OBJECTIVE: To set up a novel protocol of sperm head in vitro decondensation that obviates the problematic effect of the variable degree of sperm chromatin packaging on DNA staining needed for flow cytometric analysis. DESIGN: Development of a new cytofluorimetric assay. SETTING: University laboratory. PATIENT(S): Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Università Politecnica delle Marche. INTERVENTION(S): Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content. MAIN OUTCOME MEASURE(S): Mean fluorescent channel, DNA content, percentage diploid sperm. RESULT(S): Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated C-value (approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier. CONCLUSION(S): This study establishes a simple method for in vitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.
OBJECTIVE: To set up a novel protocol of sperm head in vitro decondensation that obviates the problematic effect of the variable degree of sperm chromatin packaging on DNA staining needed for flow cytometric analysis. DESIGN: Development of a new cytofluorimetric assay. SETTING: University laboratory. PATIENT(S): Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Università Politecnica delle Marche. INTERVENTION(S): Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content. MAIN OUTCOME MEASURE(S): Mean fluorescent channel, DNA content, percentage diploid sperm. RESULT(S): Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated C-value (approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier. CONCLUSION(S): This study establishes a simple method for in vitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.