were associated with three to five times higher odds of any prevalent HPV and two to three times higher for high-risk HPV and dysplasia/cervical cancer compared with Lactobacillus crispatus. These findings suggest an association between certain bacterial community types of the vaginal microbiota and HPV infection and HPV-related disease. This may be useful for guiding treatment options or serve as biomarkers for HPV-related disease. This network meta-analysis suggests an association between different vaginal bacterial community types and the risk of HPV.

Association between the cervicovaginal microbiome, BRCA1 mutation status, and risk of ovarian cancer: a case-control study.

Various factors-including age, family history, inflammation, reproductive factors, and tubal ligation-modulate the risk of ovarian cancer. In this study, our aim was to establish whether women with, or at risk of developing, ovarian cancer have an imbalanced cervicovaginal microbiome. We did a case-control study in two sets of women aged 18-87 years in the Czech Republic, Germany, Italy, Norway, and the UK. The ovarian cancer set comprised women with epithelial ovarian cancer and controls (both healthy controls and those diagnosed with benign gynaecological conditions). The BRCA set comprised women with a BRCA1 mutation but without ovarian cancer and controls who were wild type for BRCA1 and BRCA2 (both healthy controls and those with benign gynaecological conditions). Cervicovaginal samples were gathered from all participants with the ThinPrep system and then underwent 16S rRNA gene sequencing. For each sample, we calculated the proportion of lactobacilli species (ie, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii), which are essential for the generation of a protective low vaginal pH, in the cervicovaginal microbiota. We grouped samples into those in which lactobacilli accounted for at least 50% of the species present (community type L) and those in which lactobacilli accounted for less than 50% of the species present (community type O). We assessed the adjusted association between BRCA1 status and ovarian cancer status and cervicovaginal microbiota community type, using a logistic regression model with a bias reduction method. Participants were recruited between Jan 2, 2016, and July 21, 2018. The ovarian cancer set (n=360) comprised 176 women with epithelial ovarian cancer, 115 healthy controls and 69 controls with benign gynaecological conditions. The BRCA set (n=220) included 109 women with BRCA1 mutations, 97 healthy controls wild type for BRCA1 and BRCA2 and 14 controls with a benign gynaecological condition wild type for BRCA1 and BRCA2. On the basis of two-dimensional density plots, receiver-operating characteristic curve analysis, and age thresholds used previously, we divided the cohort into those younger than 50 years and those aged 50 years or older. In the ovarian cancer set, women aged 50 years or older had a higher prevalence of community type O microbiota (81 [61%] of 133 ovarian cancer cases and 84 [59%] of 142 healthy controls) than those younger than 50 years (23 [53%] of 43 cases and 12 [29%] of 42 controls). In the ovarian cancer set, women younger than 50 years with ovarian cancer had a significantly higher prevalence of community type O microbiota than did age-matched controls under a logistic regression model with bias correction (odds ratio [OR] 2·80 [95% CI 1·17-6·94]; p=0·020). In the BRCA set, women with BRCA1 mutations younger than 50 years were also more likely to have community type O microbiota than age-matched controls (OR 2·79 [95% CI 1·25-6·68]; p=0·012), after adjustment for pregnancy (ever). This risk was increased further if more than one first-degree family member was affected by any cancer (OR 5·26 [95% CI 1·83-15·30]; p=0·0022). In both sets, we noted that the younger the participants, the stronger the association between community type O microbiota and ovarian cancer or BRCA1 mutation status (eg, OR for community type O for cases aged <40 years in the ovarian cancer set 7·00 [95% CI 1·27-51·44], p=0·025; OR for community type O for BRCA1 mutation carriers aged <35 years in the BRCA set 4·40 [1·14-24·36], p=0·031). The presence of ovarian cancer, or factors known to affect risk for the disease (ie, age and BRCA1 germline mutations), were significantly associated with having a community type O cervicovaginal microbiota. Whether re-instatement of a community type L microbiome by using, for example, vaginal suppositories containing live lactobacilli, would alter the microbiomial composition higher up in the female genital tract and in the fallopian tubes (the site of origin of high-grade serous ovarian cancer), and whether such changes could translate into a reduced incidence of ovarian cancer, needs to be investigated. EU Horizon 2020 Research and Innovation Programme, EU Horizon 2020 European Research Council Programme, and The Eve Appeal.

Sex Work and Sexual Behaviour: Associations with Vaginal Microbiome and Cytokine Profiles in Young Women from Mombasa, Kenya

Background: Certain cervicovaginal microbiota and inflammatory cytokines are strongly associated with each other and with HIV acquisition, but the upstream drivers of this milieu remain partially defined. Methods: We characterized the vaginal microbiome and cytokine profiles of sexually active young women aged 14-24 years from Mombasa, Kenya (n=168) who were recruited from venues or hotspots associated with sex work. Vaginal secretions were collected via self-inserted SoftCupTM, and assayed for cytokines and vaginal microbiome via multiplex ELISA and 16S rRNA sequencing, respectively. Findings: The median age of participants was 20 (IQR: 18-22). In 63% of women (105/168) the vaginal microbial communities were characterized by Gardnerella and/or Prevotella spp.-dominance; a further 29% (49/168) were predominantly Lactobaccillus iners. Microbiome cluster explained a large proportion of cytokine variation (>50% by the first 2 principle components). Age was not associated with vaginal microbial profiles in univariate or multivariate analyses. Women self-identifying as sex workers had increased alpha (intra-individual) diversity, independent of age, recent sexual activity, HIV and other STIs (beta = 0.55, 95% CI: 0.13-0.97, p = 0.01). Recent sex (number of partners or vaginal sex acts last week, time since last vaginal sex) correlated with increased alpha diversity, even in participants who were not involved in sex work. Interpretation: HIV risk-associated vaginal microbial profiles and inflammation were highly associated with each other and both highly prevalent in young women from Mombasa, Kenya. These risk profiles did not differ by age, but were increased in those engaging in formal sex work, and in all participants engaging in recent and/or more frequent sexual activity. Funding Statement: The Transitions study was funded by an operating grant (MOP-13044) from the Canadian Institutes of Health Research (CIHR). Biological aspects of the study were funded by the Canadian Institute of Health Research (CIHR), grant numbers TMI 138658 and PJT-148796. AS is funded by the European & Developing Countries Clinical Trials Partnership (EDCTP) Career Development Fellowship; SM is supported by a CIHROHTN New Investigator Award; MB and LRM are supported by CIHR New Investigator Awards. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The study was approved by the ethical review boards of the University of Manitoba and Kenyatta National Hospital.

Lipschütz genital ulcer revisited: is juvenile gangrenous vasculitis of the scrotum the male counterpart?

Since its first description as ulcus vulvae acutum by Benjamin Lipschütz in 1912, the etiopathogenesis of this peculiar genital ulcer remains incompletely understood. In his original description, two different types of genital ulcers were observed and proposed, which were not precisely defined and distinguished in most subsequent reports. The first type is characterized by acute excruciating genital ulcers of first-time onset with self-limited non-recurrent course in association with gravely symptomatic systemic infections, in which a primary Epstein-Barr virus (EBV) infection is later identified to be probably the most common aetiology. The second type of ulcer usually refers to little painful ulcers of unknown etiopathogenesis in the absence of fever or chills and with a slow torpid progression and recurrent nature. Differentiation from idiopathic aphthous ulcers is unclear. The changes of the cervicovaginal microbiota and microbiome in diseased state deserve further clarification. Acute genital ulcers associated with primary EBV infection in women have drawn attention since 1970s, while the corresponding penile ulcers in men were already known in 1950s. First presented in 1973, juvenile gangrenous vasculitis of the scrotum with an acute painful scrotal ulcer preceded by symptomatic pharyngeal infections can be considered as the male counterpart of ulcus vulvae acutum, and the future clinical survey should include primary EBV infection.

Defining characteristics of genital health in South African adolescent girls and young women at high risk for HIV infection.

The genital tract of African women has been shown to differ from what is currently accepted as ‘normal’, defined by a pH≤4.5 and lactobacilli-dominated microbiota. Adolescent girls and young women (AGYW) from sub-Saharan Africa are at high risk for HIV, and we hypothesized that specific biological factors are likely to be influential. This study aimed to compare characteristics of vaginal health in HIV-negative AGYW (16-22-years-old), from two South African communities, to international norms. We measured plasma hormones, vaginal pH, presence of BV (Nugent scoring), sexually transmitted infections (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoea, Trichomonas vaginalis, Mycoplasma genitalium) and candidiasis (Gram stain) in AGYW (n = 298) from Cape Town and Soweto. Cervicovaginal microbiota was determined by 16S pyrosequencing; 44 genital cytokines were measured by Luminex; and cervical T-cell activation/proliferation (CCR5, HLA-DR, CD38, Ki67) was measured by multiparametric flow cytometry. 90/298 (30.2%) AGYW were negative for BV, candidiasis and bacterial STIs. L. crispatus and L. iners were the dominant bacteria in cervicovaginal swabs, and the median vaginal pH was 4.7. AGYW with L. crispatus-dominant microbiota (42.4%) generally had the lowest cytokine concentrations compared to women with more diverse microbiota (34/44 significantly upregulated cytokines). Frequencies of CCR5+CD4+ T-cells co-expressing CD38 and HLA-DR correlated positively with interleukin (IL)-6, TNF-α, GRO-α, macrophage inflammatory protein (MIP)-1α, and IL-9. While endogenous oestrogen had an immune-dampening effect on IL-6, TNF-related apoptosis-inducing ligand (TRAIL) and IL-16, injectable hormone contraceptives (DMPA and Net-EN) were associated with significantly lower endogenous hormone concentrations (p<0.0001 for oestrogen and progesterone) and upregulation of 34/44 cytokines. Since genital inflammation and the presence of activated CD4+ T cells in the genital tract have been implicated in increased HIV risk in South African women, the observed high levels of genital cellular activation and cytokines from AGYW may point towards biological factors increasing HIV risk in this region.

Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery

Failure to predict and understand the causes of preterm birth, the leading cause of neonatal morbidity and mortality, have limited effective interventions and therapeutics. From a cohort of 2000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Using innovative Bayesian modeling of cervicovaginal microbiota, seven bacterial taxa were significantly associated with increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of β-defensin-2 lowered the risk of sPTB associated with cervicovaginal microbiota in an ethnicity-dependent manner. Surprisingly, even in Lactobacillus spp. dominated cervicovaginal microbiota, low β-defensin-2 was associated with increased risk of sPTB. These findings hold promise for diagnostics to accurately identify women at risk for sPTB early in pregnancy. Therapeutic strategies could include immune modulators and microbiome-based therapeutics to reduce this significant health burden.

The Evolving Facets of Bacterial Vaginosis: Implications for HIV Transmission

Bacterial vaginosis (BV) is a common yet poorly understood vaginal condition that has become a major focus of HIV transmission and immunology research. Varied terminologies are used by clinicians and researchers to describe microbial communities that reside in the female reproductive tract (FRT), which is driven, in part, by microbial genetic and metabolic complexity, evolving diagnostic and molecular techniques, and multidisciplinary perspectives of clinicians, epidemiologists, microbiologists, and immunologists who all appreciate the scientific importance of understanding mechanisms that underlie BV. This Perspectives article aims to clarify the varied terms used to describe the cervicovaginal microbiota and its “nonoptimal” state, under the overarching term of BV. The ultimate goal is to move toward language standardization in future literature that facilitates a better understanding of the impact of BV on FRT immunology and risk of sexually transmitted infections, including HIV.

Cervical microbiota in women with cervical intra-epithelial neoplasia, prior to and after local excisional treatment, a Norwegian cohort study

BackgroundLocal treatment for cervical intraepithelial neoplasia (CIN) by Loop Electrosurgical Excision Procedure (LEEP) has been correlated with reproductive morbidity, while the cervicovaginal microbiota is also known to affect the risk of preterm delivery. CIN and treatment by LEEP might change the cervical microbiota. The main aim of this study was to describe the cervical microbiota before and after LEEP and assess its associaton with cone depth and HPV persistence. Further, we aimed to compare the microbiota to references with normal cervical cytology.MethodsBetween 2005 and 2007, we prospectively identified 89 women planned for LEEP in a Norwegian hospital and recruited 100 references with a normal cervical cytology. Endocervical swabs were collected prior to treatment and at six (n = 77) and 12 months (n = 72) post LEEP for bacterial culture and PCR, and post LEEP for DNA testing for human papillomavirus (HPV). We compared the cervical microbiota composition before and after treatment and between women planned for LEEP vs references.ResultsThere was a reduction in the number of non-Lactobacillus bacterial species six and 12 months after LEEP compared to before treatment and a tendency towards a concomitant increase in Lactobacillus. No association between the detection of cervical bacteria, HPV persistence or cone depth was found.Women planned for LEEP carried significantly more Bacteroides spp., Gardnerella vaginalis, Mycoplasma hominis and Ureaplasma parvum as well as a greater number of bacterial species than the references.ConclusionsLocal excisional treatment appears to alter the cervical microbiota towards a less diverse microbiota. Women with CIN have a more diverse cervical microbiota compared to women with normal cervical cytology.

Biofilm in Genital Ecosystem: A Potential Risk Factor for Chlamydia trachomatis Infection.

In healthy women, the cervicovaginal microbiota is mostly populated by Lactobacillus spp., the main host defense factor of the female genital tract. In addition to Lactobacilli, other microorganisms populate the cervicovaginal microbiota, like Candida spp. and Gardnerella vaginalis. The overgrowth of Candida spp. or G. vaginalis, known as biofilm-producing microorganisms in the genital ecosystem, may lead to microbial dysbiosis that increases the risk of acquiring sexually transmitted infections, like Chlamydia trachomatis. C. trachomatis, the leading cause of bacterial sexually transmitted diseases, is still considered an important public health problem worldwide because of the impact of asymptomatic infections on long-term reproductive sequelae, including pelvic inflammatory disease and infertility. The aim of our study was to investigate the interaction between C. trachomatis and the biofilm produced by Candida albicans or Gardnerella vaginalis, evaluating whether the biofilm can harbor C. trachomatis and influence its survival as well as its infectious properties. In order to do so, we developed an in vitro coculture transwell-based biofilm model. Our findings proved, for the first time, that C. trachomatis, an intracellular obligate pathogen, survived, for up to 72 hours after exposure, inside the biofilm produced by C. albicans or G. vaginalis, retaining its infectious properties, as evidenced by the typical chlamydial inclusions observed in the cell monolayer (chlamydial inclusion-forming units at 72 h: 9255 ± 1139 and 9873 ± 1015, respectively). In conclusion, our results suggest that the biofilm related to Candida or Gardnerella genital infections may act as a reservoir of C. trachomatis and, thus, contribute to the transmission of the infection in the population as well as to its dissemination into the upper genital tract, increasing the risk of developing severe reproductive sequelae.

523: Distinct cervicovaginal space microbiota are associated with SPTB in women undergoing cervical cerclage

523: Distinct cervicovaginal space microbiota are associated with SPTB in women undergoing cervical cerclage

560: Are cervicovaginal microbial communities early in pregnancy associated with a mid-trimester short cervix?

The cervix functions as a physical and biochemical barrier to ascending pathogens in pregnancy. Short cervical length is a major risk factor for spontaneous preterm birth (sPTB). While the lack of lactobacillus species has been associated with an increased risk of sPTB, the relationship between cervicovaginal (CV) microbial communities and cervical length is unknown. We sought to examine the association between CV microbial communities and short cervix. This was a secondary analysis of a pregnancy cohort study, “Motherhood and Microbiome.” Cervical swabs were collected at 16-20 weeks’ gestation and cervical length was measured by transvaginal ultrasound at the time of the level 2 ultrasound (mean 20 weeks). We analyzed the CV microbiota with 16S rRNA sequencing and classified microbial communities into CSTs as previously reported (Ravel et al. PNAS 2011). CST IV is characterized by a preponderance of anaerobes and paucity of lactobacillus species. In women with cervical length measured, CV microbial data were available on 67 sPTBs, 47 medically-indicated PTBs (mPTBs), and 358 term births. We used log-binomial regression to model associations of CST IV and short cervix (<25 mm) and calculate adjusted risk ratios (aRR). Mean cervical length was 34.6 mm, 34.3 mm, and 26.2 mm among term births, mPTBs, and sPTBs, respectively (p<0.0001) and did not differ significantly among women with CST IV versus all other women (32.9 mm and 33.6 mm respectively, p=0.3); however, the prevalence of a short cervix was significantly higher (11.3% compared to 6.1%, p=0.04). After adjusting for race/ethnicity, body mass index, insurance status, and prior sPTB, women with CST IV had higher risk of short cervix (aRR 1.9, CI: 1.03-3.6). CST IV, which is characterized by anaerobes and a paucity of lactobacillus species, is associated with increased risk of short cervix during pregnancy, a major risk factor for sPTB. Determining the cascade of events that lead to premature cervical remodeling, potentially including dysbiosis, could be critical in preventing sPTB (R01NR014784; ME).

Lactobacillus crispatus BC5 Interferes With Chlamydia trachomatis Infectivity Through Integrin Modulation in Cervical Cells.

Lactobacilli play a crucial role in maintaining the ecological equilibrium of the vaginal niche, preventing the colonization of exogenous microorganisms. Although many studies have discussed the mechanisms displayed by lactobacilli in counteracting several urogenital pathogens, a few data are available on the interaction between lactobacilli and Chlamydia trachomatis. This study aimed to elucidate the molecular bases of the interaction among vaginal lactobacilli, the sexually transmitted pathogen C. trachomatis and the epithelial cervical cells. We evaluated the in vitro activity of 15 Lactobacillus strains, belonging to different species (i.e., L. crispatus, L. gasseri, L. vaginalis), against C. trachomatis. In particular, we evaluated the capability of lactobacilli cells to interfere with C. trachomatis infection in HeLa cells, by exclusion assays. Lactobacilli significantly reduced C. trachomatis infectivity, being L. crispatus the most active species. Although a dose-dependent effect was noticed, a significant antagonistic activity was maintained even at lower doses. As other Gram-positive bacteria (i.e., Streptococcus agalactiae, Enterococcus faecalis, and Bacillus subtilis) failed to interfere with C. trachomatis infectivity, Lactobacillus activity proved to be specific. The potential mechanism of protection was investigated in Lactobacillus crispatus BC5, chosen as the model strain. The incubation of HeLa cell line with BC5 cells induced important modifications in the epithelial plasma membrane, by altering lipid composition and α5 integrin subunit exposure. When α5 integrin subunits were masked by a specific blocking antibody or ITGA5 gene expression was silenced, Chlamydia infection was significantly reduced. It follows that α5 integrin subunit is crucial for the pathogen infection process, and the anti-Chlamydia activity can be directly linked to membrane properties modifications in cervical cells. The three Gram-positive bacteria used as controls failed to modify the expression of α5β1 integrin. In conclusion, we identified a potential molecular mechanism at the basis of the protection exerted by L. crispatus BC5 against C. trachomatis, getting insights into the role of the cervico-vaginal microbiota for the woman’s health.

Cervicovaginal Fungi and Bacteria Associated With Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus Infections in a Hispanic Population.

The human cervicovaginal microbiota resides at an interface between the host and the environment and may affect susceptibility to disease. Puerto Rican women have high human papillomavirus (HPV) infection and cervical cancer rates. We hypothesized that the population structure of the cervicovaginal bacterial and fungal biota changed with cervical squamous intraepithelial lesions and HPV infections. DNA was extracted from cervix, introitus, and anal sites of 62 patients attending high-risk San Juan clinics. The 16S rRNA V4 region and ITS-2 fungal regions were amplified and sequenced using Illumina technology. HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. HPV prevalence was 84% of which ∼44% subjects were infected with high-risk HPV, ∼35% were co-infected with as many as 9 HPV types and ∼5% were infected with exclusively low-risk HPV types. HPV diversity did not change with cervical dysplasia. Cervical bacteria were more diverse in patients with CIN3 pre-cancerous lesions. We found enrichment of Atopobium vaginae and Gardnerella vaginalis in patients with CIN3 lesions. We found no significant bacterial biomarkers associated with HPV infections. Fungal diversity was significantly higher in cervical samples with high-risk HPV and introitus samples of patients with Atypical Squamous Cells of Undetermined Significance (ASCUS). Fungal biomarker signatures for vagina and cervix include Sporidiobolaceae and Sacharomyces for ASCUS, and Malassezia for high-risk HPV infections. Our combined data suggests that specific cervicovaginal bacterial and fungal populations are related to the host epithelial microenvironment, and could play roles in cervical dysplasia.

Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health.

Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis on the cervical epithelial barrier function and determined whether L. crispatus mitigates the effects of lipopolysaccharide (LPS) and G. vaginalis on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with L. crispatus, L. iners, and G. vaginalis bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after L. iners and G. vaginalis exposure. Soluble epithelial cadherin increased after exposure to L. iners but not G. vaginalis or L. crispatus. A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with L. iners and G. vaginalis having more diverse inflammatory effects. L. iners and G. vaginalis altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs. L. crispatus mitigated the LPS or G. vaginalis-induced disruption of the cervical epithelial barrier and reversed the G. vaginalis-mediated increase in miRNA expression. G. vaginalis colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that L. iners and G. vaginalis alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that L. crispatus confers protection to the cervical epithelial barrier by mitigating LPS- or G. vaginalis-induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.

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

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. 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. 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/β. 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. 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.

Abstract 5143: Characterization of the cervicovaginal, endometrial and anorectal microbiota and mycobiota of post-menopausal women with endometrioid and serous endometrial cancers

Abstract Objectives: Dysbiosis is thought to promote endothelial dysfunction and chronic inflammation, resulting in many chronic human diseases including cancer. This pilot study sought to characterize the cervicovaginal, endometrial and anorectal micro- and mycobiota of post-menopausal women with endometrioid endometrial adenocarcinoma (EAC), uterine serous carcinoma (USC), and controls without cancer. Methods: Post-menopausal patients undergoing hysterectomy for endometrial cancer or non-cancerous disease were enrolled after informed consent. Women were excluded if they reported history of prior cancer, recent urinary or gynecologic infection, antibiotic or probiotic use. Microbial swabs were obtained from the cervicovaginal, the anorectal, and the endometrial cavity after removal of the uterus. DNA was extracted and PCR amplified using barcoded 16SV4 (bacterial) and ITS1 (fungal) primers. Next generation libraries were prepared and sequenced on an Illumina MiSeq 2x300. Sequence reads were processed using our internally developed bioinformatics pipeline and publically available software. Results: Next-generation sequencing of bacterial and fungal DNA revealed substantial differences of cervicovaginal, endometrial and anorectal microbiota in patients with USC (n= 8) compared to those with EAC (n= 13) or benign disease (n= 6). Uterine fungal reads were much higher in patients with USC than EAC (average of 3237 reads versus 938 reads respectively, p = 0.05 Wilcox test). Candida species were reduced, but not eliminated in cervicovaginal samples, and there was a dominance of Malassezia restrica in USC and EAC. A non-significant predominance of Porphyromonas species was noted in USC specimens compared to EAC specimens (p = 0.063). Conclusions: Alpha-diversity of 16S bacterial rRNA and fungal species of the cervicovaginal microbiome was significantly associated with endometrial cancer in our study. USC had a fundamentally different microbiome from control samples and demonstrated a predominance of Porphyromonas species. Further sampling and analysis is needed to confirm these results and to identify rare pathogens. Characterization of microbiota in women with endometrial cancer may help identify risk factors for gynecologic malignancy, and drive investigation regarding preventative and therapeutic intervention. Citation Format: Gregory M. Gressel, Marina Frimer, Christine P. Zolnik, Mykhaylo Usyk, Devin T. Miller, Eirwen M. Miller, D. Y.S. Kuo, Robert D. Burk. Characterization of the cervicovaginal, endometrial and anorectal microbiota and mycobiota of post-menopausal women with endometrioid and serous endometrial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5143.