IMMUNOLOGICAL CHARACTERIZATION OF SEVERE INFERTILE MEN REVEALED ALTERATIONS OF THE LYMPHOID AND MYELOID COMPARTMENTS

Background: Infertility is the inability of a sexually active, non-contraceptive couple to achieve spontaneous pregnancy in one year. It is estimated to affect worldwide up to 15% of couples; overall, a male infertility factor has been found in at least 40% of infertile couples. Several studies have shown that infertile men have a higher incidence and prevalence of cancer, chronic non-malignant morbidities, endocrinological and autoimmune disorders, and overall a higher mortality risk compared to fertile age-matched men. Various hypotheses have been considered to explain the factors underlying male health status and male factor infertility, including genetic, endocrinological, and metabolic abnormalities. Recent evidence has shown that the immune testicular cells are different among infertile and fertile individuals. Testicular tissue of infertile men showed a pro-inflammatory phenotype which was not found in fertile controls. Moreover, a senescent phenotype was observed in azoospermic somatic cells of infertile men but not in those from the control group. The previously reported findings in infertile men documented a shift from the immune-privileged status to a proinflammatory testicular environment, eventually associated with iNOA. From a translational point of view, the pro-inflammatory status of testicular cells and the senescent phenotype characterized by decreased T cells number and higher level of inflammatory cytokines observed in infertile men could be associated with the infertility status per se, an exhausted pattern of circulating immune cells and it could predispose to future risk of developing malignancies and chronic diseases. However, a detailed characterization of the local (e.g. testicular) and systemic immune status of infertile and fertile men has never been investigated. Our main hypothesis that infertile men have alterations/defect of the immune system has been pursued with the following specific aims: AIM 1. To assess lymphoid and myeloid cell distribution in seminal fluid and peripheral blood of fertile and infertile men. AIM 2. To evaluate the presence of regulatory cells in seminal fluid and peripheral blood. AIM 3. To evaluate the presence of potential biomarkers of an inflammatory/exhausted immune status of infertile men. Methods. We enrolled 38 infertile men with a diagnosis of oligo-astheno-teratozoospermia (OAT), 13 infertile men with idiopathic non-obstructive azoospermia (iNOA) and 40 age-matched fertile controls that provided comprehensive medical data, peripheral blood (PB) and seminal fluid (SF) samples. Clinical data was collected. We defined the leukocyte composition and the presence of different subsets of myeloid cells in the peripheral blood and seminal fluid by multiparameter flow cytometry. The levels of several cytokines and chemokines were measured in serum and seminal plasma by multi-beads array, and T cell responsiveness was performed on peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation. Results. Groups were comparable in terms of age, testosterone level and metabolic parameters. As expected, testicular volume and FSH values were worse in infertile men. Seminal plasma Myeloid cells represented the major leukocyte subsets in the seminal fluid. OAT and iNOA men had higher neutrophil cells compared to fertile controls. A significant higher rate of pro-inflammatory cDC2 was observed in OAT participants compared to fertile controls. Of the cytokines analyzed, we observed an overall upregulation of myeloid-derived pro-inflammatory cytokines and of IL-10 in the seminal fluid of infertile compared to fertile men. On the contrary, chemokines involved in leukocyte recruitment, such as MCP-1, MIP1b, RANTES, Eotaxin, and IL-8, were detected at lower or comparable concentration in samples from infertile men compared to fertile ones. Peripheral blood The analysis of leucocyte subsets in the peripheral blood revealed a higher frequency of neutrophils in infertile men compared to fertile controls reaching statistical significance for OAT. Of note, CD4+ and CD8+ cells were lower in OAT compared to fertile controls. Of note, the analysis of the pro-inflammatory DC subset revealed that the rate of cDC1 was higher in OAT men as compared to fertile controls while the proportion of cDC2 was comparable between infertile and fertile men. CD4+ and CD8+ cells of infertile men showed a higher expression of markers of exhaustion compared to fertile controls. Conclusion. Although still preliminary, this study highlights novel evidence that male infertility is associated with a deregulation of the immune system without defect of the regulatory compartment (DC-10 and Tr1 cells) and with a pro-inflammatory signature that leads to the expansion of exhaustion T cell compartment. Moreover, the reduced levels of CD4+ and CD8+ T cells, along with the higher pro-inflammatory and exhausted signatures seem to depict a senescent phenotype for infertile men. These data could help the understanding of the pathophysiological mechanism underlying male factor infertility and the possible link behind the lower overall health status of infertile men compared to fertile individuals.