A Preliminary Study Of Isolated Teratozoospermia With Imprinted Genes Of DNA Methylation And DNA Damage

Infertility is one of the major health problems facing mankind today, affecting10-15%of couples of reproductive age, male factors account for about50%. Clinically,male factor infertility is usually based on abnormal sperm parameters (such asoligozoospermia, asthenospermia, teratozoospermia, etc.) to diagnosis. As one of gametes,sperm relates to human reproduction. It is reported that70%of male infertility is causedfrom the abnormal sperm. Many studies show that sperm morphology has better predictivevalue in potential of embryonic fertilization and clinical pregnancy outcomes, and thisconclusion has been verification in vivo and in vitro experiments, compared with othersemen parameters (concentration, vitality, viability, etc.). It is because of importantpredictive value of sperm morphology and the status of declining semen quality. In recentyears, WHO regarding the identification of teratozoospermia patients threshold has beenupdated. The percentage of normal sperm morphology in1978by50%to30%in1992to15%in1999, especially in the fifth edition WHO of the standard semen analysis issued in2010, it was set at4%, which triggered a reproductive scientists at home or abroadextensively widespread concern and controversy. But the current study, the vast majorityof the correlation analysis of sperm morphology and clinical outcomes after assistedreproductive technology, and few mechanisms for further analysis. Imprinted genes are the only one of parental origin homologous gene expression, and the other parent is notexpression.This difference expression is mainly composed of the DNA sequence ofimprinting control region of covalent modification to control, involved in the normalgrowth of the embryo, developmental and behavioral development in children. The spermDNA damage is closely related to embryonic development, clinical pregnancy andabortion etc. So we choose isolated teratozoospermia for the research objects, there arethree aspects that the clinical outcome of pregnancy, DNA methylation and DNA integrity.ObjectiveA retrospective analysis of isolated teratozoospermia and clinical pregnancyoutcomes are whether or not correlated; detection sperm imprinted genes (paternal H19,maternal SNRPN and Line-1) DNA methylation changes and sperm cells apoptosis.MethodsDuring the period of the April2012and May2013, in the Center for ReproductiveMedicine of Tang Du Hospital, we choose patients which due to isolated teratozoospermiacause to accept ART for the study. First retrospective analysis of sperm morphology andclinical pregnancy outcome correlation; Further selected40cases of isolatedteratozoospermia with semen samples, DNA was extracted by centrifugal purificationmethod after washing sperm with Isolate, and then it took bisulfite sequencing PCRmethod to analyze paternally imprinted genes H19, maternal imprinted genes SNRPN andLINE-1’s DNA methylation status, and further detected by flow cytometry of spermapoptosis (DNA damage) cases.Results1、After undergoing IVF assisted reproduction, normal fertilization rate, cleavage rate,quality embryo rate, clinical pregnancy rate, miscarriage rate in the control group were83.09%,94.52%,49.58%,62%,5.14%, in the study group with isolated teratozoospermianormal fertilization rate, cleavage rate, quality embryo rate, clinical pregnancy rate,miscarriage rate were83.64%,95.26%,41.88%,64.41%,6.95%, the indicators were notsignificantly different (P＞0.05). After undergoing ICSI assisted reproduction, normal fertilization rate, cleavage rate, quality embryo rate, clinical pregnancy rate in controlgroup were86.54%,96.80%,43.08%,56%, corresponding to the study group withisolated teratozoospermia were87.78%,98.13%,41.67%,56.25%, there was also nostatistically significant difference (P＞0.05). But abortion rate of patients with isolatedteratozoospermia was significantly increased, the difference was statistically significant(6.19%Vs19.5%, P＜0.05).2、DNA methylation results:①Paternally imprinted gene H19: the loss rate clone ofabnormal sperm group was36.05±2.86%, CpG islands lost was3.85±0.41%, CTCF6binding site region CpG islands loss rate was2.43±0.19%, while the control group was13.41±3.21%,0.75±0.17%,0.25±0.06%, the differences were all statisticallysignificant (P＜0.01);②Maternal imprinted gene SNRPN: the changes of DNAmethylation in abnormal sperm group could be found no significant difference comparedwith the control group(P＞0.05);③Line-1: in the abnormal sperm and the control group itshowed a high degree of methylation status (control group80.54±2.9%Vs abnormalsperm group79.67±3.2%), and there was no significant difference methylation betweenthe two groups (P＞0.05).3、The degree of apoptosis in isolated teratozoospermia group than the control group(3.01±0.38Vs0.65±0.19, P＞0.05), and with the increased malformation rate, there is anincreasing trend of apoptosis.Conclusion1、Sperm deformity rate and is closely related to clinical assistant reproductionmethods selection and pregnancy outcome.2、Sperm deformity rate and extent of hypomethylation H19imprinting controlregion showed a significant positive correlation.3、The higher sperm deformity rate, the higher the degree of sperm DNA apoptosis.But it needs large samples to be confirmed.