Abstract: Objective　To establish twotailed comet assay methodology for sperm DNA integrity detection and analyze the type of sperm DNA damage.   Methods　The neutral and alkaline twophase single cell gel electrophoresis was used to detect sperm DNA damage.  Results　After the twotailed comet assay, 9 kinds of comet forms were obtained, which representing the 9 kinds of sperm DNA damage types. H   2O   2 and AluI restriction endonucleases were used to induce singlestrand DNA and doublestrand DNA productions. With the increasing concentration of H   2O   2, the singlestrand DNA breaks gradually increased. By the microscopy, the number of comet tail in Y axis gradually increased, and the difference was statistically significant(P<0.05). With AluI digestion time extending, the doublestrand DNA breaks increased. By the microscopy, the number of comet tail in X axis increased, and the difference was statistically significant (P<0.05). In addition, the singlestrand break DNA fragmentation indices (SSBDFI) in male infertility group and normal fertility group had no significant difference. The doublestrand break DNA fragmentation index (DSBDFI) in male infertility group was significantly higher than that in normal fertility group (P<0.05).  Conclusions　Twotailed comet assay could distinguish sperm DNA damage which is singlestrand break or doublestrand break, and it maybe provide a deeper and stronger laboratory evidence for male fertility.

Key words: Twotailed comet assay, Singlestrand break, Doublestrand break, Male infertility