European Journal of Cardiovascular Medicine

Preterm birth (PTB), defined as delivery before 37 completed weeks of gestation, accounts for approximately 11% of live births worldwide and remains a significant contributor to neonatal morbidity and mortality (1). Despite advancements in obstetric care, the prevention of PTB continues to be a major clinical challenge due to its multifactorial etiology and limited predictive biomarkers (2,3). In recent years, increasing attention has been directed toward the role of the vaginal microbiome in the pathogenesis of PTB, particularly during the early stages of pregnancy (4).

The vaginal microbiota plays a crucial role in maintaining reproductive tract health and immune homeostasis. In healthy pregnant women, the microbiome is typically dominated by Lactobacillus species, which produce lactic acid and contribute to a low vaginal pH, creating a protective environment against pathogenic organisms (5). However, dysbiosis, characterized by a shift toward higher microbial diversity and increased abundance of anaerobic bacteria such as Gardnerella, Atopobium, and Prevotella, has been associated with adverse pregnancy outcomes, including PTB (6,7).

Several studies have highlighted the importance of the early pregnancy vaginal microbiome in influencing pregnancy trajectory. A non-Lactobacillus-dominated microbial community during the first trimester has been linked with increased inflammatory responses and subsequent preterm labor (8). Moreover, recent advances in 16S rRNA gene sequencing have allowed for detailed characterization of microbial community state types (CSTs), which may serve as potential biomarkers for identifying women at higher risk for PTB (9,10).

Given the critical window of fetal development during the early gestational period, this study aims to explore the association between vaginal microbiome composition in the first trimester and the risk of spontaneous preterm birth in a cohort of pregnant women. By identifying microbial patterns associated with PTB, this research seeks to contribute to the development of early diagnostic tools and preventive strategies.

A total of 200 pregnant women aged 18–40 years, presenting for their first antenatal visit before 14 weeks of gestation, were enrolled after obtaining informed written consent. Women with multiple pregnancies, known cervical incompetence, history of cervical cerclage, use of antibiotics or probiotics within the last 30 days, or any active vaginal infections were excluded.

Sample Collection and Microbiome Analysis
Vaginal swab specimens were collected from each participant during the first trimester (≤14 weeks) using sterile flocked swabs. Samples were immediately placed in DNA stabilization buffer and stored at –80°C until processing. Bacterial DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany), following the manufacturer’s protocol. Amplification of the V3–V4 hypervariable regions of the 16S rRNA gene was performed using universal primers, and sequencing was carried out on the Illumina MiSeq platform.

Bioinformatics and Microbial Classification
Raw sequence data were filtered and processed using QIIME2 pipeline. Operational taxonomic units (OTUs) were clustered at 97% similarity and assigned taxonomy using the SILVA reference database. Community state types (CSTs) were classified based on dominant bacterial taxa into Lactobacillus-dominated (e.g., L. crispatus, L. iners) and non-Lactobacillus-dominated types (e.g., Gardnerella, Atopobium, Prevotella). Microbial diversity within samples was assessed using Shannon and Simpson indices.

Follow-Up and Outcome Assessment
Participants were followed up throughout pregnancy, and delivery outcomes were recorded. The primary outcome was spontaneous preterm birth, defined as delivery before 37 weeks of gestation without medical or obstetric intervention (e.g., induction for preeclampsia or fetal distress). Secondary data such as maternal age, body mass index (BMI), parity, and obstetric history were also collected from medical records.

Statistical Analysis
Categorical variables were summarized as frequencies and percentages, while continuous variables were expressed as mean ± standard deviation. Comparisons between term and preterm birth groups were made using the chi-square test for categorical data and Student’s t-test or Mann–Whitney U test for continuous data, as appropriate. Multivariate logistic regression was employed to identify microbial predictors of PTB, adjusting for potential confounders including maternal age, BMI, parity, and smoking status. A p-value of <0.05 was considered statistically significant. Data analysis was performed using SPSS version 25.0 (IBM Corp, Armonk, NY, USA).

Out of the 200 pregnant women enrolled in the study, 38 (19%) experienced spontaneous preterm birth (PTB), while the remaining 162 (81%) delivered at term. The baseline characteristics of the study participants are summarized in Table 1. There was no significant difference in mean maternal age or BMI between the two groups. However, a higher proportion of women with a history of prior PTB was observed in the preterm group (p = 0.032).

The analysis of vaginal microbiome profiles revealed notable differences in community state types (CSTs) between term and preterm birth groups (Table 2). Lactobacillus-dominated microbiota (CST I and III) were more prevalent in term deliveries (78.4%), whereas non-Lactobacillus-dominated CST IV was significantly more common among women who delivered preterm (68.4% vs. 21.6%, p < 0.001).

Alpha diversity indices indicated increased microbial diversity in the preterm group. The mean Shannon diversity index was 2.82 ± 0.29 in women with PTB, significantly higher than 1.54 ± 0.41 in the term group (p < 0.001). Similarly, Simpson’s index values confirmed greater species richness and evenness in the PTB group (Table 3).

Multivariate logistic regression analysis identified CST IV (non-Lactobacillus-dominated) as an independent risk factor for PTB (adjusted OR = 3.51; 95% CI: 1.78–6.91; p = 0.002), while dominance of Lactobacillus crispatus was associated with a significantly reduced risk (adjusted OR = 0.42; 95% CI: 0.19–0.91; p = 0.021), as shown in Table 4.

Table 1. Baseline Demographic and Clinical Characteristics of the Study Population

Table 2. Distribution of Vaginal Microbiome Community State Types (CSTs)

Table 3. Alpha Diversity Indices of Vaginal Microbiota

Table 4. Logistic Regression Analysis for Predictors of Preterm Birth

These findings highlight a significant association between vaginal microbial composition in early pregnancy and subsequent risk of spontaneous preterm birth (Tables 2–4).

This prospective cohort study demonstrates a significant association between early pregnancy vaginal microbiome composition and the risk of spontaneous preterm birth (PTB). Specifically, a predominance of non-Lactobacillus-dominated microbiota (CST IV), characterized by increased microbial diversity and higher abundance of anaerobic bacteria, was observed in women who experienced PTB. Conversely, the presence of Lactobacillus crispatus was associated with a reduced risk of preterm delivery. These findings are consistent with previous studies highlighting the protective role of L. crispatus and the pathogenic potential of vaginal dysbiosis in pregnancy outcomes (1–3).

The vaginal microbiome in pregnancy typically shifts toward a stable, Lactobacillus-dominated state, which plays a vital role in maintaining an acidic environment and preventing colonization by pathogenic organisms (4). L. crispatus, in particular, has been shown to produce high levels of lactic acid and bacteriocins, which inhibit the growth of opportunistic microbes such as Gardnerella vaginalis and Atopobium vaginae (5,6). Our study supports these protective properties, as women with CST I microbiota had significantly lower odds of PTB compared to those with CST IV.

Higher microbial diversity, as reflected in elevated Shannon and Simpson indices among the preterm group, suggests a disruption of the normal microbial balance (7). Increased diversity and reduced Lactobacillus dominance have been associated with subclinical inflammation and impaired mucosal barrier function, both of which are implicated in the pathophysiology of preterm labor (8,9). Inflammatory mediators such as interleukins and prostaglandins produced in response to microbial imbalance may trigger uterine contractions and cervical ripening prematurely (10).

Several longitudinal studies have previously indicated that early pregnancy is a critical window for microbiome-mediated influence on pregnancy outcomes (11,12). DiGiulio et al. showed that microbial profiles established early in gestation often persist and can be predictive of PTB risk (13). Moreover, the use of high-throughput sequencing in our study allowed for comprehensive characterization of microbial community state types (CSTs), corroborating earlier reports that CST IV is disproportionately represented in women with adverse pregnancy outcomes (14,15).

Our findings highlight the potential of microbiome-based risk stratification tools for early identification of women at risk for PTB. Interventions such as probiotics, antimicrobial therapies, or vaginal microbiota transplantation could be explored in future trials to restore a protective Lactobacillus-dominated environment (6,10). However, the clinical implementation of such strategies will require rigorous validation and ethical considerations.

The study’s strengths include its prospective design, use of 16S rRNA sequencing, and follow-up through delivery. Nonetheless, some limitations must be acknowledged. The sample size, though adequate for detecting major associations, limits the power to explore interactions with other factors such as ethnicity, sexual activity, or diet. Additionally, causality cannot be established due to the observational nature of the study.

This study highlights the significant association between early pregnancy vaginal microbiome composition and the risk of spontaneous preterm birth. A predominance of non-Lactobacillus-dominated communities, particularly CST IV, and increased microbial diversity were strongly linked to higher PTB risk, whereas Lactobacillus crispatus dominance appeared protective. These findings suggest that early microbiome screening could serve as a valuable tool for identifying women at risk and guiding future preventive strategies.

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