Comparison of Storage Conditions for Human Vaginal Microbiome Studies

Abstract

BackgroundThe effect of storage conditions on the microbiome and metabolite composition of human biological samples has not been thoroughly investigated as a potential source of bias. We evaluated the effect of two common storage conditions used in clinical trials on the bacterial and metabolite composition of the vaginal microbiota using pyrosequencing of barcoded 16S rRNA gene sequencing and 1H-NMR analyses.Methodology/Principal FindingsEight women were enrolled and four mid-vaginal swabs were collected by a physician from each woman. The samples were either processed immediately, stored at −80°C for 4 weeks or at −20°C for 1 week followed by transfer to −80°C for another 4 weeks prior to analysis. Statistical methods, including Kolmogorovo-Smirnov and Wilcoxon tests, were performed to evaluate the differences in vaginal bacterial community composition and metabolites between samples stored under different conditions. The results showed that there were no significant differences between samples processed immediately after collection or stored for varying durations. 1H-NMR analysis of the small molecule metabolites in vaginal secretions indicated that high levels of lactic acid were associated with Lactobacillus-dominated communities. Relative abundance of lactic acid did not appear to correlate with relative abundance of individual Lactobacillus sp. in this limited sample, although lower levels of lactic acid were observed when L. gasseri was dominant, indicating differences in metabolic output of seemingly similar communities.Conclusions/SignificanceThese findings benefit large-scale, field-based microbiome and metabolomic studies of the vaginal microbiota.

Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy

“…In summary, consistent with results of studies of the fecal and vaginal microbiome, we observed minimal effects on microbial composition and structure when samples were held at different temperatures (13,14).…”

“…In contrast, a comparison of fecal samples from two patients with irritable bowel syndrome and two healthy controls showed that whether stored at room temperature for 24 h or at Ϫ80°C for 6 months, the microbial communities clustered with their host of origin (13). Similarly, a study of vaginal swabs from 8 women found no significant differences in vaginal bacterial community compositions among samples stored under different conditions (14).…”

“…Previous studies have evaluated the effect of storage conditions on 16S metagenomic analysis on fecal specimens (12,13) and vaginal swabs (14). For example, a study of 11 fecal samples suggested that shortterm storage conditions affected DNA integrity and resulting microbial compositions (12).…”

Maternal microbiomes in preterm birth: Recent progress and analytical pipelines

“…The vagina, or a particular region in the vagina, is sampled by circumferential swabbing using sterile technique. The swabs, which are also used in the microbiology clinic for aerobic and anaerobic cultures, can either be stored long term at −80 °C or kept on ice for direct DNA extraction 42,43 . During sampling, most investigators also measure the vaginal pH 42 .…”

Are Infants Born by Elective Cesarean Delivery Without Labor at Risk for Developing Immune Disorders Later in Life?

“…Birth constitutes a critical stage for the acquisition of the first microbiota. During the process of vaginal delivery, the conceptus is exposed to the vaginal microbiota [9][10][11][12][13][14][15] , and such bacteria become the pioneer microorganisms that invade the formerly sterile body of the infant and establish the first microbiota 16 . Using sequence-based techniques, Dominguez-Bello et al demonstrated that infants born by cesarean section are colonized with bacteria similar to that found on maternal skin 17 , while those born by vaginal delivery had flora close to that of the vagina.…”