A Study Of The Correlation Between Gene Methylation,Diurnal Variation And Sperm DNA Integrity

Background:Sperm DNA integrity damage is a common semen abnormality in males and it is considered to be the main cause of male fertility decline and infertility.DNA fragmentation index(DFI)is a common index to assess DNA damage of sperm.However,the correlation between gene methylation,diurnal variation and sperm DNA integritthe is unclear.Part I Abnormal methylation of imprinted genes in human sperm is associated with sperm DNA damageObjection:To analyze the correlation between sperm DNA damage and imprinted gene methylation by detecting the methylation state of imprinted genes.Methods and materials:DNA methylation levels were determined at seven imprinted genes loci(H19,INS-IGF2,KCNQ1,MEG3,MEST,PEG3,and SNRPN)in 66 semen samples using the MSRE-qPCR method.The semen samples were divided into two groups according to the threshold value(25%)of DFI.The correlation between sperm DNA damage and imprinted gene methylation was analyzed.Results:We found that the mean methylation level at IGF2(cg17037101)in the group with DFI?25%was lower than that in the group with DFI<25%(13.7 ±3%vs.31.5 ±5.3%,P=0.0053).However,the methylation levels of other CpGs did not differ from the imprinted genes.Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2(cg17037101)methylation level was negatively correlated with sperm DFI(r=-0.448,P=0.0038),and the KCNQ1(cg24932449)methylation level was positively correlated with sperm DFI(r=0.354,P=0.0273).Conclusion:These results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.Part ? Genome-wide DNA methylation detection of human sperm with DNA damage and functional study of BRCA1 geneObjection:To screen the differential methylation genes associated with sperm DNA damage using Genome-wide methylation chip and investigate the role of BRCA1 in sperm DNA damage.Methods and materials:Semen samples of 20 infertility men were collected.Genomic DNA of sperm was extracted,bisulfite converted,and hybridized with the Infinium Methylation EPIC BeadChip to analyze differential methylation sites.Genome-wide DNA methylation profiles of the 20 samples were analyzed by unsupervised hierarchical clustering.Quantitative PCR was used to analyze the expression of BRCA1 gene in different DFI semen samples,and siRNA was used to knock out the expression of BRCA1 in GC-2 cells to study its function.Results:This analysis demonstrated the presence of three demarcated clusters in the population of undiagnosed infertility couples according to the DNA methylation profiling with differential levels of DFI.20 samples were divided into three groups accordingly:high(H),medium(M)and low(L)DFI groups and 959,738,and 937 different methylation regions were found in L vs.M,M vs.H,and L vs.H groups,respectively.GO enrichment analysis revealed that differentially methylated regions are mainly involved in DNA methylation involved in gamete generation,male meiotic nuclear division,male meiosis I,P granule and pi-body,etc Targeted Region Bisulfite Sequencing found that there were significant differences in methylation of multiple CpG loci of BRCA1 gene between high and low DFI groups.Correlation analysis found that CpG4,CpG11 and CpG20 methylation levels were significantly correlated with semen DFI.In addition,the expression of BRCA1 was decreased in the high DFI group.Knockdown of BRCA1 expression in GC-2 cells had a slight effect on cell growth,significantly reduced the survival rate of damaged cells after H2O2 treatment,and increased the apoptosis and yH2AX foci formation induced by H2O2 treatment.Conclusion:It is suggested that high sperm DFI may be associated with abnormal sperm DNA methylation.Abnormal methylation and decreased expression of BRCA1 gene may play an important role in DNA damage of male germ cells.Part ? The effect of diurnal variation on sperm DNA integrityObjection:Circadian rhythms have been found in some reproductive functions phenotypes but remain unclear for DFI.The present study aims to investigate the diurnal variation of DFI in mice model and men spermMethods and materials:Adult male mice were sacrificed for sperm DFI with Sperm Chromatin Structure Assay(SCSA)in 24 hours at 6 evenly distributed time points.A total of 11382 semen samples from two independent populations,including a panel dataset of visitors to Reproductive Medical Center in the First Hospital of Wenzhou Medical University and a cross-sectional dataset from the community population were recruited.Semen samples were collected between 7 AM and 11 AM in Reproductive Medical Center,between 8 AM and 20 PM in the community population.DFI was measured with Sperm Chromatin Dispersion(SCD)test or SCSA.Linear regression and multi-level model were used to analyze the association between ejaculation timepoint and DFI.Results:A cosinor pattern of DFI was observed with a nadir at zeitgeber time 10 AM in mouse models.In a community population with 630 semen samples collected between 8 AM and 20 PM,the temporal variation of DFI also fit a cosinor pattern with a-343° acrophase and a nadir at 11 AM(P=0.031).In a reproductive-medical-center dataset of 10752 semen samples collected between 7 AM and 11 AM,the decreasing trend of DFI was also confirmed.For the males with multiple samples,intra-individual comparison between different timepoints was performed,and each consecutive hour after 7 AM was also associated with 2.5(95%CI:-1.0,5.9)%lower DFI by SCSA or 4.9(1.9,7.8)%lower DFI by SCD.Conclusion:Our study reveals a daily diurnal variance in sperm DFI which may suggest a practical approach to get more qualified sperms for natural or assisted reproduction.