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Freezing of Vaginal Swabs Prior to DNA Purification Does Not Statistically Significantly Affect Microbiome Composition

Year: 2025

Type: article

Abstract: Vaginal microbiome profiles may be affected by storage conditions, especially after human DNA depletion, yet systematic evaluations are limited. To assess the short-term impact of storage temperature on vaginal microbiome composition following host DNA depletion. Vaginal swabs were stored at 5°C (48 h), -20°C (3 weeks), or -80°C (3 weeks). DNA was extracted using the MolYsis Complete5 kit and profiled with MetaPhlAn4. Alpha and beta diversity metrics and differential abundance tests (CLR-transformed, Kruskal-Wallis and Wilcoxon with FDR correction) were applied. Internal quality controls (IQCs) assessed reproducibility and storage bias. No significant differences were found in alpha diversity, relative abundances (Kruskal-Wallis p = 0.786), or global beta diversity (ANOSIM R = - 0.042, p = 0.937). PERMANOVA showed a trend (F = 3.51, p = 0.061), but ANCOM-BC2 found no differentially abundant taxa. IQCs revealed variation in low-abundance Gram-negative species after freezing. Vaginal microbiome composition remained largely stable under short-term freezing conditions (-20°C and -80°C), supporting their use in clinical and research workflows. Nonetheless, subtle shifts in low-abundance or fragile taxa may occur and should be interpreted with caution in studies emphasizing microbial fine structure or biomarker discovery.

Source: MicrobiologyOpen

DOI: 10.1002/mbo3.70053