Cell-type-specific determination of reactive oxygen species by flow cytometry

BackgroundSeminal leukocyte-generated reactive oxygen species may have a significant impact on sperm intracellular reactive oxygen species levels, therefore contributing to oxidative damage and consequent functional impairment of spermatozoa. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics. ObjectiveTo obtain seminal cell-specific, reactive oxygen species-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying reactive oxygen species overproduction (oxidative burst) from normozoospermic seminal samples. Material and methodsEjaculates gained by masturbation were obtained from patients in the framework of andrology consultations. The results published in this paper were generated from samples for which the attending physician requested spermatograms and seminal reactive oxygen species laboratory tests. Routine seminal analyses were performed according to World Health Organization guidelines. Samples were divided into normozoospermic non-inflamed, and leukocytospermic groups. The semen was stained by 2 ',7 '-dichlorodihydrofluorescein diacetate and the reactive oxygen species-related fluorescence signal and the percentage of reactive oxygen species-positive spermatozoa within the living population were quantified by flow cytometry. ResultsReactive oxygen species-related mean fluorescence intensity was higher in both spermatozoa and leukocytes from leukocytospermic samples than in those from normozoospermic samples. Mean fluorescence intensity in spermatozoa was positively and linearly correlated with mean fluorescence intensity measured in leukocytes in both groups. DiscussionThe capacity of spermatozoa to generate reactive oxygen species is at least three log lower than that of granulocytes. The question is whether the reactive oxygen species-producing machinery of spermatozoa is capable of causing autologous oxidative stress or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the reactive oxygen species production of leukocytes may have a significant impact on the overall reactive oxygen species levels measured in spermatozoa. ConclusionReactive oxygen species-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on reactive oxygen species mean fluorescence intensity measurement.

Automated summary

The study aims to provide a reliable method for diagnosing oxidative stress caused by male urogenital inflammation. Seminal cell-specific fluorescence intensity of oxidative stress can differentiate leukocytospermic samples with excessive reactive oxygen species from normozoospermic samples. Results showed that both spermatozoa and leukocytes in leukocytospermic samples had higher oxidative stress-related fluorescence intensity compared to normozoospermic samples. The study suggests that leukocyte-generated oxidative stress may be the main source of oxidative stress in semen.