| ObjectiveSpontaneous abortion in early pregnancy,the incidence of fetal stop development have reached to 12.54%,and which is increasing.Fetal stop development is closely related to factors such as environment,endocrine,chromosome,immune system and mental health,but 50% of the factors are unclear.The epigenetic etiology of adverse pregnancy outcomes has become a frontier in reproductive and developmental study in recent years.Genomic imprinting refers to the fact that a particular gene or gene cluster in a progeny expresses only one parent's allele,while the other parental allele is being silence.Animal studies have shown that knockout of imprinted genes can lead to abnormal energy metabolism,embryonic developmental delay,and even embryonic lethality.A study shows that imprinted genes related to fetal stop development include IGF2 and H19.DNA methylation is one of the most widely studied epigenetic modifications that control the expression of imprinted genes.It's principle is that the 5' carbon position of the cytosine of the CpG dinucleotide of the DNA differential methylation region(DMR)is covalently bonded to the methyl group to form an imprinted control region(ICR)to regulate the expression of the imprinted gene.Many studies have noted the relationship between embryonic development and DNA methylation in mothers and fetuses.Methylation of imprinted genes in sperm may be involved in the function of the placenta and the development of the fetus,but little has been done on the methylation of sperm imprinted genes.Therefore,this study explore the relationship between the methylation level of sperm imprinted genes H19,Peg3 and Meg3 and fetal stop development,to provide clues for the mechanism of fetal stop development,and to provide a theoretical basis for theprevention of fetal stop development.MethodsA total of 440 men were enrolled from reproductive center of the Maternal and Child Health Hospital of Shanxi Province during March-April,2015 and March,2016.General information,semen samples and clinical semen analysis results were collected.Each subject signed the informed consent.The sperm DNA was extracted.And the methylation level was determined in the Differentially Methylated Regions(DMRs)of H19,PEG3 and MEG3 by pyrosequencing.As of March 2018,394 follow-up results were obtained by hospital archival records and telephone follow-up.In order to study the relationship between sperm imprinted genes methylation and fetal stop development,(1)21 men with fetal stop development after natural conception were selected as the fetal stop development group of natural conception;21 men with normal live births after natural conception were selected as its control group,and a 1:1 match was made based on father's age,BMI,smoking and drinking.(2)26 men with fetal stop development after assisted reproduction were selected as fetal stop development group of assisted reproduction;26men with normal live births after assisted reproduction were selected as its control group,and a 1:1 match was made based on father's age,BMI,smoking and drinking.The analysis includes:(1)We compared the semen parameters(including semen volume,sperm density,sperm motility and sperm normal morphology rate)and the methylation level of sperm imprinted genes in the fetal stop development group of natural conception and its control group;(2)We compared the semen parameters and the methylation level of sperm imprinted genes in the fetal stop development group of assisted reproduction and its control group;(3)general demographic characteristics,semen parameters,imprinted genes methylation of sperm in the natural pregnancy stop group and the assisted reproductive fetus group.Statistical methods include normality test,t-test,Wilcoxon signed rank sum test,Wilcoxon rank sum test,and Spearman rank correlation.The statistical software used IBM SPSS 24.0.? = 0.05.Results1.Comparing the fetal stop development group and the control group in natural pregnancy,we found that there was no significant difference in semen parameters(P >0.05).It is also no different that the methylation level of the mean and each CpG site of H19,Peg3 and Meg3 in sperm(P > 0.05).2.Comparing the fetal stop development group and the control group in assisted reproduction technology,we found that there was no significant difference in semen parameters(P > 0.05).It is also no significantly different that the average methylation level of H19,Peg3 and Meg3 in sperm(P > 0.05).The methylation levels of CpG5(P = 0.028),CpG6(P = 0.036)and CpG8(P = 0.033)of Meg3 were statistically different in the two groups.Further correlation analysis revealed that the methylation levels of these three loci were negatively correlated with those in fetal stop development group(assessment: 0 = control group,1 = fetal stop development group),the correlation coefficients were-0.281(P = 0.058),-0.318(P = 0.031)and-0.310(P = 0.036).But it is no significantly different that each CpG site methylation level of H19 and Peg3 in sperm(P > 0.05).3.Comparing the fetal stop development group of assisted reproduction technology and the fetal stop development group of natural pregnancy,we found that there was no significant difference in age,BMI,smoking,alcohol consumption and semen parameters.The methylation levels of CpG2(P = 0.032)of Meg3 was significantly increased,and the methylation levels of CpG3(P = 0.005),CpG4(P = 0.042)and average methylation level(P = 0.006)of Peg3 were significantly decreased in the fetal stop development group of assisted reproduction technology.It is no significantly different that the other CpGs methylation and the average methylation of H19 and Peg3 in sperm in the two group(P >0.05).Conclusions1.Compared with its control group,the methylation levels of CpG5,CpG6 and CpG8 of Meg3 in sperm DNA were significantly decreased in the fetal stop development group of assisted reproduction technology.2.Compared with fetal stop development group of natural pregnancy,the methylation levels of CpG2 of Meg3 was significantly increased,and the methylation levels of CpG3,CpG4 and average methylation level of Peg3 were significantly decreased in the fetal stop development group of assisted reproduction technology.3.This study did not find that methylation levels of H19 and Peg3 in sperm were associated with fetal stop development. |
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