# Optimising Vaginal Microbiome Profiling for Clinical Translation: A Comparative Assessment of Sample Storage Methods and a Vagina-Specific 16S rRNA Gene Database

## TL;DR

This study evaluates storage methods and a specialized database for vaginal microbiome profiling to improve preterm birth risk assessment.

## Contribution

A curated vagina-specific 16S rRNA gene database (VagDB) is introduced to enhance microbiome profiling accuracy.

## Key findings

- Amies-stabilized samples yielded higher DNA but did not affect diversity or community structure.
- VagDB improved species-level resolution of vaginal microbiome data.
- Storage method had minimal impact on community state type (CST) allocation, with over 90% concordance.

## Abstract

Vaginal microbiome composition has been linked to risk of preterm birth (PTB), a persistent global health challenge. 16S rRNA microbial profiling has identified specific vaginal community state types (CSTs) that have been associated with PTB risk. Diagnostic profiling requires standardised pre-analytical protocols. We evaluated two storage methods and validated a curated, vagina-specific 16S rRNA gene database (VagDB) to enhance annotation. Paired Copan FLOQ swabs from 22 women at high PTB risk were processed for either (a) dry/immediate freezing or (b) Amies-stabilisation/refrigeration. Amplicon sequence variants were generated via 16S rRNA gene (V4) PCR and Illumina sequencing. We assessed diversity, composition, and community state type (CST) allocation. Amies-stabilised samples yielded significantly higher DNA (p = 0.003), but this did not alter species richness, evenness, or community structure. VagDB enhanced species-level resolution. PCoA showed robust clustering by participant and CST (p < 0.001), irrespective of storage; CST concordance exceeded 90%. Routinely collected vaginal swabs in stabilisation medium with an 8–72 h refrigeration window yield reliable data, supporting the integration of vaginal microbiome profiling into clinical PTB risk assessment.