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Freezing of Vaginal Swabs Prior to DNA Purification Does Not Statistically Significantly Affect Microbiome
Khaled Saoud Ali Ghathian1,2, Julie Elm Heintz1, Sarah Mollerup1
1Department of Clinical Microbiology, Copenhagen University Hospital - Amager and Hvidovre, Hvidovre, Denmark.
Microbiologyopen
|August 29, 2025
Summary
Short-term freezing of vaginal swabs, even after DNA depletion, largely preserves the vaginal microbiome. This stability supports using frozen samples in research, though minor shifts in rare bacteria warrant careful interpretation.
Area of Science:
- Microbiology
- Genomics
- Biobanking
Background:
- Vaginal microbiome analysis is crucial for understanding reproductive health.
- Storage conditions can impact microbiome profiles, particularly after host DNA depletion.
- Limited data exists on the effects of short-term storage temperatures on the vaginal microbiome.
Purpose of the Study:
- To evaluate the short-term impact of different storage temperatures (5°C, -20°C, -80°C) on vaginal microbiome composition after host DNA depletion.
- To determine the stability of vaginal microbiome profiles under common biobanking conditions.
Main Methods:
- Vaginal swabs were stored at 5°C (48 hours), -20°C (3 weeks), or -80°C (3 weeks).
- Host DNA was depleted, and microbial DNA was profiled using MetaPhlAn4.
- Alpha and beta diversity, along with differential abundance, were analyzed using statistical methods (Kruskal-Wallis, Wilcoxon, PERMANOVA, ANCOM-BC2).
- Internal quality controls assessed reproducibility and storage bias.
Main Results:
- No significant differences were observed in alpha diversity, overall relative abundances, or global beta diversity across storage conditions.
- PERMANOVA indicated a non-significant trend towards altered community structure with freezing (p=0.061).
- ANCOM-BC2 identified no differentially abundant taxa between groups.
- Internal quality controls showed minor variations in low-abundance Gram-negative species after freezing.
Conclusions:
- Vaginal microbiome composition remains largely stable under short-term storage at -20°C and -80°C following host DNA depletion.
- These freezing conditions are suitable for clinical and research workflows involving vaginal microbiome analysis.
- Subtle shifts in low-abundance or sensitive taxa may occur and require cautious interpretation, especially in studies focusing on microbial fine structure or biomarker discovery.
