Lactobacillus-dominated Microbiota Research Articles

Changes in Vaginal Bacteria and Inflammatory Mediators From Periconception Through the Early Postpartum Period in a Cohort of Kenyan Women Without HIV.

Women's increased risk of HIV acquisition during pregnancy and postpartum may be mediated by changes in vaginal microbiota and/or cytokines. A cohort of 80 Kenyan women who were HIV-1 seronegative contributed 409 vaginal samples at 6 pregnancy time points: periconception, positive pregnancy test result, first trimester, second trimester, third trimester, and postpartum. Concentrations of vaginal bacteria linked with HIV risk and Lactobacillus spp were measured using quantitative polymerase chain reaction. Cytokines were measured by immunoassay. Based on Tobit regression, later pregnancy time points were associated with lower concentrations of Sneathia spp (P = .01), Eggerthella sp type 1 (P = .002), and Parvimonas sp type 2 (P = .02) and higher concentrations of Lactobacillus iners (P < .001), Lactobacillus crispatus (P < .001), Lactobacillus vaginalis (P < .001), interleukin 6 (P < .001), TNF (P = .004), C-X-C motif chemokine ligand 10 (CXCL10; P < .001), C-C motif ligand 3 (P = .009), C-C motif ligand 4 (P < .001), C-C motif ligand 5 (P = .002), interleukin 1β (P = .02), and interleukin 8 (P = .002). Most cervicovaginal cytokines and vaginal bacteria clustered separately in principal component analysis, except for CXCL10, which did not group with either cytokines or bacteria. The shift toward a Lactobacillus-dominated microbiota during pregnancy mediated the relationship between pregnancy time point and CXCL10. Increases in proinflammatory cytokines, but not vaginal bacterial taxa linked with higher HIV risk, could provide an explanation for increased HIV susceptibility during pregnancy and postpartum.

Role of short interpregnancy interval, birth mode, birth practices, and the postpartum vaginal microbiome in preterm birth.

There have been widely documented beneficial role of vaginal Lactobacillus species as an important biomarker for vaginal health and healthy pregnancy progression. When translating this to clinical settings, pregnant women with low proportions of Lactobacillus and commensurately high proportion of rich and highly diverse abnormal microbiota are most likely to encounter negative pregnancy outcome such as preterm birth and postpartum complications. However, multiple literatures have also addressed this notion that the absence of a Lactobacillus-dominated microbiota does not appear to directly imply to a diseased condition and may not be a major determinant of negative obstetric outcome. Caesarian delivery is notably a risk factor for preterm birth and postpartum endometritis, yet recent data shows a trend in the overuse of CS across several populations. Growing evidence suggest the potential role of vaginal/uterine cleaning practice during CS procedures in influencing postpartum infections, however there is a controversy that this practice is associated with increased rates of postpartum endometritis. The preponderance of bacterial vaginosis associated bacteria vagitype at postpartum which persist for a long period of time even after lochia regression in some women may suggest why short interpregnancy interval may pose a potential risk for preterm birth, especially multigravidas. While specifically linking a community of microbes in the female reproductive tract or an exact causative infectious agent to preterm birth and postpartum pathologies remains elusive, clinical attention should also be drawn to the potential contribution of other factors such as short interpregnancy interval, birth mode, birth practices and the postpartum vaginal microbiome in preterm birth which is explicitly described in this narrative review.

Age- and endometrial microbiota-related delay in development of endometrial receptivity.

We evaluated factors affecting the development of endometrial receptivity according to age and changes in the endometrial microbiota. We recruited patients with infertility who underwent transcriptomic analyses of endometrial receptivity and the endometrial microbiome prior to frozen embryo transfer. An endometrial biopsy was performed 108 h after initial progesterone administration. In 185 tests from 185 eligible patients, the results of endometrial receptivity analysis were receptive in 111 (60.0%) patients and pre-receptive in 74 (40.0%) patients. Compared with receptive patients, pre-receptive patients had significantly older ages (36.0 ± 0.5 vs. 38.2 ± 0.5, p = 0.0021), a smaller proportion of normal Lactobacillus-dominant microbiota (27.9% vs. 12.2%), and a greater proportion of microbiota with ultralow biomass (22.5% vs. 41.9%) (p = 0.0074). Patient age (adjusted odds ratio: 1.08, 95% confidence interval: 1.01-1.16, p = 0.0351) and a microbiome with ultralow biomass (adjusted odds ratio: 3.82, 95% confidence interval: 1.49-9.82, p = 0.0039) were independent predictive factors for pre-receptive endometrium. Older age was accompanied by a decrease in Lactobacillus-dominant microbiota; aging and endometrial microbiota with ultralow biomass were significantly associated with pre-receptive endometrium. Our findings suggest that the quantity (rather than proportion) of Lactobacillus in the endometrium is important in the development of endometrial receptivity.

Therapeutic intervention based on gene sequencing analysis of microbial 16S ribosomal RNA of the intrauterine microbiome improves pregnancy outcomes in IVF patients: a prospective cohort study.

A Lactobacillus-dominated microbiota in the endometrium was reported to be associated with favorable reproductive outcomes. We investigated in this study whether 16S ribosomal RNA (rRNA) gene sequencing analysis of the uterine microbiome improves pregnancy outcomes. This prospective cohort study recruited a total of 195 women with recurrent implantation failure (RIF) between March 2019 and April 2021 in our fertility center. Analysis of the endometrial microbiota by 16S rRNA gene sequencing was suggested for all patients who had three or more failed embryo transfers (ETs). One hundred and thirty-one patients underwent microbial 16S rRNA gene sequencing (study group) before additional transfers, while 64 patients proceeded to ET without that analysis (control group). The primary outcome was to compare the cumulative clinical pregnancy rate of two additional ETs. An endometrial microbiota considered abnormal was detected in 30 patients (22.9%). All but one of these 30 patients received antibiotics according to the bacterial genus detected in their sample, followed by treatment with probiotics. As a result, the cumulative clinical pregnancy rate (study group: 64.5% vs. control group: 33.3%, p = 0.005) and the ongoing pregnancy rate (study group: 48.9% vs. control group: 32.8%, p = 0.028) were significantly increased in the study group compared to the control group. Personalized treatment recommendations based on the microbial 16S rRNA gene sequencing of the uterine microbiota can improve IVF outcomes of patients with RIF. The University Hospital Medical Information Network (UMIN) Clinical Trial Registry: UMIN000036050 (date of registration: March 1, 2019).

O-029 The Vaginal Microbial Predictors of the Endometrial Microbiota

Abstract Study question Can we find specific biomarkers in vaginal microbiota predicting the state of endometrial microbiota? Summary answer The higher levels of Eubacterium spp. in the vagina could serve as a predictor of the non-Lactobacillus-dominated microbial community in the uterus. What is known already The absence of microorganisms or the Lactobacillus-dominated community in the uterus cavity is considered more favorable than the non-Lactobacillus-dominated community. The latter is associated with poor pregnancy outcomes in women undergoing in vitro fertilization. However, endometrial microbiota analysis is challenging due to the possibility of contaminating the samples with vaginal microbiota during transvaginal sampling. Also, there is always a risk of upper genital tract inflammation after the sampling. For these reasons, the idea of finding markers predicting the state of endometrial microbiota is promising. Study design, size, duration It is a cross-sectional study of the vaginal and endometrial microbiota in 61 reproductive-age women with the normal histological pattern of endometrial samples. Endometrial and vaginal samples were collected simultaneously on days 7–10 of the menstrual cycle. Depending on the type of endometrial microbiota, the women were divided into two groups: Group 1 – the Lactobacillus-dominated microbiota or no microbiota (n = 43), Group 2 – the non-Lactobacillus-dominated microbiota (n = 18). Participants/materials, setting, methods All women came to the “Garmonia” Medical Center (Yekaterinburg, Russia) seeking infertility treatment from 2019 to 2021. Vaginal and endometrial microbiota was analyzed using Femoflor and Androflor real-time PCR kits (DNA-Technology, Russia). For the vaginal samples, the quantities and proportions of 22 microorganism groups were compared in Group 1 and Group 2 using Mann-Whitney test. For the variables with significant differences, ROC curves were constructed to assess the diagnostic value of the tests. Main results and the role of chance When comparing vaginal microbiota in Groups 1 and 2, we registered the statistically significant differences for 6 out of 44 variables (quantities and proportions of 22 microorganism groups). These variables were the quantities of Eubacterium spp. (10° vs. 104.7, p = 0.002) and Gardnerella vaginalis (10° vs. 104.0, p = 0.033), the proportions of obligate anaerobes (0% vs. 6.0%, p = 0.010), Peptostreptococcus spp./Parvimonas spp. (0% vs. 0%, p = 0.011), G. vaginalis (0% vs. 0.2%, p = 0.012), Eubacterium spp. (0% vs. 0.6%, p = 0.033). “Eubacterium spp.’ quantity” variable demonstrated the highest discriminatory power (the area under the ROC curve value — 0.737 [CI 0.593-0.882], p = 0.004). The AUC value was considered as acceptable to divide patients into Group 1 and Group 2. The cutoff value of Eubacterium spp. 103.15 genome-equivalent/ml (GE/ml)) allowed to predict the non-Lactobacillus-dominated microbiota in the uterus cavity with 77.8% sensitivity and 62.8% specificity. Limitations, reasons for caution The results were obtained on a small population group of women with the normal histological pattern of endometrial samples. It may be different if the larger pool of samples is investigated. It is also possible, that the results will not apply to the patients with endometrial abnormalities. Wider implications of the findings Further research should determine the diagnostic value of Eubacterium spp. identified in vaginal samples as the predictor for the endometrial microbiota composition in women with endometrial abnormalities. The next step is research dedicated to the link between different signatures in vaginal microbiota and endometrial abnormalities. Trial registration number Protocol № 7 dated September 20, 2019

P-384 Efficacy of endometrial microbiome metagenomic analysis with recurrent implantation failure and recurrent pregnancy loss: multicenter study in Japan

Abstract Study question Does the result of endometrial microbiome metagenomic analysis (EMMA), a next generation sequencing (NGS)-based test of the intrauterine microbiome, have an impact on pregnancy rate after the test? Summary answer After recommend treatment with antimicrobial and probiotic therapy, the group diagnosed as dysbiosis by EMMA achieved pregnancy significantly earlier than the group with Normal result. What is known already Using NGS technology, EMMA testing can determine the composition of the endometrial microbiome by analysing bacterial 16S ribosomal RNA with a focus on the lactobacillus population. Endometrial flora in patients undergoing in vitro fertilization (IVF) is often composed of pathogenic microorganisms which decrease implantation rates, such as the Enterobacteriaceae family, Staphylococcus spp., Escherichia coli and Gram-negative bacteria. Other studies also indicate that Lactobacillus spp. is a major microorganism in the endometrium. Especially, lactobacillus-dominated microbiota (LDM, defined as &amp;gt; 90% Lactobacillus spp.) in the endometrium has been reported to lead to better pregnancy outcomes than non-LDM (&amp;lt;90% Lactobacillus spp.). Study design, size, duration This study was a prospective, multicenter cohort study of 527 patients (under 42 years old) with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) at 14 IVF treatment facilities in Japan from June 2019 to August 2021. Defining RIF as three or more failed implantation attempts and RPL as two or more miscarriages, we examined the prognosis of two additional embryo transfers (ETs) after the EMMA test in patients who underwent the test. Participants/materials, setting, methods Endometrial tissue was obtained by aspiration from patients in day 15-25 of their menstrual cycles, and sample tissues were analyzed by NGS for EMMA. Participant centers treated patients according to the therapies specified in the reports, including antibiotic treatments, probiotic treatments, re-analysis, and embryo transfer. Multivariate analysis was performed using a generalized linear model with the endpoint of ongoing pregnancy. For the time-to-event analyses, we used Kaplan-Meier survival analysis to compare time to ongoing pregnancy. Main results and the role of chance The results of the first EMMA were as follows: 229 patients (43.4%) were normal with Lactobacillus spp. &amp;gt;90% (Normal group), 110 patients (20.9%) were abnormal with less than 90% Lactobacillus spp. and predominantly pathogenic bacteria (Abnormal group), and 188 patients (35.7%) were mild with low absolute amounts of bacteria and ultralow biomass indicating almost sterile (Mild + Ultralow group). There were no significant differences in background factors such as age, duration of infertility, number of previous ETs, or history of deliveries among the three groups. Gardnerella was the most pathogenic bacteria detected in patients with Abnormal EMMA results. All patients in the Abnormal group were treated with antimicrobials and probiotics, and those in Mild + Ultralow group were treated with probiotics. Odds ratio for ongoing pregnancy rate was 1.10(95%CI 0.67-1.82, p = 0.699) in Abnormal group and 1.23(95%CI 0.80-1.89, p = 0.342) in Mild + Ultralow group, respectively. After the intervention, ongoing pregnancies were comparable to those in Normal group.Analysis of time to pregnancy using Kaplan-Meier survival curves showed that Abnormal group had a significantly higher rate of ongoing pregnancies during the observation period than the other groups (p = 0.031). Limitations, reasons for caution Since this study was not necessarily limited to euploid embryos transferred after testing, an aging bias cannot be excluded. Since this study was conducted with all patients receiving EMMA, the effectiveness of the test needs to be further validated by comparison to patients without EMMA testing. Wider implications of the findings This study is the first multicenter study to demonstrate that the intervention based on EMMA reports improve pregnancy outcome in the patients with RIF and RPL. We suggest that the EMMA procedure, which aims at establishing an appropriate uterine microbiome, may be important for implantation and pregnancy continuation. Trial registration number UMIN000036917

O-256 Effect of an endometrial microbiota on pregnancy outcome of frozen embryo transfer (FET) cycles

Abstract Study question Is specific microbiota in the endometrial fluid (EF) associated with pregnancy outcome in a frozen embryo transfer (FET) cycles? Summary answer Lactobacillus had high abundance in the microbiota of endometrial fluid of patients with successful pregnancy after FET treatment. What is known already Recently, the relationship between endometrial microbiota and repeated implantation failure (RIF) has been reported. While Lactobacillus-dominated microbiota (LDM, defined as &amp;gt; = 90 % Lactobacillus species) in the EF of the receptive phase was reported to be associated with favorable reproductive outcome, non-LDM (&amp;lt; 90 % Lactobacillus species) was found to decrease implantation, clinical pregnancy, ongoing pregnancy, and live birth rates. However, it is still unclear the effect of the microbiota on pregnancy outcome of the patients with the assisted reproductive technology, especially a frozen embryo transfer (FET) treatment. Study design, size, duration We included 802 cycles with clinical results of pregnancy outcome after FET treatment at our clinic from December 2018 to January 2021. Endometrium fluid was collected before FET and microbiota was examined. We examined the relationship between endometrial microbiota and pregnancy outcome in 463 cycles in which the endometrial microbiome test results were available, pregnancy outcome was known, CD138 negative, and less than 38years (229 cycles were positive pregnancy and 234 cycles were negative pregnancy). Participants/materials, setting, methods Sampling was performed carefully avoiding contamination before FET treatment. Extracted genomic DNA was sequenced for the region of 16S ribosomal RNA using next-generation sequencer (Endometrial microbiome test, Varinos, Japan). The sequencing data was assigned to bacterial taxonomy and the background-contaminated bacteria were excluded from the microbiome profile. Lactobacillus abundance was calculated. After concordance of patient background, Lactobacillus abundance between successful and unsuccessful pregnancy group was compared. Main results and the role of chance We investigated the effect of microbiota in EF, especially Lactobacillus, for the infertile female with frozen embryo transfer (FET) as an assisted reproductive treatment with large amount of cycles (802 cycles). First, all of cycles were divided into a successful pregnancy group (305/802) and an unsuccessful pregnant group (497/802), and then the abundance of Lactobacillus was analyzed (the pregnancy ratio was 38.0%). The mean abundance of Lactobacillus in EF was significantly higher in the successful pregnant group than in the unsuccessful pregnant group (70.2% 40.5 versus 63.5% 43.1, p = 0.007). To further refine the conditions of each cycle, patient backgrounds of each cycle were compared. There was a significant difference in age and CD138 results between the successful pregnant group and unsuccessful pregnant group, so the analysis was performed with the limited cycles with CD138-negative and under 38 years (229/463 versus 234/463, respectively). However, even under this strict condition, the mean abundance of Lactobacillus was significantly higher in the pregnant group than in the unsuccessful pregnant group (72.1% 39.7 versus 61.1% 44.4, p = 0.003). These results suggest that the endometrial environment with high Lactobacillus abundance is more conducive to pregnancy in FET treatment. Limitations, reasons for caution The limitation of this study is that the design is not a randomized controlled one, although it is prospective. Wider implications of the findings The results of this study suggest that the endometrial microbiota at the time of embryo implantation, especially Lactobacillus genus, is highly relevant to pregnancy outcome, implying that disruption of the intrauterine microbiota may cause infertility. We need further studies to improve the endometrial microbiota of infertile women. Trial registration number not applicable

The Impact of Over-The-Counter Lactic Acid Containing Vaginal Gels on the Integrity and Inflammatory State of the Vaginal Epithelium in vitro.

The vaginal microbiome influences a wide range of health outcomes in women, where a microbiome dominated by Lactobacillus spp. is considered optimal and associated with reduced risk of pre-term birth and acquisition of sexually transmitted infections including HIV. Conversely, replacement of lactobacilli by non-optimal bacteria leads to the development of bacterial vaginosis, which is associated with increased risk of these outcomes. Lactobacilli produce the metabolite lactic acid (LA) which is a potent antibacterial and antiviral agent. The potential therapeutic benefits of LA have prompted the development of numerous over-the-counter LA-containing gels for use in the vagina, although a comprehensive analysis of the impact of these formulations on the cervicovaginal epithelium and pro-inflammatory cytokine/chemokine responses, has not been assessed. Here, we evaluated the properties of 11 over-the-counter gels, including 9 containing LA, marketed for use in the vagina. Ten of the 11 gels had an osmolality greater than vaginal fluid from women with Lactobacillus-dominated microbiota (370 ± 40 mOsmol/kg in women with Nugent score 0–3), with six gels that were hyperosmolal >2,000 mOsmol/kg. Using a reconstructed primary cell model of the vaginal epithelium, we found hyperosmolal gels had a detrimental impact on epithelial barrier integrity, resulting in substantial cellular toxicity (<10% viability as compared to untreated cells) and reduced epithelial barrier integrity [≈30% of untreated cells, assessed by transepithelial electrical resistance (TEER)]. Treatment of vaginal tissues with most of the gels elicited the production of pro-inflammatory factors including IL-1α (8 of 11) and IL-1β (10 of 11) which are associated with heightened risk of HIV acquisition in vivo. The majority of the OTC gels elicited moderate tissue damage as determined by histology. The detrimental effects of these gels on the human vaginal epithelium in vitro may predict compromised epithelial barrier integrity and genital inflammation in vivo, which has implications for sexual and reproductive health. This study highlights the importance of evaluating the impact of intravaginal products on the integrity and inflammatory status of the mucosal epithelium to avoid unfavorable off target effects.

Endometrial and vaginal microbiomes influence assisted reproductive technology outcomes.

The role of Lactobacillus-dominant microbiota in the endometrium in reproductive function is unclear. We therefore aimed to explore the impact of the balance of Lactobacillus and pathological bacteria in the endometrial and vaginal microbiomes on the pregnancy outcomes of women treated with assisted reproductive technology (ART). This study included 35 women with infertility submitted to good-quality embryo transfers. The cutoff values for abundance of Lactobacillus species (spp.) and pathological bacteria in the endometrium and vagina were calculated. Women with Lactobacillus spp. and pathological bacteria abundance above the cutoff values were categorized in the high-abundance group, whereas those with abundance below cutoff values were categorized in the low abundance group. We divided the patients into four groups based on the combination of high/low abundance of Lactobacillus spp. and pathological bacteria. The 35 cases of good-quality embryo transfer resulted in 21 pregnancies. Pregnant women were present in significantly higher proportions in the high Lactobacillus spp. abundance and low pathological bacteria abundance group, whereas the opposite combination (i.e., low Lactobacillus spp. abundance and high pathological bacteria abundance) saw a significantly higher proportion of nonpregnant women (p=0.022). The balance between Lactobacillus and pathological bacterial abundance in the endometrial and vaginal microbiomes is associated with pregnancy from ART.

The Vaginal Microbiome: II. Vaginal Dysbiotic Conditions.

ObjectiveThis series of articles, titled The Vaginal Microbiome (VMB), written on behalf of the International Society for the Study of Vulvovaginal Disease, aims to summarize the recent findings and understanding of the vaginal bacterial microbiota, mainly regarding areas relevant to clinicians specializing in vulvovaginal disorders.Materials and MethodsA search of PubMed database was performed, using the search terms “vaginal microbiome” with “dysbiosis,” “bacterial vaginosis,” “cytolytic vaginosis,” “desquamative inflammatory vaginitis,” and “aerobic vaginitis.” Full article texts were reviewed. Reference lists were screened for additional articles.ResultsThe second article in this series focuses on vaginal dysbiotic conditions. Dysbiosis is a term describing imbalances in bacterial communities. Given that lactobacillus-dominated microbiota are thought to be the most optimal, vaginal dysbiosis is usually considered as lactobacilli-depleted VMB. Bacterial vaginosis (BV), the most common vaginal dysbiotic condition, is a polymicrobial disorder, considered the leading cause for vaginal discharge in women worldwide. In addition, we review the VMB in other vaginal conditions associated with lactobacilli depletion: desquamative inflammatory vaginitis and aerobic vaginitis. We also discuss the controversial diagnosis of cytolytic vaginosis, related with lactobacilli overgrowth.ConclusionsBacterial vaginosis displays complex microbiology. The heterogeneity and diversity within the genus Gardnerella may impact the progression of BV. Bacterial biofilms may contribute to the etiology and persistence of BV, and various bacteria may affect its clinical presentation and pathogenicity. Lack of lactobacilli is not always accompanied by an overgrowth of anaerobes.

O-128 Intra-cycle alterations of the uterine microbiota in patients with recurrent miscarriage or recurrent implantation failure and healthy controls

Abstract Study question Uterine microbiota: are there differences within three major time points of the menstrual cycle in healthy controls, recurrent miscarriage (RM) and recurrent implantation failure (RIF) patients? Summary answer Compared to controls, RM and RIF patients showed an altered uterine microbiota throughout the menstrual cycle, with a lower dominance of lactobacilli. What is known already In contrast to the former notion of a sterile womb, bacterial colonization in the uterus and the placenta has been demonstrated. Studies showed that Lactobacillus-dominated endometrial microbiota correlate with reproductive success. Moreover, the presence of non-Lactobacillus-dominated microbiota, especially with detection of Gardnerella and Streptococcus in the endometrial fluid, seems to be associated with lower implantation-, ongoing pregnancy- and live birth-rates. However, intra-cycle variations in healthy women as well as possible alterations in patients with RM or RIF remain unknown. Study design, size, duration In total, n = 20 RM patients (≥ 3 consecutive miscarriages), n = 20 RIF patients (≥3 fresh or frozen embryo transfers with negative serum hCG, good quality embryos) and n = 10 healthy controls (no pregnancy) were included in this study. All patients had a 28 day menstrual cycle. During follicular, ovulatory and luteal-phase, after a thorough cleaning of the cervix, a flexible catheter was introduced into the uterine cavity and a uterine flushing with 1ml of NaCl was performed. Participants/materials, setting, methods Bacterial DNA was extracted using a QIAamp DNA kit (Qiagen) in combination with a PrecellysR24 homogenizer (Peqlab, Erlangen, Germany) according to the manufacturer’s instructions. The V3-V4 region of the bacterial 16S rRNA gene was amplified. Samples were pooled in equimolar ratios and progressed to pyrosequencing using an Illumina MiSeq se-quencer with MiSeq Kit V2 (250 bp paired-end). Analysis of 16S rRNA data, including alpha- and beta-diversity, were calculated using the phyloseq package in R. Main results and the role of chance For the Shannon index (species richness and evenness) a significant decrease during the ovulation period was shown in the control group, indicating a more uniform microbiota (p &amp;lt; 0.05). This loss of diversity was not shown in RIF and RM patients. Overall, we could observe a higher similarity in taxonomic distribution in RM compared to the RIF patients. Longitudinal dynamics included increases in Firmicutes (CTRL and RM only) and a concomitant loss of Proteobacteria. Notably, significant amounts of bacteroides were only detected in the RIF patients. Actinobacteria were more frequent in both, RM and RIF as compared to controls. Limitations, reasons for caution To minimize the impact of a potential contamination, we performed pre-experiments with paired samples both from the vaginal fornix and the endometrial cavum and could show a significant difference in overall microbiome configuration. However, the route of sample can still be prone to contamination. Wider implications of the findings For the first time, we were able to show cycle-dependent alterations in the endometrial microbiome. These findings underline the role of an altered endometrial microbiome as a cause for RM and RIF and can contribute to the future establishment of therapeutic strategies in cases of a dysbalanced microbiome. Trial registration number Drks00020803

Assessing the Concordance Between Urogenital and Vaginal Microbiota: Can Urine Specimens Be Used as a Proxy for Vaginal Samples?

BackgroundThe vaginal microbiota play a key role in defense against reproductive tract infections; however, many population-based women’s health studies do not collect vaginal samples. Molecular examinations of urine samples have revealed common vaginal bacteria. We sought to assess the extent that community state type assignments of archived random-catch and clean-catch urine samples agreed with the paired vaginal samples in both reproductive-age and peri/post-menopausal women.ResultsUsing archived samples, we evaluated the microbiota concordance among women in three studies: two with paired mid-vaginal/random-catch urine (N=91 reproductive-age participants and N=13 peri/post-menopausal participants), and one with paired mid-vaginal/clean-catch urine (N=99 reproductive-age participants). Microbiota composition was characterized by sequencing amplicons of the 16S rRNA gene V3-V4 regions and assigned to community state types. Similarity of paired samples was gauged using agreement of community state types and Yue-Clayton θ indices. Analysis of Composition of Microbiomes II indicated which taxa were differently relatively abundant in paired vaginal and urine samples. In reproductive-age women, random-catch and clean-catch urines were 89.0% and 86.9% concordant on five community state types with paired mid-vaginal swabs, and Kappa statistics indicated almost perfect agreement (κrandom-catch=.85, κclean-catch=.81, p<0.0001). A small number of pairs of samples were discordant (23/190, 12%), and discordant pairs tended to be between samples classified to L. iners-dominated and/or low-Lactobacillus states. Concordance and agreement remained similar when dichotomizing the microbiota to Lactobacillus-dominated versus low-Lactobacillus microbiota, as well as when evaluating separately the three subtypes of the low-Lactobacillus community state type IV. Median similarity of paired urine/vaginal samples was high (θrandom-catch=.85, θclean-catch=.88), and a comparison of the random-catch and clean-catch similarity scores showed no significant difference (p=.80). Concordance and similarity were lower for peri/post-menopausal women, but agreement remained substantial (76.9% concordant, κrandom-catch= 0.64, θrandom-catch=.62). Taxonomic-level analysis confirmed these findings.ConclusionsRandom-catch and clean-catch urine samples showed substantial agreement on bacterial composition to paired mid-vaginal samples, indicating that the genitourinary microbiota may be a reliable proxy for assessing the overall composition of the vaginal microbiota via community state types. This data suggests that urine samples can, with proper interpretation, be utilized as a surrogate for developing preliminary data and hypothesis-generating studies.

Vaginal microbiota diversity and paucity of Lactobacillus species are associated with persistent hrHPV infection in HIV negative but not in HIV positive women

The vaginal microbiota is thought to play a role in modulating risk of high-risk human papillomavirus (hrHPV) infection. We examined the relationship between the vaginal microbiota and persistent hrHPV infection in HIV-negative and HIV-positive women. We used 16S-rRNA sequencing to characterize the vaginal microbiota of two serial samples taken six months apart from 211 Nigerian women (67%, 142/211 HIV-positive and 33%, 69/211 HIV-negative) and evaluated the association between the vaginal microbiota and persistent hrHPV infection using generalized estimating equation logistic regression models and linear discriminant analysis effect size (LEfSe) algorithm to identify phylotypic biomarkers of persistent hrHPV infection. The high diversity microbiota, Community State Type IV-B, was the most prevalent in both HIV-negative (38% at baseline, 30% at the follow-up visit) and HIV-positive (27% at baseline, 35% at the follow-up visit) women. The relationship between the vaginal microbiota and persistent hrHPV was modified by HIV status. In HIV-negative women, women with Lactobacillus dominant microbiota had lower odds (OR: 0.35, 95% CI 0.14–0.89, p = 0.03) of persistent hrHPV compared to women with Lactobacillus deficient microbiota. While among HIV-positive women, the odds of being persistently infected with hrHPV was higher in women with Lactobacillus dominant microbiota (OR: 1.25, 95% CI 0.73–2.14 p = 0.41). This difference in effect estimates by HIV was statistically significant (p = 0.02). A high diversity vaginal microbial community with paucity of Lactobacillus species was associated with persistent hrHPV infection in HIV-negative women but not in HIV-positive women.

Association Between Vaginal Bacterial Microbiota and Vaginal Yeast Colonization.

Vaginal yeast is frequently found with Lactobacillus-dominant microbiota. The relationship between vaginal yeast and other bacteria has not been well characterized. These analyses utilized data from the Preventing Vaginal Infections trial. Relative abundance of vaginal bacteria from 16S ribosomal ribonucleic acid gene amplicon sequencing and quantities of 10 vaginal bacteria using taxon-directed polymerase chain reaction assays were compared at visits with and without detection of yeast on microscopy, culture, or both. Higher relative abundances of Megasphaera species type 1 (risk ratio [RR], 0.70; 95% confidence interval [CI], 0.52-0.95), Megasphaera species type 2 (RR, 0.81; 95% CI, 0.67-0.98), and Mageeibacillus indolicus (RR, 0.46; 95% CI, 0.25-0.83) were associated with lower risk of detecting yeast. In contrast, higher relative abundances of Bifidobacterium bifidum, Aerococcus christensenii, Lactobacillus mucosae, Streptococcus equinus/infantarius/lutentiensis, Prevotella bivia, Dialister propionicifaciens, and Lactobacillus crispatus/helveticus were associated with yeast detection. Taxon-directed assays confirmed that increasing quantities of both Megasphaera species and M indolicus were associated with lower risk of detecting yeast, whereas increasing quantities of L crispatus were associated with higher risk of detecting yeast. Despite an analysis that examined associations between multiple vaginal bacteria and the presence of yeast, only a small number of vaginal bacteria were strongly and significantly associated with the presence or absence of yeast.

Distinct Immune Responses Elicited From Cervicovaginal Epithelial Cells by Lactic Acid and Short Chain Fatty Acids Associated With Optimal and Non-optimal Vaginal Microbiota.

Non-optimal vaginal microbiota, as observed in bacterial vaginosis (BV), is typically characterized by a depletion of beneficial lactobacilli and an abundance of numerous anaerobes. These non-optimal conditions are associated with subclinical cervicovaginal inflammation and an increased risk of HIV infection compared to women colonized with optimal vaginal microbiota dominated by lactobacilli. Lactic acid (LA) is a major organic acid metabolite produced by vaginal lactobacilli that elicits anti-inflammatory effects from cervicovaginal epithelial cells and is dramatically depleted during BV. However, it is unclear if LA retains its anti-inflammatory activity in the presence of vaginal microbiota metabolites comprising short chain fatty acids (SCFAs) and succinic acid, which are also produced by an optimal vaginal microbiota. Furthermore, the immunomodulatory effect of SCFAs and succinic acid on cervicovaginal epithelial cells at higher concentrations present during BV is unknown. Here we report that in the presence of physiologically relevant concentrations of SCFAs and succinic acid at pH 3.9 (as found in women with lactobacillus-dominated microbiota) LA induced an anti-inflammatory state in cervicovaginal epithelial cells and inhibited inflammation elicited by the toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid and Pam3CSK4. When cervicovaginal epithelial cells were treated with a vaginal microbiota metabolite mixture representative of BV, containing a lower concentration of LA but higher concentrations of SCFA/succinic acid at pH 7, no anti-inflammatory was observed. Rather, the vaginal microbiota metabolite mixture representative of BV dysregulated the immune response of cervicovaginal epithelial cells during prolonged and sustained treatments. This was evidenced by increased basal and TLR-induced production of pro-inflammatory cytokines including tumor necrosis factor-α, but decreased basal production of chemokines including RANTES and IP-10. Further characterization of individual components of the BV vaginal microbiota mixture suggested that acetic acid is an important vaginal microbiota metabolite capable of eliciting diverse immunomodulatory effects on a range of cervicovaginal epithelial cell targets. These findings indicate that elevated levels of SCFAs are a potential source of cervicovaginal inflammation in women experiencing BV, and support the unique anti-inflammatory properties of LA on cervicovaginal epithelial cells as well as a role for LA or LA-producing lactobacilli to reverse genital inflammation associated with increased HIV risk.

Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status

Recent research on vaginal microbiota relies on high throughput sequencing while microscopic methods have a long history in clinical use. We investigated the correspondence between microscopic findings of Pap smears and the vaginal microbiota composition determined by next generation sequencing among 50 asymptomatic women. Both methods produced coherent results regarding the distinction between Lactobacillus-dominant versus mixed microbiota, reassuring gynaecologists for the use of Pap smear or wet mount microscopy for rapid evaluation of vaginal bacteria as part of diagnosis. Cytologic findings identified women with bacterial vaginosis and revealed that cytolysis of vaginal epithelial cells is associated to Lactobacillus crispatus-dominated microbiota. Education and socio-economic status were associated to the vaginal microbiota variation. Our results highlight the importance of including socio-economic status as a co-factor in future vaginal microbiota studies.

Quantitation of all Four Gardnerella vaginalis Clades Detects Abnormal Vaginal Microbiota Characteristic of Bacterial Vaginosis More Accurately than Putative G.vaginalis SialidaseA Gene Count.

BackgroundBacterial vaginosis (BV) is a vaginal disorder characterized by a depletion of the normal lactobacillus-dominant microbiota and overgrowth of mainly anaerobic bacteria.ObjectivesThe study aimed to evaluate the distribution and abundance of the Gardnerella vaginalis clades and sialidase A gene in vaginal samples from Russian women, and investigate if the G. vaginalis sialidase A gene count detects an abnormal vaginal microbiota characteristic of BV more accurately than G. vaginalis load.MethodsVaginal samples from 299 non-pregnant patients of gynecological clinics were examined using Nugent scores and G. vaginalis clade and sialidase A gene quantitative real-time polymerase chain reactions (PCRs). Discriminatory power for BV microbiota was evaluated with receiver operating characteristic (ROC) analysis.ResultsThe vaginal microbiota was characterized by Nugent scores as normal, intermediate, and BV microbiota in 162, 58, and 79 women, respectively. G. vaginalis clades 1, 2, 3, 4, and the sialidase A gene were detected in 56% (51–62%), 40% (34–45%), 20% (16–25%), 94% (91–96%), and 70% (64–75%) of vaginal samples, respectively. The frequency and abundance of clades 1, 2, 4, and the sialidase A gene as well as clade multiplicity were significantly associated with abnormal microbiota. The sialidase A gene was present in all multi-clade samples, in all single-clade samples comprising clades 1, 2, and 3, and in four of 84 (5% [2–12%]) samples comprising clade 4 only. Total G. vaginalis load showed significantly higher discriminatory power for abnormal microbiota than sialidase A gene count (areas under ROC curves 0.933 vs. 0.881; p = 0.0306).ConclusionsQuantifying all four G. vaginalis clades discriminates between BV microbiota and normal microbiota more accurately than measuring G. vaginalis sialidase A gene. Clade 4 is strongly associated with BV microbiota, despite most clade 4 strains lacking the sialidase A gene.

A pilot study and case reports on endometrial microbiota and pregnancy outcome: An analysis using 16S rRNA gene sequencing among IVF patients, and trial therapeutic intervention for dysbiotic endometrium.

PurposeThe present study aimed to analyze the pregnancy outcomes of IVF patients presenting Lactobacillus‐dominated microbiota (LDM) or non‐Lactobacillus‐dominated microbiota (NLDM) of their endometrium and to report cases who were treated for NLDM concurrently with antibiotics and prebiotic/probiotic supplements in a Japanese infertile population.MethodsNinety‐two IVF patients were recruited from August 2017 to March 2018. Endometrial fluid samples for sequencing were collected using an IUI catheter. The bacterial status of the endometrium and the pregnancy outcomes were analyzed. For cases with NLDM, antibiotics and prebiotics/probiotics were administered according to their individual microbial conditions.ResultsForty‐seven cases (51.1%) presented LDM and 45 cases (48.9%) presented NLDM at initial analysis. Nine Patients with NLDM were treated by antibiotics and prebiotics/probiotics, and successfully became Lactobacillus‐dominant. Pregnancy rates by single vitrified‐warmed blastocyst transfers were higher in the LDM group (58.9% per patient and 36.3% per FBT) than in the NLDM group (47.2% per patient and 34.7% per FBT) but not significantly different.ConclusionThe results of this study could not necessarily prove the clear benefit of establishing Lactobacillus‐dominated endometrium in terms of pregnancy outcome, but there is significance in searching for endometrial microbial status of infertile patients and recovering Lactobacillus‐dominated endometrium might benefit implantation.

Associations between improvement in genitourinary symptoms of menopause and changes in the vaginal ecosystem.

The aim of the study was to identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen, and serum estrogen. Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs venlafaxine vs placebo) who reported GSM and provided vaginal swabs at 0, 4, and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3-V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn, or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using χ, Wilcoxon signed-rank test, or Hotelling's t test. Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared with placebo (54%; P = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, P = 0.08). Vaginal glycogen, serum estradiol, and estrone significantly increased in women whose MBS improved. A larger proportion of women whose MBS improved had a Lactobacillus dominant microbiota at enrollment than those who had no improvement during the trial, though this difference was not statistically significant. Larger trials are needed to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.

Vaginal microbiota and susceptibility to HIV.

: Bacterial vaginosis, characterized by the replacement of the Lactobacillus-dominant microbiota with anaerobic bacteria and facultative Gram-negative rods, has been associated with adverse reproductive health outcomes including HIV acquisition. With the advent of newer molecular techniques, the vaginal microbiota can be investigated in more detail and the association with HIV examined more thoroughly. This review examines recent evidence suggesting that vaginal dysbiosis with increased microbial diversity, specific vaginal bacterial communities, and the presence and concentrations of some individual bacterial species, may increase HIV susceptibility. Potential mechanisms through which vaginal microbiota could impact HIV susceptibility are discussed. On the basis of the available data, this review finds that there is a modest, but growing, body of evidence linking vaginal microbiota to HIV susceptibility in women. The evidence could be strengthened through two main pathways. First, laboratory studies such as ex-vivo or animal experiments are needed to move from plausible mechanisms towards proven mechanisms that explain an effect of the vaginal microbiota on HIV susceptibility. Second, experimental evidence could directly test the hypothesis that sustaining optimal microbiota reduces HIV risk, though there are important obstacles to conducting such studies. Finally, this review examines strong evidence from a recent publication suggesting that deviations from an optimal vaginal microbiome, and particularly the presence of some bacterial communities with high relative abundance of Gardnerella vaginalis, reduces the efficacy of vaginal tenofovir-based microbicides.