School of Health, Leeds Beckett University, Leeds, United Kingdom.
School of Health and Life Sciences, Teesside University, Middlesbrough, United Kingdom.
PLoS One. 2022 Mar 17;17(3):e0265441. doi: 10.1371/journal.pone.0265441. eCollection 2022.
Formalin-Fixed Paraffin-Embedded (FFPE) tissues are routinely collected, archived, and used for clinical diagnosis, including maternal and neonatal health. Applying FFPE samples to microbiota research would be beneficial to reduce preparation, storage and costs associated with limited available frozen samples. This research aims to understand if FFPE fetal membrane samples are comparable to frozen tissues, which are the current gold standard for DNA microbiota analysis. Extracted DNA from nine matched paired patients were sequenced by Illumina sequencing of the V4 16S rRNA gene region. This included duplicate frozen amnion and chorion fetal membrane rolls or FFPE combined amniochorionic samples. Negative controls of surrounding wax blocks and DNA extraction reagents were processed alongside samples using identical methods. DNA quality and quantity was assessed by NanoDrop, agarose gel electrophoresis and Bioanalyzer. Decontam and SourceTracker were integrated into microbiota analysis to identify the presence of contaminating sources. The bacterial profile and nine genera differed between FFPE and frozen fetal membranes. There were no differences in bacterial profiles between FFPE samples and corresponding wax negative controls, with 49% of bacteria in FFPE fetal membrane samples matched to the source origin of paraffin wax, and 40% originating from DNA extraction reagent sources. FFPE samples displayed high fragmentation and low quantity of extracted DNA compared to frozen samples. The microbiota of FFPE fetal membrane samples is influenced by processing methods, with the inability to differentiate between the microbiota of the tissue sample and the surrounding wax block. Illumina sequencing results of FFPE and frozen fetal membrane samples should not be compared using the methods employed here. Variation could be influenced by limitations including storage time, DNA extraction and purification methods. To utilise FFPE fetal membrane samples in microbiota research then contamination prevention and detection methods must be included into optimised and standardised protocols, with recommendations presented here.
福尔马林固定石蜡包埋（FFPE）组织通常被收集、存档，并用于临床诊断，包括母婴健康。将 FFPE 样本应用于微生物组研究将有助于减少与有限的可用冷冻样本相关的准备、储存和成本。本研究旨在了解 FFPE 胎膜样本是否与冷冻组织具有可比性，冷冻组织是目前用于 DNA 微生物组分析的黄金标准。从 9 名配对患者的 FFPE 样本中提取 DNA，通过 Illumina 测序 V4 16S rRNA 基因区域进行测序。这包括重复的冷冻羊膜和绒毛膜胎膜卷或 FFPE 联合羊膜绒毛膜样本。使用相同的方法，对周围蜡块和 DNA 提取试剂的阴性对照进行了与样本相同的处理。通过 NanoDrop、琼脂糖凝胶电泳和 Bioanalyzer 评估 DNA 质量和数量。将 Decontam 和 SourceTracker 整合到微生物组分析中，以识别污染来源的存在。FFPE 和冷冻胎膜之间的细菌谱和 9 个属不同。FFPE 样本与相应的蜡阴性对照之间的细菌谱没有差异，49%的 FFPE 胎膜样本中的细菌与石蜡的来源一致，40%的细菌与 DNA 提取试剂的来源一致。与冷冻样本相比，FFPE 样本的 DNA 提取片段化程度更高，数量更少。FFPE 胎膜样本的微生物组受处理方法的影响，无法区分组织样本和周围蜡块的微生物组。不能使用这里采用的方法来比较 FFPE 和冷冻胎膜样本的 Illumina 测序结果。变异可能受到包括储存时间、DNA 提取和纯化方法在内的限制因素的影响。要在微生物组研究中使用 FFPE 胎膜样本，必须在优化和标准化的方案中包含污染预防和检测方法，并在此提出建议。
