Genome-Wide DNA Methylation Profiles And SncRNAs Signatures Study Of High DFI Sperm

Part 1 Genome-wide DNA methylation profiles study of high DFI spermPurpose: The integrity of sperm DNA is critical for fertilization and offspring health,and an increasing number of studies have shown a direct relationship between sperm DNA damage and male infertility.The sperm DNA fragmentation index(DFI)can assess sperm quality and predict fertility more accurately than traditional semen analysis.Recent studies have shown that changes in sperm DNA methylation are associated with abnormal semen parameters and male infertility.However,no studies have reported the effect of DFI levels on sperm methylation,so we hoped to search for new sperm quality biomarkers by comparing genome-wide DNA methylation profiles of high DFI and low DFI sperm samples.Methods: We collected 6 weak group(DFI≥30%)sperm samples and 7 normal group(DFI≤15%)sperm samples in Changhai Hospital.A pure sperm pellet was obtained by processing the semen by somatic cell lysis.Sperm genomic DNA was extracted and subjected to whole-genome bisulfite sequencing(WGBS),followed by differential analysis of genome-wide DNA methylation profiles.In addition,we constructed sperm DNA methylation correlation matrix using WGBS data to compare the differences in the spatial conformation of sperm chromosomes between the two groups.The TPM data of gene expression in all normal human tissues were downloaded from the GTEx database,and the differential genes whose expression level in the testis was more than 2 times that of any other tissue were identified as testis-specific genes.We also compared the relative expression of MOK mRNA in 10 pairs of sperm samples,and performed targeted bisulfite sequencing(TBS)on the differentially methylated regions(DMRs)of the MOK gene in 15 pairs of sperm samples.Finally,by constructing Mok gene knockout mice,the effects of Mok gene knockout on the reproductive system and reproductive ability of male mice were analyzed.Results: Compared with normal group sperm,the overall DNA methylation level of weak group sperm showed a downward trend.A total of 4939 DMRs were identified in weak group sperm,of which 2072 DMRs were located in the promoter region.In different gene regions,the proportion of hypermethylated DMRs was higher than that of hypomethylated DMRs.By cluster analysis of the top 300 DMRs,these DMRs were found to be able to distinguish the two groups of sperm samples,so these 300 DMRs may be potential biomarkers for assessing sperm quality.Gene ontology(GO)and pathway enrichment analysis was performed on 3083 hypermethylated DMRs,and it was found that they were mainly enriched in neuronal and microtubule regions.Using the WGBS data to construct DNA methylation correlation matrix,it was found that compared with the normal group,the correlation matrix of the five chromosomes(13,4,5,21 and Y)in the weak group sperm changed,and the correlation of methylation was weakened,suggesting that the structure of these five chromosomes in weak group sperm became loose.We screened2186 human testis-specific genes from the GTEx database,intersected them with 1090 promoter hypermethylated genes in weak group sperm,and obtained a total of 79 genes.Then,the gene MOK with the most prominent promoter hypermethylation was selected from these 79 genes for further study.We found that the MOK mRNA expression of sperm in the weak group was significantly lower than that in the normal group.A total of 12 Cp G sites were detected in DMR-MOK,and 8 Cp G sites had significantly higher methylation levels in weak group sperm than in normal group sperm.The remaining 4 Cp G sites were not significantly different.There were no significant differences in reproductive function between Mok knockout male mice and wild male mice.Conclusion: Our study is the first to analyze the effect of DFI levels on the genome-wide methylation profiles of human sperm.The genome-wide DNA methylation profile of sperm is significantly correlated with sperm DFI level.The chromosomes of sperm with high DFI become loose and thus more vulnerable to reactive oxygen species(ROS)attack and more prone to DNA breakage.Elevated levels of sperm DFI may affect the development of the embryonic nervous system and may also reduce sperm motility by affecting sperm microtubule structure.Although Mok knockout had no obvious effect on male reproductive function,DMR-MOK of high DFI sperm was hypermethylated and inhibited the expression MOK.Therefore,DMR-MOK is likely to be a biomarker for sperm quality assessment,and has good clinical significance for improving the success rate of natural pregnancy and assisted reproductive technology(ART).Part 2 sncRNAs signatures study of high DFI spermPurpose: A growing number of studies have shown that mammalian spermRNA is another source of paternal genetic information besides DNA.Environmental factors,including toxins,mental stress,and unhealthy diet,can reshape spermRNA signatures and induce offspring phenotypes associated with paternal environmental stress.SpermRNA plays a very important role in embryonic development and offspring phenotype.In recent years,more and more types ofRNA have been found in sperm.Existing research mainly focuses on small noncodingRNAs(sncRNAs).However,no studies have reported the effect of sperm DNA fragmentation index(DFI)levels on the expression of sperm sncRNAs,so we hope to find new sperm quality biomarkers by comparing the sncRNAs characteristics of high DFI and low DFI sperm samples.Methods: We collected 13 weak group(DFI≥30%)sperm samples and 17 normal group(DFI≤15%)sperm samples in Changhai Hospital.A pure sperm pellet was obtained by processing the semen by somatic cell lysis.Total spermRNA was extracted for sncRNAs library construction and deep sequencing.Statistical analysis was performed using the R package.DESeq2 was used to analyze the differentially expressed miRNAs,tsRNAs and rsRNAs between the two groups of sperm,only the sncRNAs with P<0.05 were considered as differentially expressed sncRNAs.Student’s test was used to analyze the ratios of various sncRNAs.If P<0.05,the correlation of miRNAs,tsRNAs or rsRNAs between the two groups could be established.Cluster analysis was performed using Pheatmap,principal component analysis(PCA)was performed using the procomp package,and graphs were plotted using ggbiplot.The target genes of miRNAs were predicted by Target Scan(http://www.targetscan.org),and Gene Ontology(GO)function enrichment analysis was performed on the target genes of up-regulated and down-regulated miRNAs in sperm of the weak group.Results: On average,40.5% of the sncRNAs in sperm were annotated as rsRNA,19.3% as tsRNA,10.4% as yRNA,and 7.1% as miRNA.The proportion of various sncRNAs between the two groups of sperm had no significant difference.A total of 632 miRNAs(average RPM>10)were detected in sperm,among which 27 differentially expressed miRNAs(9 up-regulated,18 down-regulated),PCA analysis showed that these27 differentially expressed miRNAs could effectively distinguish the two groups of sperm(PC1 48.72%,PC2 19.14%).A total of 3612 tsRNAs(average RPM>10)were detected in sperm,of which 151 differentially expressed tsRNAs(76 up-regulated,75 down-regulated).PCA analysis showed that these 151 differentially expressed tsRNAs could also differentiate sperm samples(PC1 28.79%,PC2 23.57%).A total of 10,707 rsRNAs(average RPM>100)were detected in sperm samples,of which 70 were differentially expressed rsRNAs(42 up-regulated,28 down-regulated).PCA analysis showed that these70 differentially expressed rsRNAs could also differentiate the two groups of sperma to a certain extent(PC1 47.06%,PC2 18.13%),but the distinguishing effect was not as good as miRNA and tsRNA.Finally,we identified 9 sncRNAs as candidate sperm quality biomarkers,and PCA analysis showed that these 9 sncRNAs could better differentiate the two groups of sperm(PC1 34.02%,PC2 26.28%).Conclusion: Our study is the first to analyze the effect of DFI levels on the sperm sncRNAs signature.The most abundant sncRNAs we detected in human sperm were rsRNA,followed by yRNA and tsRNA.Although there was no significant difference in the proportion of various sncRNAs between high DFI and low DFI sperm,we detected many differentially expressed miRNAs,tsRNAs and rsRNAs between the two groups.These differentially expressed sncRNAs had a good discriminative effect on the two groups of sperm samples.The sncRNAs signatures of sperm were significantly correlated with sperm DFI levels.We finally screened 9 sncRNAs as candidate sperm quality biomarkers,which are more effective in distinguishing the two groups of sperm,and a large sample size verification of these 9 sncRNAs is needed in the future.