(187) T LYMPHOCYTES IN HUMAN SEMEN: INSIGHTS INTO IMMUNE TOLERANCE OF SPERMATOGENESIS

Abstract Introduction Emerging preclinical work has demonstrated the requisite role of the adaptive immune system in maintaining tolerance toward “foreign” spermatozoa. As seen in animal models, breaks in this system directly causes male factor infertility. Depletion of tolerogenic T lymphocytes induces severe autoimmune orchitis, immune cell infiltration, and reduced sperm number and motility. Objective There exists no investigation of the adaptive regulatory immune system in the context of human male fertility. We interrogated our seminal fluid cryobank which consists of 1700 patient samples to study this highly relevant question in male fertility. Methods We identified 15 semen samples with increased round cells on semen analysis. Patient samples were thawed, fixed and permeabilized, and stained with the nuclear fluorescent stain DAPI. This differentiates DAPI dim spermatozoa [haploid] from DAPI bright leukocytes [diploid]. Cells were then stained with fluorescently conjugated antibodies (CD45, CD3, CD4, CD25 and Foxp3) to identify leukocytes and specifically, tolerogenic CD4 T Regulatory Cells (CD4 Tregs). Multiparameter flow cytometric analysis was performed (BD LSRFortessa) and data analyzed using GraphPad Prism Software. Results CD45 positive leukocytes as identified by flow cytometry ranged in frequency from 0.003% to 2.93% +/- 0.88% and correlated positively with WBC counts on semen analyses. We were able to reliably identify Foxp3+ CD4 Tregs in semen samples. Increased frequencies of CD4 Tregs were observed in patients with higher seminal fluid WBC counts. Interestingly, CD4 Treg frequencies were inversely correlated with sperm density and motility [p < 0.05 linear regression]. Conclusions We describe the first report of tolerogenic T lymphocytes studied in human semen. We reliably identified and documented increased frequencies of CD4 Tregs in patients with lower sperm density and motility. CD4 Tregs expand in inflammatory conditions to quell ongoing autoimmunity. Overall, we hypothesize that inadequate control of autoreactive anti-sperm lymphocytes by tolerogenic CD4 Tregs leads to poor sperm quality. Further mechanistic investigation of this paradigm remains highly warranted. Disclosure No.