Objective: To assess the effect of cryoprotectant, frozen process and cryopreservation duration on DNA methylation status of paternal imprinted gene H19 and maternal imprinted gene MEST Imprinting Control Region (ICR) in human sperm.
Methods: The semen samples from 10 qualified donors were collected at Beijing Human Sperm Bank. The samples 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 ICR was determined by Bisulfite Sequencing PCR (BSP) method.
Results: H19 ICR DNA methylation degree analyzed by clone numbers was 58.70%, 53.85%, 49.46% and 45.74%, while analyzed by CpG island numbers, it was 97.57%, 97.33%, 97.13% and 96.56% in group A, B, C and D, respectively. The results from both analytical methods showed a decrease trend, but no significant difference was found among the four groups (P>0.05). MEST ICR DNA methylation degree analyzed by clone numbers in the four groups was 41.10%, 45.33%, 47.30% and 50.68%, while analyzed by CpG island numbers, it was 1.77%, 1.74%, 2.00% and 2.26% in group A, B, C and D, respectively. There was also no significant difference among the groups (P>0.05) despite an increasing trend.
Conclusions: The lack of significant difference in the DNA methylation degree of H19 and MEST ICR in our study indicates that DNA methylation of human sperm is not affected by cryopreservation and the cryopreservation technology.
