Human sperm cryopreservation is a common technique which is used in assisted reproductive technologies. Despite the existence of evidence supporting the production of ROS and DNA fragmentation during sperm cryopreservation, there is little and equivocal information about the cryopreservation effects on methylation 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. Semen samples from 10 healthy normozoospermic men were collected and each sample was divided into four equal aliquots: fresh, cryoprotectant, cryopreservation, and H₂O₂. We measured the ROS levels and DNA fragmentation using DCFH-DA and TUNEL assay respectively by flow cytometry. DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11–q13 region were evaluated by quantitative methylation-specific PCR technique. Intracellular levels of ROS and percentage of TUNEL-positive spermatozoa significantly increased in cryopreservation group compared to fresh group. Exposure to cryoprotectant had no significant effect on 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. In conclusion, based on our study, clinical use of sperm cryopreservation for fertility treatments appear to be safe in regard to DNA methylation in the chromosome 15q11–q13 region.

中文翻译：

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冷冻保存对人精子染色体15q11-q13区域DNA甲基化模式的影响。

人类精子冷冻保存是一种用于辅助生殖技术的常用技术。尽管有证据支持在精子冷冻保存过程中产生ROS和DNA片段，但是关于冷冻保存对印迹基因和印迹控制区域甲基化的影响的信息很少，也不清楚。在这项研究中，我们研究了低温冷冻对DNA甲基化在启动子区域的影响SNURF - SNRPN和UBE3A基因印记，PWS-ICR和AS-ICR染色体15q11-Q13区域。收集了来自10个健康的正常精子症男性的精液样本，并将每个样本分为四个等份：新鲜，冷冻保护剂，冷冻保存和H ₂ O₂。我们通过流式细胞术分别使用DCFH-DA和TUNEL测定法测量ROS水平和DNA片段化。DNA甲基化在启动子区域SNURF - SNRPN和UBE3A通过定量甲基化特异性PCR技术评估了染色体15q11–q13区中的印迹基因PWS-ICR和AS-ICR。与新鲜组相比，冷冻保存组的细胞内ROS水平和TUNEL阳性精子百分比显着增加。接触冷冻保护剂对ROS水平和DNA片段化没有显着影响。冷冻保存或不暴露于冷冻保护剂都不会显着影响所选基因区域的DNA甲基化。然而，DNA片段化与AS-ICR的DNA甲基化呈正相关。总之，根据我们的研究，就15q11–q13染色体区域的DNA甲基化而言，精子冷冻保存用于生育治疗的临床应用似乎是安全的。