Study On The Relationship Between Sperm DNA Single,Double Strand Damage And Male Infertility Induced By Mycoplasma And Chlamydia

Objective 1.To establish the optimization conditions and effect analysis of two-tailed comet assay;2.To study sperm DNA single and double strand damage on mycoplasma and chlamydia infection in male urogenital tract of male infertility patients;3.To analysis the relationship between sperm DNA single,double strand damage and male infertility of mycoplasma and chlamydia infection.Methods 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.2.30 patients with mycoplasma and chlamydia infection in urogenital tract and 30 normal males were collected randomized.We analysis the routine semen parameters with Weili color sperm quality detection system,and the sperm DNA fragmentation index was detected by using two-tailed comet assay,analyzed the DNA single and double strand damage in male infertility patients who infects mycoplasma and chlamydia and the difference with normal men.3.Choose the sensitive antibiotics according to the drug sensitivity results.After 2 months,We analysis the routine semen parameters with Weili color sperm quality detection system,and detect sperm DNA integrity by two-tailed comet assay.Then compare the results between pre-and post-treatment,to investigate the effect of infection on sperm DNA single and double strand damage.4.To statistical analyse the sperm DNA damage of mycoplasma and chlamydia infection in urogenital tract of male infertility and normal fertile men,and to investigate the relationship between sperm DNA single and double strand damage and male infertility.Results I.By optimizing the experimental conditions of the two-tailed comet assay,we selected 1%low melting point agarose gel as the experimental gel concentration.In the experiment,we chose to adjust the sperm density to 5×106/ml in order to prepare for the follow-up experiment.The DTT concentration of 0.8mol/L was chosen as the cell lysis solution,and 20 minutes was used as the best condition for neutral and alkaline comet assay at the same time.2.The semen volume of mycoplasma and chlamydia infection in urogenital tract of male infertility(patient group)and normal fertile men(control group)was at the normal range,there was no significant difference.The sperm density and motility of male infertility patients who infected mycoplasma and chlamydia in urogenital tract were lower than the control group,and the rate of sperm deformity was higher than the control group,the difference was statistically significant.The sperm DNA DSB-DFI of infertile male who infected mycoplasma and chlamydia in urogenital tract was significantly higher than the normal control group,but SSB-DFI in the two groups was no significant difference.3.Compared with the normal control group,the severe SSB infection was significantly increased in mycoplasma and chlamydia infection in urogenital tract of male infertility patients(P<0.05),and severe DSB was significantly higher(P<0.01)than that in normal control group.And mild DSB,SSB accounted for no statistically significant difference(P>0.05).4.After anti infection treatment,We compared mycoplasma and chlamydia infection in urogenital tract of male infertility pre-with post-treatment in semen parameters and sperm DNA integrity.After treatment,normal morphology sperm percentage of infertility group with mycoplasma,chlamydia infection was significantly improved(P<0.01),and the sperm concentration,sperm survival rate,the percentage of sperm motility were not obvious.After treatment,the DSB-DFI,DFI and DSB-DFI/DFI of the sterile group had a certain improvement rate(P<0.01),while SSB-DFI/DFI and SSB-DFI showed no significant change.5.Compared with pre-treatment,the severe SSB infection was significantly reduced in mycoplasma and chlamydia infection in urogenital tract of male infertility patients(P<0.05),and severe DSB was significantly lower(P<0.01)than that in normal control group.And mild DSB,SSB accounted for no statistically significant difference(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 DNA single and double strand damage,and has greater reference value in its assessment of male reproductive ability.2.The sperm DNA double strand damage of male infertility patients who infected mycoplasma and chlamydia in urogenital tract was significantly higher than that of healthy fertile men.The appropriate antibiotic therapy can reduce sperm DNA damage which caused by infection,and the sperm DNA integrity analysis in the diagnosis and treatment of infertile men who infected mycoplasma and chlamydia was a great significance.