Round cells do not contaminate or mask human sperm proteome in proteomic studies using cryopreserved samples

Semen contains leucocytes and round cells, besides spermatozoa. The objective of this study was to identify whether the proteins from round cells and leucocytes affect the proteomic analysis of spermatozoa. Cryopreserved human sperm samples were divided into four groups: (1) samples with ≥1 × 10⁶/ml leucocytes unprocessed; (2) samples with ≥1 × 10⁶/ml leucocytes processed by 65% density centrifugation; (3) samples with round cells <1 × 10⁶/ml unprocessed; and (4) samples with round cells <1 × 10⁶/ml processed by 65% density centrifugation. Samples from each group (1, 2, 3 and 4) were pooled (n = 5) for quantitative proteomic analysis using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Comparative analysis revealed nine differentially expressed proteins (DEPs) groups 1 and 2. Moreover, five DEPs were identified between groups 3 and 4. We observed that cylicin‐1, Atlastin‐1 and vesicle transport protein SFT2B are specific to spermatozoa, and none of them were associated with leucocytes. The number of DEPs in spermatozoa of processed and unprocessed cryopreserved semen samples was negligible. Our results indicate that the presence of round cells (<1 × 10⁶/ml) in the seminal ejaculation does not interfere in the accurate detection of spermatozoa proteome by LC‐MS/MS.