title: [Optimization of sperm alkaline single-cell gel electrophoresis].
abstract: OBJECTIVE
  To investigate the main factors that influence the results of sperm alkaline single-cell gel electrophoresis (SCGE), optimize the conditions, and standardize its procedures.
  
  
  METHODS
  Using alkaline SCGE, we detected the DNA fragments of sperm treated with different concentrations of H2O2 and determined the influences of the number of agarose gel layers, pH during DNA unwinding and electrophoresis, the time of DNA unwinding and electrophoresis, and cumulative sperm number on the results of sperm alkaline SCGE. Then we optimized the procedures, analyzed the repeatability of the optimized method, and examined 40 semen samples using the method.
  
  
  RESULTS
  Three agarose gel layers could reduce the background. The optimal pH during DNA unwinding and electrophoresis was 10, and the best times for DNA unwinding and electrophoresis were 40 min and 30 min, respectively. Fifty sperm were adequate to ensure the reliability of the results. Based on the percentage of tail DNA, the intra- and inter-assay repeatabilities of the optimized sperm alkaline SCGE were 3.12% and 7.13%, and by the DNA damage score, they were 2.38% and 6.09%, respectively. Sperm DNA fragments were significantly increased in the infertile patients with oligoasthenoteratozoospermia as compared with healthy fertile males (P <0.05).
  
  
  CONCLUSIONS
  The optimized sperm alkaline SCGE, highly repeatable and easy to be standardized, can be applied to the clinical detection of sperm DNA fragmentation in infertile men.
authors: Shuang
authors: S
authors: Deng
authors: Lang
authors: L
authors: Fan
authors: Xi-yan
authors: XY
authors: Wu
authors: Yan
authors: Y
authors: Zhu
authors: Ke-qian
authors: KQ
authors: Xu
mesh_terms: D053627:Asthenozoospermia / Q000235:genetics*; D020552:Comet Assay / Q000592:standards*; D004249:DNA Damage; D053938:DNA Fragmentation; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D008297:Male; D009845:Oligospermia / Q000235:genetics*; D016877:Oxidants / Q000633:toxicity; D015203:Reproducibility of Results; D013076:Sperm Count; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D013997:Time Factors
chemical_list: D016877:Oxidants; D006861:Hydrogen Peroxide