A comparative investigation into the seminal microbiome of men with obesity has revealed distinctive bacterial profiles between infertile and fertile individuals, uncovering specific bacterial associations with sperm motility, total antioxidant capacity, and sperm DNA integrity. Led by researchers Elzem Nisa Alkan, Neslihan Hekim, Sezgin Gunes, Ramazan Asci, and Ralf Henkel, the study evaluated microbial diversity and composition using next-generation sequencing. The primary objective was to evaluate the microbial content in semen specimens obtained from men presenting with clinical obesity, clarify compositional differences separating fertile and infertile cohorts, and examine how specific seminal bacterial taxa correlate with standard sperm parameters, sperm chromatin condensation quality, sperm DNA fragmentation, and seminal plasma total antioxidant capacity.

Male infertility represents a multifaceted physiological condition shaped by both genetic background and environmental factors, occurring alongside documented worldwide reductions in seminal parameters. Lifestyle modifications, nutritional shifts, and environmental conditions have been recognized as meaningful contributors to this rising incidence. Nutrition significantly modulates the human microbiome, which governs vital immunological and metabolic pathways that frequently intersect with chronic inflammatory conditions such as obesity. Obesity itself has been clinically connected to impaired reproductive potential through endocrine disruptions, impaired spermatogenesis, elevated scrotal temperatures, decreased semen parameters, increased sperm DNA fragmentation, and erectile dysfunction. While historical medical dogma long maintained that the male urogenital tract was entirely sterile, advancements in high-throughput sequencing technologies have confirmed that semen harbors diverse, non-pathogenic bacterial communities even in healthy men, prompting researchers to examine whether seminal dysbiosis directly tracks with fertility status.

To investigate this biological intersection, the research team recruited eighteen male volunteers with obesity, defined by a body mass index exceeding thirty kilograms per square meter, who presented at the Urology Clinic of Ondokuz Mayis University Faculty of Medicine Application and Research Hospital in the Black Sea Region of Turkiye. The patient group comprised thirteen infertile men with obesity seeking in vitro fertilization treatment, where clinical evaluations verified that reproductive impairment was solely attributable to male factors while female partners demonstrated normal obstetric evaluations. Men presenting with established genetic causes of infertility, such as karyotype anomalies, Y-chromosome microdeletions, or cystic fibrosis, were systematically excluded. For the control group, five fertile men with obesity who had fathered children under two years of age and had a body mass index above thirty kilograms per square meter were enrolled as age-matched controls within a five-year margin.

Semen samples were collected via masturbation following a strict abstinence period of three to five days and liquefied at thirty-seven degrees Celsius for twenty to thirty minutes before undergoing manual semen analysis in accordance with World Health Organization guidelines. Fixed smears stained by the Papanicolaou technique were used to quantify sperm morphology and leukocyte counts. Each participant specimen was separated into defined aliquots: one fraction was allocated to assess sperm chromatin condensation via aniline blue staining, measure sperm DNA fragmentation using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and quantify total antioxidant capacity using an enzyme-linked immunosorbent assay. The second fraction underwent centrifugation at five hundred times gravity for seven minutes to separate the seminal plasma supernatant from the cellular pellet, after which bacterial genomic DNA was extracted from the seminal plasma and stored at minus eighty degrees Celsius.

High-throughput taxonomic profiling was executed using next-generation sequencing on an Illumina NovaSeq 6000 platform. The hypervariable V3 to V4 regions of the bacterial 16S ribosomal RNA gene were specifically amplified by polymerase chain reaction following DNA isolation, and sequence data were bioinformatically processed through the QIIME2 analytical platform. Normality of data distribution was established using the Shapiro-Wilk test, after which continuous variables were evaluated using independent samples t-tests or Mann-Whitney U tests depending on distribution patterns, and correlational relationships were quantified using Spearman rank correlation coefficients with significance set at a p-value below 0.05. Although eighteen individuals originally participated, sixteen provided sufficient microbial DNA concentrations for successful sequencing, while chromatin condensation and DNA fragmentation indexes were successfully obtained from fourteen participants, and antioxidant metrics were acquired for seventeen.

Conventional semen analysis revealed substantial baseline physiological divergences between the fertile and infertile cohorts despite identical body mass index and age brackets. Infertile participants demonstrated significantly lower sperm concentrations with a p-value of 0.043, reduced total sperm counts with a p-value of 0.032, lower progressively motile sperm counts with a p-value of 0.028, and a significantly lower percentage of normally formed sperm morphology with a p-value of 0.019. Conversely, the proportion of immotile sperm was significantly elevated in infertile men with a p-value of 0.012. Within functional analyses, aniline blue staining indicated that dark blue aniline-positive cells corresponded to abnormal histone retention and compromised condensation, whereas pale or unstained aniline-negative cells indicated mature protamine packaging. A strong negative correlation was observed between the percentage of aniline-positive sperm and sperm motility, sperm concentration, and total sperm count.

Taxonomic classification across all sequenced semen specimens identified four dominant bacterial phyla: Bacillota, Pseudomonadota, Actinomycetota, and Bacteroidota. At the phylum level, Bacillota and Pseudomonadota displayed comparable relative abundances between infertile and fertile cohorts, and while Actinomycetota appeared more pronounced in infertile patients and Bacteroidota showed higher representation among fertile controls, these broad phylum differences did not achieve statistical significance. Across all samples, 206 individual bacterial genera were identified. The most abundant genera across both groups consisted of Pseudescherichia, Staphylococcus, Paenibacillus, Streptococcus, Klebsiella, and Moraxella. In infertile patients, Pseudescherichia accounted for 16.6 percent, Staphylococcus 13.4 percent, Paenibacillus 9.3 percent, Streptococcus 6.7 percent, Moraxella 6.1 percent, and Klebsiella 5.4 percent of relative abundance, while controls exhibited 13.8 percent Pseudescherichia, 9.0 percent Staphylococcus, 6.7 percent Paenibacillus, 5.4 percent Faecalibacterium, 5.2 percent Streptococcus, 4.4 percent Moraxella, 4.2 percent Corynebacterium, and 4.0 percent Klebsiella.

Statistically significant differences emerged when comparing relative abundances at the genus level between the two cohorts. Seven genera showed significantly higher relative abundance in the infertile cohort: Acinetobacter with a p-value of 0.0009, Kaistella with a p-value of 0.0032, Delftia with a p-value of 0.0091, Stenotrophomonas with a p-value of 0.0109, Psychrobacillus with a p-value of 0.0140, Staphylococcus with a p-value of 0.0275, and Aerococcus with a p-value of 0.0380. Conversely, fertile controls possessed significantly greater abundances of Gemmobacter with a p-value of 0.012, Clostridium with a p-value of 0.0242, and Tahibacter with a p-value of 0.0381. Furthermore, host demographic parameters exhibited specific microbial relationships; Corynebacterium, Hydrogenophaga, and Paenalcaligenes were negatively correlated with body mass index, while Fructilactobacillus abundance demonstrated a positive correlation with participant age.

Correlation analyses between seminal microbiota and functional reproductive markers identified distinctive patterns across sperm parameters and molecular integrity indices. Brevundimonas displayed strong, highly significant positive associations with progressive motility, total sperm count, and sperm concentration, while Pseudomonas was positively correlated with progressive motility, and Lysinibacillus and Pantoea correlated positively with total sperm count. In contrast, Delftia, Bacillus, Gemmobacter, Komagataeibacter, and Massilia were correlated with the percentage of immotile spermatozoa. Regarding DNA integrity, sperm DNA fragmentation index correlated positively with Brevibacterium and Paenibacillus, but showed negative correlations with Alistipes, Lactiplantibacillus, Rhizobacter, Sphingomonas, and Veillonella. Total antioxidant capacity was positively associated with Devosia, Bacteroides, and Acidovorax, but negatively associated with Pantoea. Additionally, Fusobacterium abundance was negatively associated with the proportion of immature, histone-rich spermatozoa, indicating an association with favorable chromatin condensation.

The study authors contextualized these findings by acknowledging specific procedural and biological limitations inherent to the experimental model. The most prominent constraint was the modest cohort size of eighteen participants, driven by the practical difficulty of recruiting fertile men with obesity for voluntary semen donation and the financial expense of high-throughput sequencing. Additionally, the lack of non-obese fertile and infertile comparator groups prevented isolated attribution of these microbial patterns purely to obesity. The researchers also noted that 16S amplicon sequencing targeting hypervariable V3 to V4 regions cannot provide species-level or strain-level resolution, which would require shotgun metagenomic sequencing. Regional factors from the Black Sea Region cohort, alongside variable circumcised status, represent additional ecological influences. Nevertheless, the study demonstrates that distinct bacterial genera in semen associate with critical metrics of sperm quality, chromatin condensation, and oxidative balance in men with obesity.

Subject of Research: Seminal microbiome composition and sperm quality in men with obesity

Article Title: Evaluation of the semen microbiome for fertility in men with obesity using next-generation sequencing

Article References: Alkan, E. N., Hekim, N., Gunes, S., Asci, R., & Henkel, R. (2025). Evaluation of the semen microbiome for fertility in men with obesity using next-generation sequencing. Basic and Clinical Andrology, 35(1), Article 47. https://doi.org/10.1186/s12610-025-00294-x

Image Credits: AI Generated

DOI: 10.1186/s12610-025-00294-x

Keywords: semen microbiome, male infertility, obesity, next-generation sequencing, sperm DNA fragmentation, total antioxidant capacity, seminal microbiota, andrology, Evaluation, semen, microbiome, fertility