Citation: Veneruso, I.; Cariati, F.; Alviggi, C.; Pastore, L.; Tomaiuolo, R.; D’Argenio, V. Metagenomics Reveals Specific Microbial Features in Males with Semen Alterations. Genes 2023, 14, 1228. https://doi.org/ 10.3390/genes14061228 Academic Editor: Jose María Requena Received: 2 May 2023 Revised: 1 June 2023 Accepted: 4 June 2023 Published: 6 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article Metagenomics Reveals Specific Microbial Features in Males with Semen Alterations Iolanda Veneruso 1,2 , Federica Cariati 3 , Carlo Alviggi 3 , Lucio Pastore 1,2 , Rossella Tomaiuolo 4 and Valeria D’Argenio 2,5, * 1 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Via Sergio Pansini 5, 80131 Napoli, Italy 2 CEINGE-Biotecnologie Avanzate Franco Salvatore, Via G. Salvatore 486, 80145 Napoli, Italy 3 Department of Public Health, Federico II University, Via Sergio Pansini 5, 80131 Napoli, Italy 4 Faculty of Medicine, Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy 5 Department of Human Sciences and Quality of Life Promotion, San Raffaele Open University, Via di Val Cannuta 247, 00166 Roma, Italy * Correspondence: dargenio@ceinge.unina.it Abstract: Infertility incidence is rising worldwide, with male infertility accounting for about 50% of cases. To date, several factors have been associated with male infertility; in particular, it has been suggested that semen microbiota may play a role. Here, we report the NGS-based analyses of 20 semen samples collected from men with (Case) and without (Control) semen alterations. Genomic DNA was extracted from each collected sample, and a specific PCR was carried out to amplify the V4-V6 regions of the 16S rRNA. Sequence reactions were carried out on the MiSeq and analyzed by specific bioinformatic tools. We found a reduced richness and evenness in the Case versus the Control group. Moreover, specific genera, the Mannheimia, the Escherichia_Shigella, and the Varibaculum, were significantly increased in the Case compared to the Control group. Finally, we highlighted a correlation between the microbial profile and semen hyperviscosity. Even if further studies are required on larger groups of subjects to confirm these findings and explore mechanistic hypotheses, our results confirm the correlation between semen features and seminal microbiota. These data, in turn, may open the way to the possible use of semen microbiota as an attractive target for developing novel strategies for infertility management. Keywords: infertility; male infertility; semen microbiota; metagenomics 1. Introduction Infertility incidence is progressively increasing worldwide, and it is estimated that about 50% of cases are due to male infertility [1]. Male infertility represents a highly hetero- geneous condition that may be related to pre-testicular (i.e., alterations of the hypothalamic– pituitary axis), testicular, and post-testicular (i.e., urogenital obstructions, vasectomy, and accessory glands impairment) diseases [2]. Moreover, genetic, environmental, and micro- biological factors have also been related to this condition [3–5]. The correct identification of the possible cause of male infertility, together with the female partner assessment, is crucial for proper couples’ evaluation and ensuring the best strategy to improve couples’ reproductive outcomes [6]. The semen analysis represents a routine step in assessing male infertility since it provides considerable information regarding macroscopic and micro- scopic features that can highlight underlying diseases [7,8]. Moreover, we have recently reported that semen sample parameters’ alterations correlate with urogenital infections and sperm DNA fragmentation, suggesting that an in-depth semen evaluation may improve male infertility management [9]. In recent years, metagenomics has been widely used to characterize the taxa and gene content of the human microbiome. In particular, microbiome analyses allow for estimat- ing the taxonomic and functional composition of the different microorganisms present in Genes 2023, 14, 1228. https://doi.org/10.3390/genes14061228 https://www.mdpi.com/journal/genes