Comparative analysis of the urethral and testicular microbiota in obstructive azoospermia patients with the results of assisted reproductive technologies

Introduction. The problem of male infertility remains a pressing issue in the field of reproductive medicine, as male infertility progresses year after year. The male reproductive microbiome is a complex community of microorganisms that inhabit various parts of the male body, including the urethra, prostate, and seminal vesicles. Research shows that the microbiome plays an important role in maintaining reproductive health and may influence fertility. Objective. To identify differences in the composition of the testicular and urethral microbiota between groups of patients with positive and negative results of assisted reproductive technologies (ART). Materials and methods. A comparative analysis of the taxonomic structure of the testicular and urethral microbiota was conducted in 24 infertile men with obstructive azoospermia (OA) who underwent ART procedures. Pregnancy occurred in 13 cases (ART+ group), while pregnancy was not achieved in 11 cases (ARTgroup). To analyze the bacterial composition of the urogenital tract, biological material was collected from the urethra of all study participants, as well as a testicular biopsy. Analysis of bacterial 16S rRNA gene amplicons was performed using high-throughput next-generation sequencing (NGS). The obtained data were processed using QIIME software (version 1.9.1), which allowed for taxonomic analysis and assessment of microbiota diversity. Results. Our study revealed significant differences in the microbiota composition between testicular and urethral tissue in infertile men with OA, as well as between groups of patients with positive and negative ART results. In testicular tissue, the main microbiota families were Enterobacteriaceae and Burkholderiaceae, while in the urethra, the dominant families were Mycobacteriaceae, Staphylococcaceae, Lactobacillaceae, and Peptoniphilaceae. Despite differences in microbiota composition, no significant differences in alpha diversity indices of testicular and urethral microbiota were found between the ARTand ART+ groups. However, in the ARTgroup, a significantly increased relative representation of 19 bacterial families was observed in the testicular microbiota compared to patients in the ART+ group, including increased representation of two families: Mycobacteriales and Legionellaceae. This may indicate a possible link between microbiota composition and ART success. Correlation analysis revealed a positive association between inhibin levels and testicular microbiota composition. This suggests that testicular microbiota may influence inhibin levels, which are an important marker of testicular function and may play a role in fertility. Conclusions. The results demonstrate that OA-associated disorders lead to changes in the relative representation of individual families of the urethral and testicular microbiota, which has a significant impact on ART outcomes. The practical significance of these data is that microbiota correction may be a potential strategy for improving ART outcomes in patients with OA, opening up new approaches to treatment and enhancing fertility, and suggests the theory of «testicular fertility microbiota» and may subsequently play an important role in pre-conception cryopreservation in ART programs and the creation of new tests for sperm selection in artificial insemination programs.