Simultaneous detection of sperm membrane integrity and DNA fragmentation by flow cytometry: A novel and rapid tool for sperm analysis

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 membraneintact 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. K E Y W O R D S acridine orange, DNA damage, flow cytometry, membrane integrity, sperm DNA fragmentation test 2 | 1 | INTRODUC TI ON Since the first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported in Wuhan, China, it has rapidly spread and affected more than 21 million people worldwide as of 17 August 2020. 1 SARS-CoV-2 uses angiotensin-converting enzyme II (ACE2) to enter host cells, similar to SARS-CoV, which emerged 18 years ago. 2COVID-19 induces respiratory-predominant multiorgan dysfunction, including myocardial, renal, enteric and hepatic dysfunction, which coincides with the tissue expression of ACE2. 3 Meanwhile, several studies have shown that ACE2 is expressed in human testes (eg spermatogonia, Leydig cells and Sertoli cells), 4,5 suggesting that the testes may be another organ affected by COVID-19.Numerous viruses have been detected in human semen. 6 Viruses may persist in semen and last longer in seminal fluid than in other body fluids due to the immune privilege of the testes and the contribution of the blood-testes barrier to resistance to therapeutic agents. 7,8 Semen may also have higher loads of viruses, such as Zika virus, th...

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Leads to Impaired Semen Parameters, Increased Sperm DNA Fragmentation and Unfavorable Changes of Sperm Protamine mRNA Ratio

“…The relation of DFI to other sperm parameters or factors such as sperm membrane integrity remains controversial. Several studies found negative correlations with conventional semen parameters, while others could not reproduce these associations [44][45][46]. In this study, we could demonstrate several significant correlations with other sperm parameters.…”

Sperm characteristics in normal and abnormal ejaculates are differently influenced by the length of ejaculatory abstinence

“…[37][38][39] Further, methods of preparation of spermatozoa for the assessment of DNA damage and the selection of different detection techniques can influence DNA integrity. 40 Hence, these factors, which are often neglected, need to be addressed while deciding the clinical value of sperm DNA integrity.…”

“…In general, it is important to note that suboptimal laboratory conditions, such as handling, prolonged incubation, and centrifugation, can influence sperm DNA integrity 37–39 . Further, methods of preparation of spermatozoa for the assessment of DNA damage and the selection of different detection techniques can influence DNA integrity 40 . Hence, these factors, which are often neglected, need to be addressed while deciding the clinical value of sperm DNA integrity.…”

Diagnostics and Management of Male Infertility in Primary Ciliary Dyskinesia

“…The sperm chromatin structure assay, used to detect the DNA fragmentation index (DFI), is a routine test to estimate sperm DNA damage. Recently, a simultaneous detection of sperm membrane integrity and DNA fragmentation assay was tested using flow cytometry and co-staining, consisting of acridine orange (AO) and LIVE/DEAD™ fixable blue dead cell stain [ 75 ], to estimate sperm quality prior to selection for ART. In the case of high DNA fragmentation, testicular sperm can be selected for ICSI.…”