Lietaer Leen, Pascottini Osvaldo Bogado, Lacoere Tim, Kerckhof Frederiek-Maarten, Martens Ann, Van de Wiele Tom, Opsomer Geert
Department of Internal Medicine, Reproduction and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Department of Internal Medicine, Reproduction and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; Department of Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, University of Antwerp, Wilrijk, Belgium.
J Microbiol Methods. 2023 Feb;205:106664. doi: 10.1016/j.mimet.2022.106664. Epub 2022 Dec 29.
Recent studies have suggested that bacteria associated with the female reproductive tract - the uterine microbiota - may be important for reproductive health and pregnancy success. Therefore, uterine microbiome research gained much interest in the last few years. However, it is challenging to study late postpartum uterine samples, since they hold a low microbial biomass. Next-generation sequencing techniques are very sensitive for microbial identification, but they cannot make a distinction between actual microbiota and contaminant bacteria or their DNA. Our aim was to test a new method to sample the bovine uterine lumen in vivo, while minimizing the risk of cross-contamination. In order to evaluate this method, we performed a descriptive assessment of the microbial composition of the obtained samples. Transabdominal, laparoscopic sampling of the uterine lumen was conducted in five Holstein-Friesian cows. Uterine fluid from the uterine horns was collected by low-volume lavage. DNA from the samples was extracted using two different DNA extraction methods, and negative controls (sampling blank controls and DNA extraction blank controls) were included. Bacteria were identified using 16S rRNA gene amplicon sequencing. In this proof-of-concept study, no evidence for authentically present uterine microbiota could be found. During laparoscopic sampling, some practical challenges were encountered, and the reliability of low-volume-lavage for the collection of a low microbial biomass could be questioned. By comparing two DNA extraction methods, a significant contamination background could be noticed originating from the DNA extraction kits.
最近的研究表明，与女性生殖道相关的细菌——子宫微生物群——可能对生殖健康和妊娠成功至关重要。因此，子宫微生物组研究在过去几年中引起了广泛关注。然而，研究产后晚期子宫样本具有挑战性，因为它们的微生物生物量较低。下一代测序技术对微生物鉴定非常敏感，但它们无法区分实际的微生物群和污染细菌或其DNA。我们的目的是测试一种在体内对牛子宫腔进行采样的新方法，同时将交叉污染的风险降至最低。为了评估该方法，我们对获得的样本的微生物组成进行了描述性评估。对五头荷斯坦-弗里生奶牛进行经腹腹腔镜子宫腔采样。通过小容量灌洗收集子宫角的子宫液。使用两种不同的DNA提取方法从样本中提取DNA，并设置阴性对照（采样空白对照和DNA提取空白对照）。使用16S rRNA基因扩增子测序鉴定细菌。在这项概念验证研究中，未发现子宫中真实存在微生物群的证据。在腹腔镜采样过程中，遇到了一些实际挑战，小容量灌洗收集低微生物生物量的可靠性可能受到质疑。通过比较两种DNA提取方法，发现DNA提取试剂盒存在明显的污染背景。
