Publications by authors named "Raul Da Costa"

2 Publications

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

Andrology 2021 Apr 8. Epub 2021 Apr 8.

Centre for Reproductive Medicine and Andrology, University of Münster, Münster, Germany.

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  = 0.9959) and DNA damage (R  = 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.
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April 2021

Spectral features of nuclear DNA in human sperm assessed by Raman Microspectroscopy: Effects of UV-irradiation and hydration.

PLoS One 2018 20;13(11):e0207786. Epub 2018 Nov 20.

Centre for Reproductive Medicine and Andrology, University of Münster, Münster, Germany.

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.
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April 2019