The Study Of Cryopreservation On Sperm DNA Methylation Status Of Imprinted Genes And Structures

Objective:1. Assess the effects of cryoprotectant, frozen process and cryopreservation duration on sperm DNA methylation status of paternal imprinted gene H19 and maternal imprinted gene MEST Imprinting Control Region (ICR); 2. Explore the effects of routine cryopreservation technology in sperm bank on the integrity of DNA, membrane and acrosome; 3. Detect the effects of traditional Chinese medicine"Xue Yu Sheng Bao" on sperm in vitro.Methods:1.Semen samples from 10 qualified donors were collected at Beijing Human Sperm Bank. They were divided into four equal aliquots:A) untreated, B) diluted in cryoprotectant, C) diluted in cryoprotectant and cryopreserved for 2 days and D) diluted in cryoprotectant and cryopreserved for 2 months. The DNA methylation degree of H19 and MEST was determined by Bisulfite Sequencing PCR (BSP) method; 2.Semen samples from 10 preliminary qualified donors were collected at Beijing Human Sperm Bank. The control group:fresh sperm, the experiment group:diluted in cryopreservation and cryopreserved for 7 days. The integrity of sperm DNA, membrane and acrosome was determined by SCD,6-CFDA/PI double fluorescence staining method and PSA fluorescence staining methodrespectively; 3. Semen samples from 10 preliminary qualified donors were collected at Beijing Human Sperm Bank.The control group:the mixture of 0.5ml sperm and 0.5ml normal saline, the experiment group:the mixture of 0.5ml sperm and 0.5ml liquid medicine. The sperm activity, VAP, VCL and VSL in Oh, 0.5h,3h and 24h were measured by CASA. Acrosome integrity was determined in 0.5h by PSA fluorescence staining method. The concentration of 8-OHdG was measured in 3h by ELISA. The menbrance integrity was analized in 24h by eosin-aniline black method.Results:1. H19 ICR DNA methylation degree in group A, B, C and D was 58.70%、 53.85%、49.46% and 45.74% respectively when analyzed by clone numbers, and it was 91.51%,97.33%.97.13% and 96.56% respectively when analyzed by CpG island numbers. The results from both analytical methods showed a decrease trend although significant difference was not found among the groups(P> 0.05). MEST ICR DNA methylation degree in the four groups was 41.10%、45.33%、47.30% and 50.68% respectively when analyzed by clone numbers, and it was 1.77%、1.74%、2.00% and 2.26% when analyzed by CpG island numbers. There was also no significant difference among the groups(P> 0.05) despite the increasing trend;2. DNA integrity in the control group and the experiment group were(80.5±5.4)%and (74.3±4.6)%respectively. There were significant differences between the two groups (P< 0.05). Menbrance integrity in the two groups were (75±5.82)% and (68±6.23)%, and acrosome integrity were (82±5.71)% and (77±6.13)% respectively. There were also significant differences between the two groups (P< 0.05); 3.There were significant differences between the two groups in the aspect of CAS A parameters, acrosome integrity,8-OHdG concentrationand menbrance integrity (P< 0.05).Conclusion:1.The lack of significant difference in the DNA methylation degree of H19 and MEST in our study indicates that sperm DNA methylation is unaffected by cryopreservation and this cryopreservation technology in human sperm bank is safe in terms of DNA methylation; 2. Routine cryopreservation technology in sperm bank has negative effects in the integrity of DNA, membrane and acrosome; 3. The traditional Chinese medicine"Xue Yu Sheng Bao"has no significant protection on sperm in vitro.