Determining the contribution of different cell types in semen to the global values of the intracellular oxidants H 2O 2 and peroxynitrite using flowcytometry

Abstract

OBJECTIVE: Sperm, leukocytes and round cells contribute to total oxidants production in semen. The Chemiluminescence assay provides a global value of reactive oxygen species in semen but provides no information on the contribution of different cell types. In this study we used flowcytometry to assess the global intracellular levels of the oxidants H2O2 and peroxynitrite in semen and the relative contribution of each cell type, simultaneously. DESIGN: Prospective study. MATERIALS AND METHODS: Semen samples from 19 fertility men and 120 men undergoing fertility assessment were collected. After liquefaction, semen was centrifuged at 300 g for 7 min. The peletted cells were resuspended in phosphate buffered saline to achieve sperm concentration of 1.25×10/mL. Aliquots were incubated with DCFH-DA and DHR12 dyes to detect intracellular H2O2 and peroxynitrite, respectively, using flowcytometry. Gated analysis determined the relative contribution of sperm, leukocytes and round cells. RESULTS: The median percentage contributions of leukocytes and round cells to the global H2O2 and peroxynitrite intracellular levels were different (Table 1). For each cell type, there was significant positive interdependence between the percentage contribution to the levels of H2O2 and of peroxynitrite.Table 1Data summary.Median (25th, 75th centiles) contribution to global levels (%)Semen cell typesH2O2PeroxynitriteSpearman's r (P)∗Spearman's rank correlation.Sperm36 (19. 61)39 (18, 60)r =0.72 (P < 0.0001)Leucocytes8 (2, 19)27 (14, 44)r =0.42 (P < 0.0001)Round cells50 (29, 65)25 (19, 37)r =0.52 (P < 0.0001)∗ Spearman's rank correlation. Open table in a new tab CONCLUSIONS: This study demonstrated that each cell type in semen contributed differently to the global intracellular levels of H2O2 and of peroxynitrite. However, in a semen sample, an increased production of H2O2 by one cell type was associated with a relative increase in its peroxynitrite production.