O-305 Mapping the entire functionally active seminal microbiota and its association with semen quality parameters
Authors: Salas-Espejo E , Terrón-Camero L  C , Molina N  M , Sola-Leyva A , Pérez-Prieto I , Vargas E , Canha-Gouveia A , Folch B  A , Molina Romero M , Rodríguez Guirado S , Castilla-Alcala J  A , Andrés-León E , Altmäe S 
Published: July 2024

Abstract

Study question
Does semen harbour functionally active microorganisms, and whether the microbial composition associate with semen quality parameters?

Summary answer
Seminal fluid harbours functionally alive microorganisms including bacteria, viruses, archaea, viroids and fungi whose composition and metabolic functions associate with semen quality parameters.

What is known already
Seminal fluid has been shown to possess different microbes, where different bacteria have been associated with spermatogenesis, seminal parameters and infertility. Nevertheless, previous results are inconclusive and the core microorganismal composition in semen is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Further, there is limited knowledge of the potential host-microbe interactions.

Study design, size, duration
In total 78 men with age between 18 and 45 years donated semen for the study, 59 men with good quality semen parameters and 19 men with low semen quality and diagnosed asthenospermia, oligoasthenozoospermia or oligoasthenoteratozoospermia comprised control and case groups, respectively.

Participants/materials, setting, methods
Semen quality parameters were analysed following WHO, 2021 guidelines. Seminal fluid underwent NextSeq total RNA sequencing, separating human and non-human sequences with Hisat2 aligner. Taxonomic classification of microorganisms was obtained using Kraken2. Microbial species and human RNA-Seq analyses, comparing control and infertility groups, utilized metagenomeSeq and edgeR R packages. Human and microbial pathways were annotated using MetaCyc, followed by GSEA analysis. The integration of metabolic pathways between host and microbes was investigated.

Main results and the role of chance
With our total RNAseq analysis, we mapped the entire alive microbiota composing of 6453 microorganisms within the seminal fluid samples. Microbes such as bacteria, fungi, viruses, viroids and archea were identified. When analysing microbiota associations with seminal parameters, 404 microbial species (Lactobacillus, Gardnerella, Prevotella, Atopobium, Pseudomonas among others) were significantly differently present among infertile men vs. good quality semen group. The host transcriptomic analysis identified 2569 differentially expressed genes in infertile vs. good quality semen groups, involved in sperm differentiation, spermatid development, reproduction, cilium movement and meiosis. Metabolic pathway analysis detected possible metabolic activity in the host-microbiota crosstalk in FAO-PWY: fatty acid & beta-oxidation, LIPASYN-PWY: phospholipases, PWY66-429: fatty acid biosynthesis, PWY-3781: aerobic respiration I pathways.

Limitations, reasons for caution
Bigger sample size of infertile men would be needed to validate the study findings.

Wider implications of the findings
We confirm the presence of active microbes in semen with implications in seminal quality parameters such as motility, sperm count and morphology. Our results contribute to better understanding of seminal microbiota dysregulation in infertility with broader implications for reproductive health, suggesting functional links between seminal microbiota and sperm parameters.

Trial registration number
NA