This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

Raman Microspectroscopy represents an innovative tool for the assessment of sperm biochemical features otherwise undetectable by routine semen analysis. Previously, it was shown that induced DNA damage can be detected in smeared sperm by this technique. This novel readout may be of value for clinical settings especially if it can be transferred to living cells. Yet, starting with living sperms this study was carried-out using a variety of conditions to disclose the Raman features of sperm nuclei under different hydration conditions and UV exposure. Human sperm were immobilized and Raman spectra were obtained from individual sperm as repeated measurements. To create conditions with controlled DNA damage, sperm samples were exposed to ultraviolet light. Several media were used to evaluate their effect on Raman spectra in aqueous conditions. To substantiate differences between the experimental conditions, the spectra were analyzed by Principal Component Analysis. We observed that spectra of sperm nuclei obtained in different solutions showed a qualitatively unchanged spectral pattern showing the principal signals related to DNA. Evaluating the effect of ultraviolet light generated the finding that spectra representing DNA damage were only observed in dry conditions but not in aqueous medium. Thus, Raman microspectroscopy was successfully applied for sperm analysis in different conditions, among them in live spermatozoa in aqueous solution during the initial measurement, revealing the principle use of this technique. However, implementation of Raman spectroscopy as a technique for clinical sperm analysis and selection may be especially relevant when DNA evaluation can be established using live sperm.

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...