MP38-01 THE EFFECT OF CRYOPRESERVATION ON DNA METHYLATION PATTERNS OF THE CHROMOSOME 15Q11-Q13 REGION IN HUMAN SPERMATOZOA

Abstract

INTRODUCTION AND OBJECTIVE: Human sperm cryopreservation is a common technique which is widely used in assisted reproductive technologies (ARTs). Despite the existence of evidence supporting the production of reactive oxygen species (ROS) and DNA fragmentation during sperm cryopreservation, there is little and equivocal information about the effect of cryopreservation on methylation patterns of imprinted genes and imprinting control regions. In this study, we have investigated the effects of cryopreservation on DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11-q13 region. METHODS: Semen samples from 10 healthy normozoospermic men were collected and each sample was divided into four equal aliquots: fresh, cryoprotectant (diluted in cryoprotectant), cryopreservation (diluted in cryoprotectant and cryopreserved for 1 month), and H2O2 (treated with 4mM H2O2 after cryopreservation). We measured the intracellular ROS levels and DNA fragmentation using DCFH-DA andTUNEL assay respectively by flow cytometry. DNA methylation levels were evaluated by quantitative Methylation-Specific PCR technique. RESULTS: Intracellular levels of ROS (presented as the DCF mean fluorescence intensity) and the percentage of TUNEL-positive spermatozoa significantly increased in cryopreservation group compared to fresh group (P = 0.015 and P = 0.013 respectively). Exposure to cryoprotectant had no significant effect on intracellular ROS levels and DNA fragmentation. Neither cryopreservation nor exposure to cryoprotectant significantly affected DNA methylation of the selected gene regions. However, DNA fragmentation had positive correlation with DNA methylation of AS-ICR. CONCLUSIONS: Although cryopreservation of spermatozoa can lead to ROS production and DNA fragmentation, but this technique had no significant detrimental effect on DNA methylation status of the selected gene regions. The clinical use of sperm cryopreservation for fertility treatments appear to be safe in regard to DNA methylation. Source of Funding: The authors would like to thank the Tehran University of Medical Sciences for the financial support (grant No: 32185).