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

• Published in: Andrology (Oxford) 2023-11, Vol.11 (8), p.1558-1565
• Main Authors: Hajnal, Agnes, Bogdandi, Noemi, Marokházi, Judit, Guba, Dorina, Mikus, Endre G
• Format: Article
• Language: English
• Subjects: Flow cytometry; Leukocytes; Oxidative stress; Reactive oxygen species
• ISSN: 2047-2919; 2047-2927
• DOI: 10.1111/andr.13473

Seminal leukocyte-generated reactive oxygen species (ROS) may have a significant impact on sperm's intracellular ROS level, therefore contributing to oxidative damage and consequent functional impairment of sperm. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics. To obtain seminal cell-specific, ROS-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying ROS overproduction (oxidative burst) from normozoospermic seminal samples. Ejaculates 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 ROS laboratory tests. Routine seminal analyses were performed according to World Health Organization (WHO) guidelines. Samples were divided into normozoospermic "non-inflamed", and leukocytospermic groups. The semen was stained by 2',7'-Dichlorodihydrofluorescein diacetate and the ROS-related fluorescence signal and the percentage of ROS-positive spermatozoa within the living population was quantified by flow cytometry. ROS-related mean fluorescence intensity (MFI) was higher in both spermatozoa and leukocytes from leukocytospermic samples than from normozoospermic samples. MFI in sperm positively and linearly correlated with MFI measured in leukocytes in both groups. The capacity of sperm to generate ROS is at least three log lower than that of granulocytes. The question is whether the ROS producing machinery of sperm is capable of causing autologous oxidative stress, or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the ROS production of leukocytes may have a significant impact on the overall ROS levels measured in sperm. ROS-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on ROS MFI measurement. (269 words) This article is protected by copyright. All rights reserved.