Study On The Relationship Between Sperm Nuclear DNA Damage And Sperm Production

Objective 1.To establish the optimization conditions and effect analysis of two-tailed comet assay;2.Study on male infertility patients sperm concentration and sperm nuclear DNA single and double strand damage relationship,and to explore the little spermatozoon disease pathogenesisMethods 1.To explore each experimental condition in neutral and alkaline environment of the two phase electrophoresis of single cell gel electrophoresis to determine a more stable and accurate test method.2.Study of 120 cases of male infertility patients,aged between 25-40 years of age,according to the World Health Standard for routine examination of semen analysis of sperm concentration and motility using two tailed comet assay analysis of sperm DNA single and double strand damage index.Results 1 By optimizing the experimental conditions of the two-tailed comet assay,we selected as test specimens with double agarose gel layer number.Alkaline electrophoresis buffer pH adjusted to 10 for subsequent use.The sperm DNA unwinding time was adjusted to 20min,and the 20min of the medium and alkaline electrophoresis time were the best conditions for electrophoresis.2.120 cases of male infertile patients according to the sperm concentration is divided into three study groups,i.e.,the set of(sperm concentration "10×106ml-1),group 2(10 x 106ml-1" sperm concentration "20 x 106ml-1)and group 3(sperm concentration>*106ml-1),each group 40 cases.,DSB-DFI/DFI,DSB-DFI(.SSB-DFI 3DFI,DSB-DFI/DFI,DSB-DFI,SSB-DFI/DFI,SSB-DFI 2DFI,DSB-DFI/DFI,DSB-DFI,SSB-DFI/DFI,SSB-DFI DNADFI between the 3 groups in age,sperm motility and sperm deformity rate had no significant difference;group 1 sperm(59.48 + 15.21%)(25.86 +11.86%)(43.31 + 13.72)%(31.93 + 10.21)%(53.85 + 10.27)%,group(3.402 + 1.77%)(26.71+ 3.45%)(78.59 + 9.78)%(837 + 3.04%(2.14 + 9.79%);group(20.77 + 243)%(20.44 +239)%and(SSB-DFI/DFI 98.44 + 2.87%(132 + 0.61%(155 + 2.87%).SSB-DFI among the three groups no significant difference(P>0.05).SSB-DFI/DFI between group 1,group 2 and group 3 with the sperm concentration increased gradually increased(P<0.05).DSB-DFI with sperm concentration increased and decreased gradually(P<0.05).DSB-DFI/DFI with sperm concentration increased gradually decreased(P<0.05).There was no correlation(P>0.05)in the conventional sperm SSB DFI and SSB DFI/DFI and semen parameters,sperm DSB-DFI and DSB-DFI/DFI and sperm concentration of a negative correlation(P<0.05).Conclusion 1.After optimization of two-tailed comet assay,it becomed good reproducibility,high stability,and conducive to the standardization,it can be better to detect sperm DNAsingle and double strand damage,and has greater reference value in its assessment of male reproductive ability.2.the higher the level of DSB-DFI and DSB-DFI/DFI,the lower sperm concentration,the sperm DNA double chain injury may be the real reason for low sperm concentration.