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

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.