State-of-the-art approaches in the investigation of human seminal bacteriome using metagenomic methods

Jan Hofman^1,2Petra Brenerova¹Petra Borilova Linhartova^1*

• ¹Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, South Moravia, Czechia
• ²Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, South Moravia, Czechia

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).