(186) THE USE OF THE FLUORESCENT NUCLEAR STAIN DAPI TO IDENTIFY SEMINAL LEUKOCYTES

Abstract Introduction Emerging animal-based research highlights the requisite role of the immune system in maintaining tolerance to spermatozoa. Critically, however, there exists a paucity of studies investigating the role of the immune system in clinical male factor infertility. Current protocols investigating seminal fluid resident white blood cells (leukocytes) include peroxidase staining; however, further investigation of these cells is rarely performed due to the extremely low frequency of these cells. The AUA and ASRM have no guidelines regarding the workup or treatment of leukospermia. Objective We sought to develop a streamlined and simple assay to identify and better characterize rare leukocytes within spermatozoa-rich semen samples. Methods We identified 15 patients with “Too Numerous to Count” round cells from our seminal fluid cryobank. These semen samples were thawed and exposed to a formalin/saponin based buffer to fix and permeabilize cells. Cells were then stained with the fluorescent nuclear stain DAPI and fluorescently conjugated antibodies directed against CD45. Sample acquisition was performed on a BD LSRFortessa Flow Cytometer and data analyzed using FlowJo software. Results DAPI nuclear staining clearly separates DAPI dim spermatozoa (haploid cells) from DAPI bright leukocytes (diploid cells). Subsequent dual-channel CD45 antibody staining confirms the absence of CD45 on haploid spermatozoa (98% negative) and positivity of CD45 on diploid leukocytes (87.2% positive). Appropriate DAPI signal intensity of the leukocytes within the semen samples was confirmed by spiking semen samples with peripheral blood mononuclear cells. Overall, CD45 positive leukocytes represented 0.003% to 2.93% +/- 0.88% of the leukocytes found in the seminal plasma of infertile patients. Conclusions 50% of male factor infertility remains undefined. Preclinical evidence reveals a required role of the immune system in maintaining immunologic tolerance to “foreign” spermatozoa. Breaks in this system may perpetuate male factor infertility but clinical investigation of this paradigm remains to be pursued. Disclosure No.