Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.

Anti-chlamydial activity of vaginal fluids: new evidence from an in vitro model.

Introduction: We assessed the in vitro anti-chlamydial activity of fresh vaginal secretions, deciphering the microbial and metabolic components able to counteract Chlamydia trachomatis viability. Methods: Forty vaginal samples were collected from a group of reproductive-aged women and their anti-chlamydial activity was evaluated by inhibition experiments. Each sample underwent 16S rRNA metabarcoding sequencing to determine the bacterial composition, as well as 1H-NMR spectroscopy to detect and quantify the presence of vaginal metabolites. Results: Samples characterized by a high anti-chlamydial activity were enriched in Lactobacillus, especially Lactobacillus crispatus and Lactobacillus iners, while not-active samples exhibited a significant reduction of lactobacilli, along with higher relative abundances of Streptococcus and Olegusella. Lactobacillus gasseri showed an opposite behavior compared to L. crispatus, being more prevalent in not-active vaginal samples. Higher concentrations of several amino acids (i.e., isoleucine, leucine, and aspartate; positively correlated to the abundance of L. crispatus and L. jensenii) lactate, and 4-aminobutyrate were the most significant metabolic fingerprints of highly active samples. Acetate and formate concentrations, on the other hand, were related to the abundances of a group of anaerobic opportunistic bacteria (including Prevotella, Dialister, Olegusella, Peptostreptococcus, Peptoniphilus, Finegoldia and Anaerococcus). Finally, glucose, correlated to Streptococcus, Lachnospira and Alloscardovia genera, emerged as a key molecule of the vaginal environment: indeed, the anti-chlamydial effect of vaginal fluids decreased as glucose concentrations increased. Discussion: These findings could pave the way for novel strategies in the prevention and treatment of chlamydial urogenital infections, such as lactobacilli probiotic formulations or lactobacilli-derived postbiotics.

The vaginal microbiome composition during pregnancy in a region compromising different ethnic origins.

BACKGROUND: The vaginal microbiota plays a significant role in pregnancy outcomes and newborn health. Indeed, the composition and diversity of the vaginal microbiota can vary among different ethnic groups. Our study aimed to investigate the composition of the vaginal microbiome throughout the three trimesters of pregnancy and to identify any potential variations or patterns in the Turkish population compromising mixed ethnicities. METHOD: We conducted a longitudinal study to characterize the vaginal microbiota of pregnant women. The study included a total of 25 participants, and the samples were collected at each trimester: 11-13 weeks, 20-24 weeks and 28-34 weeks gestation. RESULTS: Lactobacillus species were consistently found to be dominant in the vaginal microbiota throughout all trimesters of pregnancy. Among Lactobacillus species, L. crispatus had the highest abundance in all trimesters (40.6%, 40.8% and 44.4%, respectively). L. iners was the second most prevalent species (28.5%, 31% and 25.04, respectively). Our findings reveal that the dominant composition of the vaginal microbiota aligns with the CST-type I, commonly observed in the European population. CONCLUSIONS: This suggests that there are shared mechanisms influencing the microbial communities in the vagina, which are likely influenced by factors such as genetics, lifestyle, and cultural behaviors rather than ethnicity alone. The complex interplay of these factors contributes to the establishment and maintenance of the vaginal microbiota during pregnancy. Understanding the underlying mechanisms and their impact on vaginal health across diverse populations is essential for improving pregnancy outcomes. The study was approved by the Koc University Ethical Committee (no:2019.093.IRB2.030) and registered at the clinical trials.

Log (Lactobacillus crispatus/ Gardnerella vaginalis): a new indicator of diagnosing bacterial vaginosis.

To explore a new marker which can detect bacterial vaginosis (BV) with high sensitivity and specificity quantitatively. According to the Nugent Score, vaginal secretions from study participants were divided into BV, healthy, and BV-intermediate groups. First, we compared the obvious differences and high abundance of bacteria in the three groups using 16S rRNA-sequencing, and screened out candidate markers. Then, quantitative detection of these candidate markers from the three groups was done using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by evaluation of the sensitivity and specificity. Finally, we verified the new markers using clinical cases. Gardnerella vaginalis, Atopobium vaginae, Lactobacillus, Megasphaera were screened out by 16S rRNA-sequencing. RT-qPCR data were transformed and analyzed through ROC curves. PCR results for these bacteria were log-transformed using Lactobacillus crispatus as the numerator and other BV-related bacteria as the denominator. Four new indicators were found. Of these, log L. crispatus/G. vaginalis (L/G) <0 was the best indicator. The sensitivity, specificity, positive predictive value, and negative predictive value of our system were 93.5%, 97.2%, 96.6 and 94.6%, respectively. Combination of data for 16S rRNA-sequencing and RT-qPCR revealed four indicators for BV detection. Of these, log L/G < 0 was the best indicator. Creating a molecular-diagnostic system independent of the Nugent Score for BV could have an important impact on the clinical management of BV.Abbreviation: log L. crispatus/G. vaginalis (logL/G); Bacterial vaginosis (BV); vaginal secretions (VSs); polymerase chain reaction (PCR); rRNA-sequencing (rRNA-seq); real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); operational taxonomic unit (OTU); non-metric multidimensional scaling (NMDS); receiver operating characteristic (ROC).

Activity of Lactobacillus crispatus isolated from vaginal microbiota against Mycobacterium tuberculosis.

Tuberculosis, an infectious disease, is caused by Mycobacterium tuberculosis. It remains a significant public health issue around the globe, causing about 1.8 million deaths every year. Drug-resistant M. tuberculosis, including multi-drug-resistant (MDR), extremely-drug-resistant (XDR), and totally drug-resistant (TDR) M. tuberculosis, continues to be a threat to public health. In the case of antibiotic-resistant tuberculosis, the treatment effect of conventional antibiotics is low. Side effects caused by high doses over a long period are causing severe problems. To overcome these problems, there is an urgent need to develop a new anti-tuberculosis drug that is different from the existing compound-based antibiotics. Probiotics are defined as live microorganisms conferring health benefits. They can be potential therapeutic agents in this context as the effectiveness of probiotics against different infectious diseases has been well established. Here, we report that Lactobacillus crispatus PMC201 shows a promising effect on tuberculosis isolated from vaginal fluids of healthy Korean women. Lactobacillus crispatus PMC201 reduced M. tuberculosis H37Rv under co-culture conditions in broth and reduced M. tuberculosis H37Rv and XDR M. tuberculosis in macrophages. Lactobacillus crispatus PMC201 was not toxic to a guinea pig model and did not induce dysbiosis in a human intestinal microbial ecosystem simulator. Taken together, these results indicate that L. crispatus PMC201 can be a promising alternative drug candidate in the current tuberculosis drug regime. Further study is warranted to assess the in vivo efficacy and confirm the mode of action of L. crispatus PMC201.

Vaginal bacterial load in the second trimester is associated with early preterm birth recurrence: a nested case-control study.

OBJECTIVE: To assess the association between vaginal microbiome (VMB) composition and recurrent early spontaneous preterm birth (sPTB)/preterm prelabour rupture of membranes (PPROM). DESIGN: Nested case-control study. SETTING: UK tertiary referral hospital. SAMPLE: High-risk women with previous sPTB/PPROM <34+0 weeks' gestation who had a recurrence (n = 22) or delivered at ≥37+0 weeks without PPROM (n = 87). METHODS: Vaginal swabs collected between 15 and 22 weeks' gestation were analysed by 16S rRNA gene sequencing and 16S quantitative PCR. MAIN OUTCOME MEASURE: Recurrent early sPTB/PPROM. RESULTS: Of the 109 high-risk women, 28 had anaerobic vaginal dysbiosis, with the remainder dominated by lactobacilli (Lactobacillus iners 36/109, Lactobacillus crispatus 23/109, or other 22/109). VMB type and diversity were not associated with recurrence. Women with a recurrence, compared to those without, had a higher median vaginal bacterial load (8.64 versus 7.89 log10 cells/mcl, adjusted odds ratio [aOR] 1.90, 95% CI 1.01-3.56, P = 0.047) and estimated Lactobacillus concentration (8.59 versus 7.48 log10 cells/mcl, aOR 2.35, (95% CI 1.20-4.61, P = 0.013). A higher recurrence risk was associated with higher median bacterial loads for each VMB type after stratification, although statistical significance was reached only for L. iners domination (aOR 3.44, 95% CI 1.06-11.15, P = 0.040). Women with anaerobic dysbiosis or L. iners domination had a higher median vaginal bacterial load than women with a VMB dominated by L. crispatus or other lactobacilli (8.54, 7.96, 7.63, and 7.53 log10 cells/mcl, respectively). CONCLUSIONS: Vaginal bacterial load is associated with early sPTB/PPROM recurrence. Domination by lactobacilli other than L. iners may protect women from developing high bacterial loads. Future PTB studies should quantify vaginal bacteria and yeasts. TWEETABLE ABSTRACT: Increased vaginal bacterial load in the second trimester may be associated with recurrent early spontaneous preterm birth.

Variability of the response of human vaginal Lactobacillus crispatus to 17ß-estradiol.

We previously showed that the physiological concentration of 17ß-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17ß-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17ß-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17ß-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17ß-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Influence of Lactobacillus crispatus, Lactobacillus iners and Gardnerella vaginalis on bacterial vaginal composition in pregnant women.

PURPOSE: To investigate associations between bacterial species in the vagina in mid-trimester pregnant women from Brazil. METHODS: The vaginal microbiome in 613 subjects was identified by analysis of the V1-V3 region of bacterial 16S ribosomal RNA and the relative prevalence of individual bacteria were determined. RESULTS: The bacterial species present in the greatest number of women were Lactobacillus crispatus (306 women), L. iners (298 women) and Gardnerella vaginalis (179 women). When present in the vagina, L. crispatus was the most abundant bacterium more than 85% of the time. In contrast, L. iners and G. vaginalis were most abundant in 63% and 41% of women who were positive for these microorganisms, respectively (p < 0.0001 vs. L. crispatus). The proportion of L. crispatus was negatively associated with the proportions of L. iners, L. jensenii, L. gasseri, G. vaginalis, Megasphaera, Atopobium vaginae and Prevotella (p < 0.0001). In contrast, the proportion of G. vaginalis was positively associated with levels of Megasphaera, A. vaginae and Prevotella (p < 0.0001) while L. iners proportion was unrelated to the proportion of L. jensenii, G. vaginalis, Megasphaera, A. vaginae or Prevotella. CONCLUSION: The composition of the vaginal microbiota in mid-trimester pregnant women is influenced by the relative concentrations of L. crispatus, L. iners and G. vaginalis.

Connecting the Dots: Translating the Vaginal Microbiome Into a Drug.

A Lactobacillus-dominated vaginal microbiota (VMB) has been associated with health and considered an important host defense mechanism against urogenital infections. Conversely, depletion of lactobacilli and increased microbial diversity, amplifies the risk of adverse gynecologic and obstetric outcomes. A common clinical condition that exemplifies dysbiosis is bacterial vaginosis (BV). BV is currently treated with antibiotics, but frequently recurs, due in part to persistent dysbiosis and failure of lactobacilli to repopulate the vagina. New treatment options are needed to address BV. The VMB is relatively simple and optimally dominated by one or several species of Lactobacillus. Lactobacillus crispatus is strongly associated with vaginal health and depleted in dysbiosis. Replenishing the dysbiotic VMB with protective L. crispatus CTV-05 is a promising approach to prevent recurrent infections and improve women's health. Here we discuss confirmation of this approach with the microbiome-based biologic drug, LACTIN-V (L. crispatus CTV-05), focusing on prevention of BV recurrence.

A specific bacterial DNA signature in the vagina of Australian women in midpregnancy predicts high risk of spontaneous preterm birth (the Predict1000 study).

BACKGROUND: Intrauterine infection accounts for a quarter of the cases of spontaneous preterm birth; however, at present, it is not possible to efficiently identify pregnant women at risk to deliver preventative treatments. OBJECTIVE: This study aimed to establish a vaginal microbial DNA test for Australian women in midpregnancy that will identify those at increased risk of spontaneous preterm birth. STUDY DESIGN: A total of 1000 women with singleton pregnancies were recruited in Perth, Australia. Midvaginal swabs were collected between 12 and 23 weeks' gestation. DNA was extracted for the detection of 23 risk-related microbial DNA targets by quantitative polymerase chain reaction. Obstetrical history, pregnancy outcome, and demographics were recorded. RESULTS: After excluding 64 women owing to losses to follow-up and insufficient sample for microbial analyses, the final cohort consisted of 936 women of predominantly white race (74.3%). The overall preterm birth rate was 12.6% (118 births); the spontaneous preterm birth rate at <37 weeks' gestation was 6.2% (2.9% at ≤34 weeks' gestation), whereas the preterm premature rupture of the membranes rate was 4.2%. No single individual microbial target predicted increased spontaneous preterm birth risk. Conversely, women who subsequently delivered at term had higher amounts of Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii DNA in their vaginal swabs (13.8% spontaneous preterm birth vs 31.2% term; P=.005). In the remaining women, a specific microbial DNA signature was identified that was strongly predictive of spontaneous preterm birth risk, consisting of DNA from Gardnerella vaginalis (clade 4), Lactobacillus iners, and Ureaplasma parvum (serovars 3 and 6). Risk prediction was improved if Fusobacterium nucleatum detection was included in the test algorithm. The final algorithm, which we called the Gardnerella Lactobacillus Ureaplasma (GLU) test, was able to detect women at risk of spontaneous preterm birth at <37 and ≤34 weeks' gestation, with sensitivities of 37.9% and 44.4%, respectively, and likelihood ratios (plus or minus) of 2.22 per 0.75 and 2.52 per 0.67, respectively. Preterm premature rupture of the membranes was more than twice as common in GLU-positive women. Adjusting for maternal demographics, ethnicity, and clinical history did not improve prediction. Only a history of spontaneous preterm birth was more effective at predicting spontaneous preterm birth than a GLU-positive result (odds ratio, 3.6). CONCLUSION: We have identified a vaginal bacterial DNA signature that identifies women with a singleton pregnancy who are at increased risk of spontaneous preterm birth and may benefit from targeted antimicrobial therapy.

Torquetenovirus Titer in Vaginal Secretions from Pregnant and Postpartum Women: Association with Absence of Lactobacillus crispatus and Levels of Lactic Acid and Matrix Metalloproteinase-8.

Vaginal samples from women with term deliveries were tested for torquetenovirus (TTV) by gene amplification, matrix metalloproteinase (MMP)-8 and D- and L-lactic acid by ELISA, and microbiome composition by analysis of the bacterial 16S ribosomal RNA gene. TTV was detected in 43.2%, 31.5%, and 41.4% of first trimester, third trimester, and postpartum samples, respectively. The viral titer was higher in postpartum than in the first (p = 0.0018) or third (p = 0.0013) trimester. The mean gestational age at delivery was lower in women positive for TTV in their first trimester (p = 0.0358). In the first and third trimester, the MMP-8 level was higher if TTV was also present (p < 0.0091). The D-lactic acid level was lower in first trimester samples if TTV was present (p = 0.0334). Lactobacillus crispatus dominance in first and third trimester samples was higher when TTV was absent (p < 0.0033). We conclude that TTV is present in the vagina in many women with normal pregnancy outcomes and that its occurrence is associated with a lack of L. crispatus dominance, an increase in vaginal MMP-8 and a decrease in D-lactic acid.

Vaginal microbiome analysis of healthy women during different periods of gestation.

To assess the vaginal microbiome throughout full-term uncomplicated pregnancy, a longitudinal study was designed for 12 healthy women who had prepared to become pregnant and then delivered at term (38-42 weeks) without complications. The vaginal microbial community was studied at pre-pregnancy, 8-12, 24-28, 37-38 weeks of gestation, and puerperium, using hypervariable tag sequencing of the V3-V4 region of the 16S rRNA gene. Sequencing produced approximately 10 million reads on the Illumina MiSeq. Members of the Firmicutes phyla were prevailing before and during pregnancy periods, and the proportion was quite as Proteobacteria until puerperium. Lactobacillus genus was abundant before and during pregnancy, but post-delivery vaginal microflora variety turned diverse. The species-level analysis revealed that a healthy vaginal microbiome before or during pregnancy was prominently dominated by Lactobacillus crispatus. Furthermore, PCoA analysis revealed for differences in the bacterial community composition between the two levels of Lactobacillus species in pre-pregnancy and pregnancy period (PC1 contribution of 58.46%, PC3 contribution of 8.64%). Based on the taxonomic and PCoA analysis, we found that L. crispatus was dominant in the vaginal microflora of healthy women before or during pregnancy, but at the puerperium, the status changed leading to decreased abundance of protective Lactobacillus species that made vaginal micro-ecological barrier vulnerable to diseases. Additionally, vaginal pH was an important environmental property affecting the vaginal microbial community.

Randomized Trial of Lactin-V to Prevent Recurrence of Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis affects 15 to 50% of women of reproductive age, and recurrence is common after treatment with an antibiotic agent. The high incidence of recurrence suggests the need for new treatments to prevent recurrent bacterial vaginosis. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 2b trial to evaluate the ability of Lactobacillus crispatus CTV-05 (Lactin-V) to prevent the recurrence of bacterial vaginosis. Women 18 to 45 years of age who had received a diagnosis of bacterial vaginosis and who had completed a course of vaginal metronidazole gel as part of the eligibility requirements were randomly assigned, in a 2:1 ratio, to receive vaginally administered Lactin-V or placebo for 11 weeks; follow-up occurred through week 24. The primary outcome was the percentage of women who had a recurrence of bacterial vaginosis by week 12. RESULTS: A total of 228 women underwent randomization: 152 to the Lactin-V group and 76 to the placebo group; of these participants, 88% in the Lactin-V group and 84% in the placebo group could be evaluated for the primary outcome. In the intention-to-treat population, recurrence of bacterial vaginosis by week 12 occurred in 46 participants (30%) in the Lactin-V group and in 34 participants (45%) in the placebo group (risk ratio after multiple imputation for missing responses, 0.66; 95% confidence interval [CI], 0.44 to 0.87; P = 0.01). The risk ratio for recurrence by week 24 (also calculated with multiple imputation for missing responses) was 0.73 (95% CI, 0.54 to 0.92). At the 12-week visit, L. crispatus CTV-05 was detected in 79% of participants in the Lactin-V group. The percentage of participants who had at least one adverse event related to Lactin-V or placebo by week 24 did not differ significantly between the groups. The percentage of participants with local or systemic adverse events was similar in the two groups. CONCLUSIONS: The use of Lactin-V after treatment with vaginal metronidazole resulted in a significantly lower incidence of recurrence of bacterial vaginosis than placebo at 12 weeks. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT02766023.).

Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study.

BACKGROUND: Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence. OBJECTIVE: This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome. STUDY DESIGN: With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated. RESULTS: Participants had a mean age of 53 years (range 22-90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture-detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence. CONCLUSION: In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.

Lactobacillus crispatus dominant vaginal microbita in pregnancy.

OBJECTIVE: To summarize current knowledge regarding Lactobacillus crispatus-dominated vaginal microbiota in pregnancy, as well as an association between the presence of Lactobacillus crispatus-dominated vaginal microbiota and pregnancy complications. DESIGN: Review. SETTING: Department of Obstetrics and Gynecology, University Hospital Hradec Kralove. MATERIAL AND METHODOLOGY: In this review, the results from literature available about the presence of L. crispatus-dominated microbiota in pregnancy are summarized. RESULTS: Pregnant women with Lactobacillus crispatus-dominated vaginal microbiota is very common in pregnancy and it is associated with a lower risk of preterm delivery. CONCLUSION: Lactobacillus crispatus-dominated vaginal microbiota represents an optimal vaginal microbiota in pregnancy.

The first glimpse of the endometrial microbiota in early pregnancy.

Investigation of the microbial community in the female reproductive tract with the use of sequencing techniques has revealed that endometrial samples obtained through a transvaginal catheter are dominated by Lactobacillus species. Dysbiotic changes in the endometrial microbiota may be associated with implantation failure or early spontaneous abortion in patients who undergo assisted reproductive technology treatment. Whether or not there is an endometrial microbiota in early pregnancy is unknown. Herein we describe, the human endometrial microbiota in a patient who subsequently had an 8th week spontaneous clinical miscarriage with euploid embryos in the next cycle and, for the first time, during a successful pregnancy in which the endometrial fluid was sampled at 4 weeks of gestation. The microbial profile found on the endometrial sample before the spontaneous abortion had higher bacterial diversity and lower Lactobacillus abundance than the endometrial fluid from the healthy pregnancy. Functional metagenomics detected different Lactobacillus species between the 2 samples. Lactobacillus crispatus was present in the endometrium before the spontaneous abortion, as were other bacteria involved in dysbiosis, which had an unstable functional pattern characterized by transposases and insertion elements. Lactobacillus iners was the most prevalent microbe found in the endometrium during early pregnancy; its presence was associated with defense mechanisms and basal functions. These novel observations prompt future investigations to understand the potential implications of microbiology on healthy and pathologic human pregnancy.

Vaginal Microbiota and Mucosal Immune Markers in Women With Vulvovaginal Discomfort.

BACKGROUND: Up to 30% of women with vaginal symptoms are not assigned a diagnosis after standard diagnostic assessment. METHODS: We compared premenopausal women with idiopathic vaginitis (IV) or vulvodynia (VVD) to healthy controls. Microbiota were characterized using rRNA sequencing. Cytokines/chemokines (IL-10, IL-1α, IL-1ß, IL-6, IL-8, IL-2, IL-18, IL-4, IL-9, and IL-13) were measured in vaginal lavage fluid using the Meso Scale Discovery platform or ELISA (IL-1ra). Immunoglobulins were measured in vaginal lavage fluid using a bead-based immunoassay (Millipore). Cases and controls were compared using Kruskal-Wallis, analysis of variance, and linear regression or (for microbiome composition) the Bray-Curtis dissimilarity statistic. RESULTS: We compared 20 women with IV, 30 with VVD, and 52 controls. Most (80%) had greater than 90% 16S rRNA gene sequences from Lactobacillus crispatus, L. jensenii, L. gasseri, or L. iners. In analyses adjusted for age and hormonal contraception (HC), Gardnerella vaginalis was less prevalent and abundant in women with VVD (2/30, 7%) versus controls (16/52, 31%) or IV (5/20, 25%) (P = 0.030). Bray-Curtis dissimilarity was not significantly different between IV and controls or VVD. Fungal sequences were only detected in 5 participants: 2 control, 1 IV, 2 VVD. In univariate analysis, cytokines were not associated with diagnosis. Median vaginal concentration of IgE (but not other immunoglobulins) was lower in women with VVD (P = 0.006). CONCLUSIONS: Minimal differences in vaginal microbiota and inflammatory markers between women with IV, VVD or controls suggest no striking association between vaginal bacteria, fungi or inflammation and diagnosis in these women.

The Vaginal Microbiota and Behavioral Factors Associated With Genital Candida albicans Detection in Reproductive-Age Women.

BACKGROUND: Vulvovaginal candidiasis is commonly diagnosed and has been associated in prospective studies with the acquisition of HIV. Little data is available on how the composition of the vaginal microbiota, and other risk factors, are associated with the molecular detection of Candida albicans-a common cause of vulvovaginal candidiasis. METHODS: In a cross-sectional study, self-collected vaginal swabs were obtained from 394 nonpregnant, reproductive-age women. C. albicans was detected using polymerase chain reaction targeting C. albicans ITS1/2 region. Vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing of the V3 to V4 hypervariable regions and clustered into community state types (CSTs). Multiple logistic regression identified factors associated with C. albicans detection. RESULTS: Twenty-one percent had C. albicans detected and 46% reported vaginal symptoms in the prior 60 days. There was a 2-fold increase in the odds of C. albicans if a woman was in a L. crispatus-dominated CST compared to CSTs with low-Lactobacillus levels (adjusted odds ratio, 2.05; 95% confidence interval, 0.97-4.37). History of self-treatment with antifungals, L. crispatus relative abundance, and receptive oral sex were also significantly associated with C. albicans detection. CONCLUSIONS: A L. crispatus-dominated vaginal microbiota is thought to protect women from both development of bacterial vaginosis and incidence of sexually transmitted infections; however, our data suggest that L. crispatus is associated with increased C. albicans detection. Receptive oral sex may also be a risk factor for vaginal C. albicans colonization.

The vaginal microbiome as a predictor for outcome of in vitro fertilization with or without intracytoplasmic sperm injection: a prospective study.

STUDY QUESTION: Is the presence or absence of certain vaginal bacteria associated with failure or success to become pregnant after an in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment? SUMMARY ANSWER: Microbiome profiling with the use of interspace profiling (IS-pro) technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. WHAT IS KNOWN ALREADY: Live-birth rates for an IVF or IVF-ICSI treatment vary between 25 and 35% per cycle and it is difficult to predict who will or will not get pregnant after embryo transfer (ET). Recently, it was suggested that the composition of the vaginal microbiota prior to treatment might predict pregnancy outcome. Analysis of the vaginal microbiome prior to treatment might, therefore, offer an opportunity to improve the success rate of IVF or IVF-ICSI. STUDY DESIGN, SIZE, DURATION: In a prospective cohort study, 303 women (age, 20-42 years) undergoing IVF or IVF-ICSI treatment in the Netherlands were included between June 2015 and March 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study subjects provided a vaginal sample before the start of the IVF or IVF-ICSI procedure. The vaginal microbiota composition was determined using the IS-pro technique. IS-pro is a eubacterial technique based on the detection and categorization of the length of the 16S-23S rRNA gene interspace region. Microbiome profiles were assigned to community state types based on the dominant bacterial species. The predictive accuracy of the microbiome profiles for IVF and IVF-ICSI outcome of fresh ET was evaluated by a combined prediction model based on a small number of bacterial species. From this cohort, a model was built to predict outcome of fertility treatment. This model was externally validated in a cohort of 50 women who were undergoing IVF or IVF-ICSI treatment between March 2018 and May 2018 in the Dutch division of the MVZ VivaNeo Kinderwunschzentrum Düsseldorf, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: In total, the vaginal microbiota of 192 women who underwent a fresh ET could be analysed. Women with a low percentage of Lactobacillus in their vaginal sample were less likely to have a successful embryo implantation. The prediction model identified a subgroup of women (17.7%, n = 34) who had a low chance to become pregnant following fresh ET. This failure was correctly predicted in 32 out of 34 women based on the vaginal microbiota composition, resulting in a predictive accuracy of 94% (sensitivity, 26%; specificity, 97%). Additionally, the degree of dominance of Lactobacillus crispatus was an important factor in predicting pregnancy. Women who had a favourable profile as well as <60% L. crispatus had a high chance of pregnancy: more than half of these women (50 out of 95) became pregnant. In the external validation cohort, none of the women who had a negative prediction (low chance of pregnancy) became pregnant. LIMITATIONS, REASONS FOR CAUTION: Because our study uses a well-defined study population, the results will be limited to the IVF or IVF-ICSI population. Whether these results can be extrapolated to the general population trying to achieve pregnancy without ART cannot be determined from these data. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that vaginal microbiome profiling using the IS-pro technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. Knowledge of their vaginal microbiota may enable couples to make a more balanced decision regarding timing and continuation of their IVF or IVF-ICSI treatment cycles. STUDY FUNDING/COMPETING INTEREST(S): This study was financed by NGI Pre-Seed 2014-2016, RedMedTech Discovery Fund 2014-2017, STW Valorisation grant 1 2014-2015, STW Take-off early phase trajectory 2015-2016 and Eurostars VALBIOME grant (reference number: 8884). The employer of W.J.S.S.C. has in collaboration with ARTPred acquired a MIND subsidy to cover part of the costs of this collaboration project. The following grants are received but not used to finance this study: grants from Innovatie Prestatie Contract, MIT Haalbaarheid, other from Dutch R&D tax credit WBSO, RedMedTech Discovery Fund, (J.D.d.J.). Grants from Ferring (J.S.E.L., K.F., C.B.L. and J.M.J.S.S.), Merck Serono (K.F. and C.B.L.), Dutch Heart Foundation (J.S.E.L.), Metagenics Inc. (J.S.E.L.), GoodLife (K.F.), Guerbet (C.B.L.). R.K. is employed by ARTPred B.V. during her PhD at Erasmus Medical Centre (MC). S.A.M. has a 100% University appointment. I.S.P.H.M.S., S.A.M. and A.E.B. are co-owners of IS-Diagnostics Ltd. J.D.d.J. is co-owner of ARTPred B.V., from which he reports personal fees. P.H.M.S. reports non-financial support from ARTPred B.V. P.H.M.S., J.D.d.J. and A.E.B. have obtained patents `Microbial population analysis' (9506109) and `Microbial population analysis' (20170159108), both licenced to ARTPred B.V. J.D.d.J. and A.E.B. report patent applications `Method and kit for predicting the outcome of an assisted reproductive technology procedure' (392EPP0) and patent `Method and kit for altering the outcome of an assisted reproductive technology procedure' by ARTPred. W.J.S.S.C. received personal consultancy and educational fees from Goodlife Fertility B.V. J.S.E.L. reports personal consultancy fees from ARTPred B.V., Titus Health B.V., Danone, Euroscreen and Roche during the conduct of the study. J.S.E.L. and N.G.M.B. are co-applicants on an Erasmus MC patent (New method and kit for prediction success of in vitro fertilization) licenced to ARTPred B.V. F.J.M.B. reports personal fees from Advisory Board Ferring, Advisory Board Merck Serono, Advisory Board Gedeon Richter and personal fees from Educational activities for Ferring, outside the submitted work. K.F. reports personal fees from Ferring (commercial sponsor) and personal fees from GoodLife (commercial sponsor). C.B.L. received speakers' fee from Ferring. J.M.J.S.S. reports personal fees and other from Merck Serono and personal fees from Ferring, unrelated to the submitted paper. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: ISRCTN83157250. Registered 17 August 2018. Retrospectively registered.

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.