Bacterial vaginosis (BV) is the most prevalent cause of vaginal discharge in reproductive-aged women and is characterized by a disturbance in the normal vaginal microbiota, predominantly a shift from Lactobacillus-dominated flora to one enriched with anaerobic bacteria such as Gardnerella vaginalis, Atopobium vaginae, and Mobiluncus species (1). Recurrent bacterial vaginosis (RBV), defined as three or more episodes per year, affects approximately 30% of women who have previously been treated for BV and represents a significant challenge in gynecological and obstetric care (2,3). This dysbiotic state leads to an altered vaginal microenvironment that has been implicated in increasing susceptibility to sexually transmitted infections and adverse obstetric outcomes, particularly preterm birth (PTB) (4).

Preterm birth, defined as delivery before 37 weeks of gestation, remains a leading cause of neonatal morbidity and mortality worldwide, accounting for an estimated 15 million births annually (5). Several studies have suggested a strong correlation between vaginal microbiota composition and PTB risk, particularly in women with a history of recurrent BV (6). The presence of specific bacterial taxa, such as G. vaginalis and A. vaginae, has been associated with pro-inflammatory responses that may compromise the integrity of the fetal membranes and trigger premature labor (7,8).

Advancements in molecular techniques, particularly 16S rRNA gene sequencing, have enabled more accurate profiling of the vaginal microbiome, allowing for a deeper understanding of microbial dynamics during pregnancy (9). Despite this, limited data exist on how these microbial alterations in women with RBV correlate specifically with pregnancy outcomes such as PTB.

Therefore, this study aims to investigate the alterations in vaginal microbiota among pregnant women with a history of recurrent bacterial vaginosis and to explore their potential association with preterm birth, thereby identifying microbial biomarkers that could aid in risk stratification and early intervention.

Study Design and Participants

A total of 120 pregnant women, aged 18–35 years, between 12 and 20 weeks of gestation, were enrolled after obtaining written informed consent.

Participants were divided into two groups:

• Group A: 60 pregnant women with a documented history of recurrent bacterial vaginosis (defined as ≥3 episodes in the past 12 months).
• Group B: 60 pregnant women with no prior history of bacterial vaginosis and no current vaginal symptoms.

Inclusion and Exclusion Criteria

Women with singleton pregnancies and no current antibiotic or antifungal therapy were included. Exclusion criteria were: multiple pregnancies, known uterine anomalies, chronic medical conditions (e.g., diabetes, hypertension), and recent use (within 4 weeks) of probiotics or vaginal products.

Sample Collection

Vaginal swabs were collected from the posterior fornix using sterile, DNA-free swabs during a speculum examination. The samples were immediately placed in DNA stabilization buffer and stored at -80°C until processing.

Microbiota Analysis

Microbial DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s protocol. The V3–V4 region of the bacterial 16S rRNA gene was amplified using region-specific primers and sequenced using the Illumina MiSeq platform. Quality filtering and taxonomic classification were performed using QIIME 2 software and the SILVA 132 database.

Outcome Assessment

Participants were followed throughout pregnancy. The primary outcome was the incidence of preterm birth, defined as spontaneous delivery before 37 weeks of gestation. Other recorded variables included gestational age at delivery, birth weight, and presence of premature rupture of membranes (PROM).

Statistical Analysis

All data were analyzed using SPSS version 25.0 (IBM Corp., USA). Continuous variables were expressed as mean ± standard deviation, and categorical variables as frequencies or percentages. The chi-square test and t-test were used to assess differences between groups. Logistic regression analysis was conducted to evaluate associations between microbiota profiles and preterm birth. A p-value <0.05 was considered statistically significant.

Out of the 120 enrolled participants, all completed follow-up until delivery. The demographic characteristics between Group A (women with recurrent BV) and Group B (controls) were comparable in terms of age, BMI, and gestational age at enrollment (p > 0.05).

Vaginal Microbiota Composition

Analysis of 16S rRNA gene sequencing revealed distinct microbial profiles between the two groups. Group A showed a significantly lower abundance of Lactobacillus crispatus (mean relative abundance: 21.5%) compared to Group B (mean: 62.8%, p < 0.001). Conversely, higher proportions of Gardnerella vaginalis and Atopobium vaginae were detected in Group A (34.2% and 18.4%, respectively) versus Group B (9.7% and 3.5%, respectively; p < 0.001 for both) (Table 1).

Table 1: Comparative Vaginal Microbial Composition Between Groups

Obstetric Outcomes

Preterm birth occurred in 17 out of 60 women (28.3%) in Group A, significantly higher than the 5 cases (8.3%) observed in Group B (p = 0.006). The mean gestational age at delivery was also lower in Group A (36.1 ± 1.3 weeks) compared to Group B (38.2 ± 1.1 weeks, p < 0.001). Incidence of PROM was greater in Group A (20%) versus Group B (6.7%) (Table 2).

Table 2: Obstetric Outcomes in Study Groups

Association with Microbial Profiles

Logistic regression analysis identified high G. vaginalis abundance (>25%) as a significant predictor of preterm birth (Odds Ratio: 3.9; 95% CI: 1.6–9.4; p = 0.002), even after adjusting for age and BMI.

These findings suggest a strong association between disrupted vaginal microbiota in women with recurrent BV and increased risk of preterm delivery (refer Tables 1 and 2).

This study demonstrates a significant association between altered vaginal microbiota and an increased risk of preterm birth in women with a history of recurrent bacterial vaginosis (RBV). Specifically, the predominance of anaerobic bacteria such as Gardnerella vaginalis and Atopobium vaginae and a reduction in Lactobacillus crispatus were observed in these women, correlating with adverse obstetric outcomes, particularly spontaneous preterm birth.

Previous literature supports the idea that a Lactobacillus-dominated vaginal ecosystem, especially one rich in L. crispatus, is a hallmark of vaginal health and is protective during pregnancy (1,2). The depletion of Lactobacillus species and overgrowth of anaerobic organisms have been associated with increased vaginal pH, biofilm formation, and local inflammation—factors that are conducive to ascending infections and chorioamnionitis, both known triggers for preterm labor (3–5).

The increased prevalence of G. vaginalis and A. vaginae in our study aligns with prior research demonstrating these organisms as key constituents of BV-associated dysbiosis and their role in immune dysregulation at the maternal-fetal interface (6,7). These bacteria have been shown to stimulate the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, which contribute to cervical remodeling and membrane weakening (8,9). Moreover, G. vaginalis has been detected in amniotic fluid of women with preterm labor, indicating its potential for intrauterine invasion (10).

Our findings regarding the higher rate of preterm birth (28.3%) among RBV-affected women are consistent with large cohort studies and meta-analyses that suggest BV increases the risk of spontaneous preterm delivery by twofold or more (11,12). The observed increased incidence of premature rupture of membranes (PROM) and low birth weight further reinforces the clinical significance of vaginal dysbiosis in pregnancy outcomes (13).

One of the key strengths of this study was the use of 16S rRNA sequencing, which provided a detailed and accurate profile of vaginal microbial communities. This molecular technique surpasses traditional Gram stain or culture-based diagnostics in sensitivity and specificity, particularly for fastidious anaerobes (14). Additionally, the prospective design and careful control group matching helped in minimizing confounding variables.

However, certain limitations must be acknowledged. First, the study was single-centered with a moderate sample size, which may limit generalizability. Second, we assessed microbiota at a single time point during mid-pregnancy; longitudinal analysis might provide better insight into microbial shifts throughout gestation (15). Finally, while associations were observed, causality cannot be definitively established due to the observational nature of the study.

Alterations in the vaginal microbiota, particularly a decrease in protective Lactobacillus species and an overrepresentation of anaerobic pathogens like Gardnerella vaginalis and Atopobium vaginae, are significantly associated with an increased risk of preterm birth in women with recurrent bacterial vaginosis. Early identification of such dysbiosis using molecular techniques may offer a valuable opportunity for preventive interventions and improved pregnancy outcomes.

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