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ObjectivesWe evaluated how storing vaginal samples at room temperature in stabilising solutions versus immediate freezing affects 16S rRNA gene amplicon sequencing-based microbiota studies, aiming to simplify home and field collection.MethodsTwenty participants self-collected six mid-vaginal swabs that were stored in two nucleic acid preservatives (three in modified Solution C2 (Qiagen) and three in Amies/RNALater (Sigma)) in January-February 2016. From each set, two were immediately frozen (−80°C) and one was shipped to the University of Idaho (Moscow, Idaho) with return shipping to the Institute for Genome Sciences (Baltimore, Maryland). Amplicon sequencing of the 16S rRNA gene was used to characterise the vaginal microbiota, VALENCIA was used to assign community state types (CSTs), and quantitative PCR (qPCR) of 16S rRNA genes was used to estimate bacterial abundance. Cohen’s Kappa statistic was used to assess within-participant agreement. Bayesian difference of means models assessed within-participant comparisons between shipped and immediately frozen samples.ResultsThere were 115 samples available for analysis. Average duration of transit for shipped samples was 8 days (SD: 1.60, range: 6–11). Within-participant comparisons of CSTs between shipped and immediately frozen samples revealed complete concordance (kappa: 1.0) for both preservative solutions. No significant differences comparing shipped and immediately frozen samples were found with taxon-level comparisons or bacterial abundances based on pan-bacterial qPCR.ConclusionsShort-term room temperature shipping of vaginal swabs placed in stabilising solutions did not affect vaginal microbiota composition. Home collection with mail-in of vaginal samples may be a reasonable approach for research and clinical purposes to assess the vaginal microbiota.

IntroductionThe vaginal microbiota is thought to play a protective role against STIs. While urine has long been used for detection of genital STIs, there have been few studies evaluating the use of urine samples in vaginal microbiome studies. We hypothesise that urine samples could serve as a surrogate for vaginal swab collection. We sought to compare mid-vaginal swabs and random catch urine samples.MethodsMid-vaginal swabs and random catch urine samples were collected in one sitting from 75 reproductive-age women. Microbiota composition was characterised by sequencing the V3-V4 regions of the 16S rRNA gene on the Illumina platform. Vaginal microbiota were targeted for classification using PECAN, a rapid and accurate taxonomic classifier designed for the vaginal environment. Hierarchical clustering was used to assign community state type (CST) to each sample. CST-I, -II, -III, and -V are dominated by L. crispatus, L. gasseri, L. iners and L. jensenni, respectively, while CSTs IV-A and IV-B represent low-Lactobacillus states with an array of strict and facultative anaerobes. Kappa statistics and Jensen-Shannon distances were used to evaluate the concordance of urine and vaginal samples. ResultsA 77% concordance and a 0.70 kappa value were observed for CST assignments, indicating substantial agreement in microbiota structure and composition between vaginal and urine samples within a woman. Out of 17 discordant pairs, 10 pairs had one sample assigned to CST-IV and the other to CST-III. These two CSTs are known to be associated with rapidly fluctuating dysbiotic states. When comparing the population structure of all urine and vaginal samples, no statistical differences were observed (PERMANOVA: F1,148=1.0815, p=0.31). ConclusionVaginal and random catch urine samples from the same participant showed substantial agreement on bacterial composition. Random catch urine samples could present another sampling option to assess the vaginal and urogenital microbiota.

IntroductionBacterial Vaginosis (BV) is a condition of the human vagina characterised in part by a paucity of Lactobacillusspp. and the presence of a wide array of strict and facultative anaerobes such as Gardnerella vaginalisand Atopobium vaginae. BV is associated with the acquisition of sexually transmitted infections such as HIV and Chlamydia trachomatis. Host microRNAs (miRNAs) are an uncharacterized factor that may control host cellular responses to Lactobacillusand BV-associated bacterial communities. Understanding the molecular mechanisms that drive or are induced by BV-associated vaginal microbiota may help identify targets and develop strategies to restore a healthy vaginal state, which would concurrently reduce the risk of STI acquisition. We hypothesised that specific miRNAs are associated with Lactobacillus-dominated and BV-associated Community State Types (CST) by affecting specific host functions.MethodsLeveraging prospectively collected daily vaginal swab samples, the types and abundance of human miRNAs were used to gain insight into host regulatory mechanisms that potentially associate with vaginal microbial community composition shifts using miRNAseq. Random Forest miRNA feature ranking was used to identify miRNAs correlated with types of vaginal microbiota. Additional in vitrocell culture experiments were performed to demonstrate the relationship between miRNA expression, vaginal bacterial culture supernatants and epithelial cell proliferation using qPCR and Western blots.ResultsmiRNASeq was performed on 100 samples from 16 unique subjects in 3 longitudinal microbiota profile groups. One of the most significant miRNAs associated with BV was miR-193b. In vitro,its expression correlated with decreased cell proliferation in cells exposed to Lactobacillusspp. culture media relative to G. vaginalisculture supernatants.ConclusionmiR-193b over expression is associated with reduced cell proliferation in non-BV samples. Control of cell proliferation could contribute to reducing the risk of STI in Lactobacillusdominated vaginal microbiota.

IntroductionStudies have confirmed that self-collected and clinician-collected mid-vaginal swabs sample the same microbial diversity. Self-collected samples shipped through the mail for PCR-based STI testing has also been validated. We sought to determine if the microbiota of shipped vaginal samples are concordant with freshly frozen samples.MethodsIn January-February 2016, 20 women self-collected six mid-vaginal swabs which were then stored in two different nucleic acid preservatives (3 E-swabs in 2 ml of modified C2 (MO BIO) and 3 in 2 ml of Amies/RNALater). Modified C2 was selected for ease of use in laboratory processing and DNA extraction. For each set of 3 swabs, 2 were immediately frozen (−80°C) and one was sent at room temperature to the University of Idaho in a FedEx “Clinical Pak” which was then return shipped to Baltimore. Meta-taxonomic analysis was performed by sequencing the V3-V4 regions of the 16S rRNA gene. Hierarchical clustering of vaginal microbiota was used to assign community state types (CST) to each sample. Bayesian hierarchical models were applied to perform within-women comparisons of shipped and freshly frozen samples.ResultsAverage duration of transit for the shipped samples was 8 days (SD: 1.60, range: 6–11). Paired comparison of CSTs between a woman’s shipped and freshly frozen samples as well as between C2 and Amies/RNALater revealed no differences (100% concordance, kappa: 1.0 for both). After correction for multiple testing, no significant differences between phylotype relative and absolute abundances were detected in C2 or Amies/RNALater groups. Similarly, there were no statistically significant differences between total bacterial loads of shipped versus freshly frozen samples in C2 (p-value: 0.47) or Amies/RNALater (p-value: 0.21) samples.ConclusionThere were no differences in vaginal microbiota composition and structure between a woman’s shipped and freshly frozen vaginal samples stored in Amies/RNAlater or C2. These data enable future studies to allow participants to self-collect and mail vaginal microbiota specimens.