Hofman Jan, Brenerova Petra, Borilova Linhartova Petra
RECETOX, Faculty of Science, Masaryk University, Brno, Czechia.
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czechia.
Front Reprod Health. 2025 Jun 5;7:1557912. doi: 10.3389/frph.2025.1557912. eCollection 2025.
Although the understanding of the causes of infertility is the key to its successful treatment, recent studies have shown that as many as 50% of male-caused infertility cases are considered idiopathic. The microbial colonization of the male reproductive system was shown to be associated with reduced male reproductive fitness. Investigation of the seminal microbiome, however, remains challenging. This article aimed to improve this situation by creating the first comprehensive review of literature on the metagenomic methods (including the pre-analytical and analytical approaches) used in the research on human seminal bacteriome (total bacterial DNA in the matrix), published in 2018-2024. A total of 29 studies addressing the analysis of the human seminal bacteriome were identified. The analysis typically involved DNA extraction from the supernatant using commercial kits, amplification of the gene for 16S rRNA, and sequencing of amplicons. Where the separation of seminal plasma was performed, centrifugation was the dominant method used for this purpose. The significant heterogeneity in individual steps of methodological approaches in the analysis of the human seminal bacteriome complicates the comparison of results among studies and the establishment of standard procedures, hindering clinical advancements. For this reason, a protocol for the analysis of the human seminal plasma bacteriome is proposed here, which could lead to improved comparability of results among studies and make future research more efficient. This protocol is founded on rigorous quality control measures, compliance with the WHO laboratory manual for sample collection, extensive pretreatment involving mechanical and enzymatic lysis, DNA extraction using the QIAamp DNA Mini Kit (Qiagen), and short-read sequencing conducted on the MiSeq platform (Illumina).
虽然了解不孕症的病因是成功治疗的关键，但最近的研究表明，多达50%的男性因素导致的不孕症病例被认为是特发性的。男性生殖系统的微生物定植与男性生殖健康下降有关。然而，对精液微生物组的研究仍然具有挑战性。本文旨在通过对2018 - 2024年发表的关于人类精液细菌组（基质中的总细菌DNA）研究中使用的宏基因组方法（包括分析前和分析方法）的文献进行首次全面综述来改善这种情况。共确定了29项针对人类精液细菌组分析的研究。分析通常包括使用商业试剂盒从上清液中提取DNA、扩增16S rRNA基因以及对扩增子进行测序。在进行精浆分离的情况下，离心是主要使用的方法。人类精液细菌组分析方法学各个步骤中的显著异质性使得研究结果之间的比较以及标准程序的建立变得复杂，阻碍了临床进展。因此，本文提出了一种人类精浆细菌组分析方案，这可能会提高研究结果之间的可比性，并使未来的研究更高效。该方案基于严格的质量控制措施、遵循世界卫生组织样本采集实验室手册、涉及机械和酶解的广泛预处理、使用QIAamp DNA Mini试剂盒（Qiagen）进行DNA提取以及在MiSeq平台（Illumina）上进行短读长测序。
