| Sperm cryopreservation is the main methods of male reproductive ability in the field of assisted reproduction and it is widely used in the human sperm bank’s sample preservation. According to reports, cryopreservation of human sperm in28years after successful resuscitation, and with the success of assisted reproductive technology successful conception, but requires us to take responsibility for the offspring of sperm, not only can bear so simple. In recent years, more and more cancer patients before radiotherapy and chemotherapy desire of fertility preservation. In the field of assisted reproduction, some oligospermia or paraplegia male patients, in order to achieve the purpose of reproduction, must be obtained sperm through the epididymal way (not including the seminal plasma) and cryopreservation further, waiting for the right time to use IVF technology to have offsprings. With widespread use of technology in human semen cryopreservation, its security has attracted more and more attention.Freezing can prolong sperm preservation time, but in the freezing process ice crystals forming, oxidative stress reaction, the solution effect caused by high solute may cause sperm cell membrane, DNA integrity, mitochondrial function and damage, further affect the fertilization ability and embryo quality. Epigenetics is a real environmental stress and genetic linked to the subject, which do not change in DNA sequence, but gene expression is changed, and this change can stably transfer in process development and cell proliferation. DNA methylation is an important part of epigenetics; it is concerneed because of its earlier found and the relative stability of the genetic basis. Studies have shown that DNA methylation of embryonic imprinted genes is vulnerable to Influence by external environmental factors (temperature, osmotic pressure, the effect of in vitro manipulation), and the abnormal DNA methylation of imprinted genes is closely related to male infertility and fetal defects diseases, such as abnormal DNA methylation of paternally imprinted gene H19imprinting control region may cause the trophoblast cell invasion ability induced decreased and the high incidence of the disease SRS and BWS. Maternal imprinting gene KvDMR1are associated with a high incidence of AS/BWS. At present, domestic rare damage assessment of imprinted gene DNA methylation on human sperm after freezing. Therefore, this study comprehensive assesses sperm freezing’s safety from the sperm DNA integrity and DNA methylation two aspects.Objective:Detection of freezing and freezing time on sperm apoptosis and DNA methylation of imprinting control region (ICR) of sperm imprinted gene (paternal H19, maternal KvDMR1).Methods:In15cases of healthy male semen as the research object, each semen samples are divided into two parts in addition to leave a little outside a control (divide the two parts into sections). Along with the seminal plasma part undergoing programmed freezing, the other part is freezed after through the Isolate method to remove the seminal plasma. Respectively after1month,3months,6months and12months freezing, through the bisulphite sequencing PCR method analysises DNA methylation status of H19ICR and KvDMRl. Further by flow cytometry testing the sperm apoptosisin which removed of seminal plasma after frozen for1month,3months and6months.Results:1. Along with the seminal plasma freezing sperm samples, H19ICR DNA methylation droppings are13.62±2.06±%、14.88±1.96%、15.89±2.26%after frozen1month,3months and6months. There is no significant differences compared with control (12.74±2.85%)(P>0.05); DNA methylation of maternal imprinted gene KvDMR1ICR without increased compared to the control, that is to say no significant difference (P>0.05).2. Removing seminal plasma freezing sperm samples, H19ICR DNA methylation droppings are15.24±1.91%(P>0.05),13.49±2.63%(P>0.05) and30.92±3.08%(P<0.01) compared with control (12.74±2.85%) after frozen1month,3months and6months. DNA methylation of maternal imprinted gene KvDMR1ICR without changing compared to the control (P>0.05) within12months of frozen.3. Further on the removal of seminal plasma frozen for1months,3months,6months to detect the apoptosis of sperm samples, there is no significant difference compared with the control group.Conclusion:Compared with the DNA damage, DNA methylation of imprinted genes in sperm, especially the paternally imprinted gene is more susceptible to influence in the cryopreservation process, and this effect showed a time dependent. |
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