Decoding the seminal microbial fingerprint and semen quality: insights from the first Greek pilot study

<h4>Background</h4>Recent evidence suggests that human semen harbors a complex microbiome that may influence semen quality and male fertility. However, comprehensive characterization of bacterial and fungal communities in semen remains limited, particularly using long-read sequencing technologies.<h4>Methods</h4>In this pilot study, 80 semen samples from 80 men were analyzed and classified based on conventional semen-analysis findings into normozoospermic (<i>n</i> = 48) and abnormal (<i>n</i> = 32) groups. The abnormal group included men with asthenozoospermia and oligoasthenozoospermia. Microbial profiling was conducted using full-length 16S rRNA gene and internal transcribed spacer (ITS) sequencing on the Oxford Nanopore MinION platform.<h4>Results</h4>Phylum-level analysis revealed distinct microbial profiles between groups. Normozoospermic samples were dominated by Bacillota (64.13%), followed by Actinomycetota (14.61%) and Pseudomonadota (12.95%). In contrast, abnormal samples showed reduced Bacillota abundance (2.32%) and enrichment of Campylobacteriota (10.82%). At the genus level, normozoospermic samples were characterized by predominance of Enterococcus, Cutibacterium, Streptococcus, Finegoldia, and Staphylococcus, whereas abnormal samples showed increased abundance of Campylobacter, Stenotrophomonas, and Agrobacterium. Species-level profiling identified <i>Enterococcus faecalis</i> as the dominant species in normozoospermic samples (41.3%), while <i>Campylobacter ureolyticus</i> predominated in abnormal samples (49.6%). ITS sequencing did not detect fungal DNA in any semen sample, whereas all fungal controls amplified successfully, confirming methodological reliability.<h4>Conclusion</h4>Our findings demonstrate significant differences in seminal microbiome composition between normozoospermic and abnormal semen samples parameters. Alterations at the phylum, genus, and species levels were associated with impaired semen parameters, suggesting a potential link between microbial dysbiosis and male reproductive health. Furthermore, the absence of detectable fungal communities indicates that fungi may be rare or absent in human semen under physiological conditions. This study provides novel insights into the bacterial and fungal landscape of the seminal microbiome using long-read sequencing technology.