title: A novel in vitro sperm head decondensation protocol for rapid flow cytometric measurement of deoxyribonucleic acid content.
abstract: 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.
  
  
  METHODS
  Development of a new cytofluorimetric assay.
  
  
  METHODS
  University laboratory.
  
  
  METHODS
  Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Università Politecnica delle Marche.
  
  
  METHODS
  Setup of the novel in vitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content.
  
  
  METHODS
  Mean fluorescent channel, DNA content, percentage diploid sperm.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  This study establishes a simple method for in vitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.
authors.affiliation: Department of Life and Environmental Sciences, Università Politecnica delle Marche, Ancona, Italy.
authors.forename: Niki
authors.initials: N
authors.lastname: Antonucci
authors.forename: Sabrina
authors.initials: S
authors.lastname: Manes
authors.forename: Bruna
authors.initials: B
authors.lastname: Corradetti
authors.forename: Gian Carlo
authors.initials: GC
authors.lastname: Manicardi
authors.forename: Andrea
authors.initials: A
authors.lastname: Borini
authors.forename: Davide
authors.initials: D
authors.lastname: Bizzaro
mesh_terms: D000818:Animals; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000032:analysis*; D004171:Diploidy; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D011419:Propidium; D013077:Sperm Head / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011419:Propidium; D004247:DNA