# Vaginal Microbiome and Functional Pathway Alterations in Preterm Premature Rupture of Membranes Revealed by 16S rRNA Sequencing

## TL;DR

This study shows that the vaginal microbiome in women with preterm rupture of membranes is significantly altered, with potential implications for preterm birth and possible therapeutic targets.

## Contribution

The study provides a comprehensive analysis of the vaginal microbiome and functional pathways in PPROM using 16S rRNA sequencing and co-occurrence networks.

## Key findings

- PPROM is associated with a shift from Lactobacillus-dominated to polymicrobial vaginal communities.
- The PPROM microbiome shows increased alpha diversity and distinct functional enrichment in amino acid biosynthesis.
- A dense network of anaerobic bacteria replaces protective Lactobacillus interactions in PPROM.

## Abstract

Preterm prelabor rupture of membranes (PPROM) is a leading cause of preterm birth and significant neonatal morbidity. The vaginal microbiome is implicated in its pathogenesis, but its detailed characteristics and functional consequences remain to be fully elucidated. This study aimed to provide a comprehensive, multi-faceted analysis of the vaginal microbiome and its functional potential in pregnant women with PPROM compared to healthy term controls. We collected vaginal fluid samples from eight PPROM and seven healthy control (HC) pregnant women. The vaginal microbiome was analyzed using 16S rRNA gene sequencing. We assessed community composition and state types (CSTs), alpha and beta diversity, co-occurrence networks, and predicted functional pathways using PICRUSt2. A molecular bacterial vaginosis (molBV) score was also calculated to determine the clinical relevance of the dysbiosis. The PPROM microbiome was characterized by a significant depletion of Lactobacillus crispatus–dominated communities (CST I) and a shift towards L. iners–dominated (CST III) or polymicrobial (CST IV) communities, which was consistent with a BV-positive molBV score. Alpha diversity was significantly higher in the PPROM group, and beta diversity analysis confirmed a distinct microbial structure between the two groups. Co-occurrence network analysis revealed a collapse of the protective, Lactobacillus-centered network in the PPROM group, which was replaced by a densely interconnected network of anaerobic bacteria with Gardnerella vaginalis as a key hub. Functionally, the PPROM microbiome was enriched for amino acid biosynthesis pathways, in contrast to the HC group, which was enriched for nucleotide and peptidoglycan biosynthesis. PPROM appears to be linked with a complex vaginal dysbiosis that encompasses significant alterations in microbial composition, diversity, interactions, and functional potential. These findings highlight the vaginal microbiome as a critical factor in the pathogenesis of PPROM and suggest its potential for risk stratification and as a therapeutic target to improve pregnancy outcomes.