Virtanen Seppo, Kalliala Ilkka, Nieminen Pekka, Salonen Anne
Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, London, United Kingdom.
PLoS One. 2017 Jul 19;12(7):e0181477. doi: 10.1371/journal.pone.0181477. eCollection 2017.
Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species.
The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001).
The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different sampling strategies should be comparable in the absence of other technical confounders. The Evalyn® self-sampling device performed equally well compared to samples taken by a clinician, and hence offers a good-quality microbiota sample without the need for a gynecological examination. The amount of collected sample as well as the DNA and protein yield varied across the sampling techniques, which may have practical implications for study design.
诸如新一代测序等分子方法正被积极用于表征健康和疾病状态下的阴道微生物群。先前的研究主要集中于表征微生物群的生物学变异，而对于与采样相关的因素如何影响结果则了解较少。我们的目的是研究采样设备和解剖采样部位对阴道微生物群研究相关的定量和定性结果的影响。我们用植绒拭子、Evalyn® 自行采样装置、无菌塑料刮匙和宫颈刷对10名具有不同临床特征的芬兰女性进行采样，这些采样工具用于从穹窿、阴道壁和宫颈采集样本。基于16S rRNA基因的Illumina MiSeq测序，对样本的DNA和蛋白质产量、细菌载量以及微生物群多样性和物种组成进行了比较。我们量化了采样变量与内在变量在整体微生物群变异中的相对贡献，并使用几种常用指标（如α和β多样性以及主要细菌属和种的丰度）评估了微生物群谱。
总DNA产量强烈依赖于采样设备，在较小程度上依赖于采样的解剖部位。采样策略不影响蛋白质产量或细菌载量。基于MiSeq测序，所有测试的采样方法产生的微生物群谱具有高度可比性。采样方法仅解释了整体微生物群变异的2%（p值 = 0.89），明显低于诸如临床状态（细菌性阴道病显微镜检查占53%，p = 0.0001）、出血（19%，p = 0.0001）以及个体间变异（11%，p值0.0001）等内在因素。
结果表明不同的采样策略产生可比的阴道微生物群组成和多样性。因此，在没有其他技术混杂因素的情况下，过去和未来采用不同采样策略的阴道微生物群研究应该具有可比性。与临床医生采集的样本相比，Evalyn® 自行采样装置表现同样良好，因此无需妇科检查就能提供高质量的微生物群样本。不同采样技术收集的样本量以及DNA和蛋白质产量各不相同，这可能对研究设计具有实际意义。
