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Simultaneous detection of sperm membrane integrity and DNA fragmentation by flow cytometry: A novel and rapid tool for sperm analysis
Author(s) -
Da Costa Raul,
Redmann Klaus,
Schlatt Stefan
Publication year - 2021
Publication title -
andrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.947
H-Index - 43
eISSN - 2047-2927
pISSN - 2047-2919
DOI - 10.1111/andr.13017
Subject(s) - dna fragmentation , sperm , acridine orange , propidium iodide , biology , andrology , staining , microbiology and biotechnology , semen , dna , fragmentation (computing) , stain , genetics , apoptosis , programmed cell death , medicine , ecology
Abstract Background Sperm DNA integrity has become one of the most discussed and promising biomarkers for the assessment of male fertility. However, an easy‐to‐apply method capable of estimating DNA fragmentation in the live fraction of spermatozoa has remained elusive, preventing this parameter from being fully applied in clinical settings. Objectives To validate a novel co‐staining for the analysis of DNA fragmentation in membrane‐intact spermatozoa. Materials and methods Normozoospermic semen samples were used to validate the co‐staining consisting of acridine orange (AO) and LIVE/DEAD™ Fixable Blue Dead Cell Stain (LD), against established methods for the evaluation of cell viability, propidium iodide stain (PI), and DNA fragmentation, the sperm chromatin structure assay (SCSA), to rule out cross‐interference. Furthermore, the accuracy of the method was tested by the evaluation of samples prepared with different amounts of membrane and DNA damage (20, 40, 60, 80, and 100%). Results No significant differences were observed between the co‐staining and the established staining procedures (membrane integrity, p = 0.755; DNA fragmentation p = 0.976). Moreover, high R square values were obtained from the analysis of samples of known membrane (R 2 = 0.9959) and DNA damage (R 2 = 0.9843). The simultaneous assaying of sperm membrane integrity and nuclear DNA fragmentation allowed the analysis of four sperm categories and thereby to assess the proportion of membrane‐intact spermatozoa with compromised DNA integrity. Discussion and Conclusion This new protocol has the potential to provide clinically relevant information about the DNA fragmentation in membrane‐intact spermatozoa. Thus, it has the potential of improving the diagnostic of male infertility and enabling a better understanding of sperm dysfunction.