Developing Noninvasive Epigenetic Biomarkers For Human Testis/Epididymis From Cell-free Seminal Nucleic Acids

Part Ⅰ Identification of the miRNAs predominately derived from testis/epididymis in human seminal plasmaBackground and objective:Developing epigenetic biomarkers for male infertility is desired. However, investigators rely heavily on the testicular and epididymal tissues to obtain their epigenetic information, thus limits the research in human. Recently, abundant miRNAs has been detected in human seminal plasma and a few reports indicated that they can be used as noninvasive epigenetic biomarkers for male infertility. Spermatogenesis and sperm maturation happen in testis and epididymis respectively. Ideally, seminal miRNAs predominately deprived from testis/epididymis should reliably reflex their expression profiles in these organs, and thus hold promise as reliable epigenetic noninvasive biomarkers for male infertility. Therefore, we identified the miRNAs predominately derived from testis and epididymis in human seminal plasma.Methods:Since the ejaculate of successfully vasectomized men does not contain any secretion from testis and epididymis, we screened seminal miRNAs that predominately derived from testis/epididymis by comparing the result of Solexa sequencing with the pooled seminal RNA of5vasectomized men and4normozoospermic donors respectively. Subsequently, the miRNAs were validated by RT-qPCR in the seminal plasma of3vasectomized men and4normozoospermic donors.Results:Totally84seminal miRNAs exhibited levels>4-fold higher in normozoospermic donors than in vasectomized men. Subsequent validation in individuals confirmed61miRNAs reliably predominately secreted from testis/epididymis. Interestingly,28miRNAs, which contain5miRNA clusters, reside on the X-chromosome. Conclusion:The present study identified seminal miRNAs that predominately derived from testis and epididymis, which could be the potential noninvasive markers for human male infertility researches on revealing the etiology and physiopathological status of impaired sperm production and maturation. Part Ⅱ The exploration study of using epididymal miRNAs in human seminal plasma as noninvasive biomarkers of idiopathic asthenospermiaBackground and objective:Cell-free miRNA could reflex diverse physiological and pathological status including pregnancy, tumor, cardiovascular diseases and tissual lesion, and might be a new kind of molecular biomarker for the research and diagnosis of disease. We have previously identified some miRNAs predominately derived from testis and epididymis in human seminal plasma. Considering miRNAs have the important role of regulating spermatogenesis and sperm mature in the testis and epididymis respectively, thus, we postulated that epididymal miRNAs correlated with sperm maturation might be dysregulated in some patients with idiopathic asthenospermia and the dysregulation could be detected in cell-free seminal miRNAs (cfs-miRNAs) by reverse transcription-quantitative PCR (RT-qPCR). Then, the dysregulated miRNAs can be used as the noninvasive biomarkers of some idiopathic asthenospermia. In the present study, this hypothesis was tested on patients with idiopathic asthenospermia.Methods:The relative amount of13epididymal cfs-miRNAs (miR-888, miR-890, miR-891a, miR-891b, miR-892a, miR-892b, miR-660, miR-421, miR-221, miR-200b, rm"R-220c, miR-187and miR-181c) was compared between20normozoospermic donors and the22patients with asthenospermia by RT-qPCR. The including patients of asthenospermia must meet the following requirements:there was no evidence of anatomical abnormalities of the genital tract, including varicocele, nor was there a history of cryptorchidism, mumps, eating raw cottonseed oil, testicular torsion, hyperpyrexia, genital tract infection, drug addiction, occupational exposure of chemical material and hyperthermia environment, etc. The culture of semen was negative, including Escherichia coli, Staphylococcus aureus, Streptococcus, Neisseria gonorrhoeae, Ureaplasma urealyticum, Chlamydia, Mycoplasma detection. The hormonal serum profile (gonadotropins, prolactin, testosterone, estradiol, follicle stimulating hormone and luteinizing hormone) was normal. Patients with abnormal semen pH or liquefaction were also excluded. The absence of known possible genetic causes (chromosome abnormality, and Y microdeletion), immune disease (including anti-sperm antibody), and other systemic diseases was also verified. Three suggested endogenous miRNAs controls (miR-30d, miR-93and miR-107) were selected, the mean of Ct value in three endogenous miRNA controls was applied to calculate the relative amount of target cfs-miRNAs.Results:The relative amount of5epididymis-specific miRNAs (miR-888, miR-890, miR-891a, miR-891b and miR-892a), which belong to the miR-890/891a cluster on the X-chromosome and have important roles in epididymal morphogenesis and sperm maturation, were obviously lower in idiopathic asthenospermia than normozoospermia (P<0.05). No obvious difference was observed in other8miRNAs (miR-892b, miR-660, miR-421, miR-221, miR-200b, miR-220c, miR-187and miR-181c) between normozoospermia and idiopathic asthenospermia (P>0.05).Conclusions:The down-regulation of epididymal miRNAs ((miR-888, miR-890, miR-891a, miR-891b and miR-892a)), which was detected by cfs-miRNA, might be responsible for some patients with the decrease of sperm vitality. Whether the dysregulation is the epigenetic cause or a molecular change of other causes for idiopathic asthenospermia deserves further investigation. Our data also suggested the potential of cfs-miRNA as a new kind of noninvasive biomarker for researching the etiology and mechanism of some idiopathic infertility. Part Ⅲ Genome-wide promoter methylation profile of human testis and epididymis:identified from cell-free seminal DNABackground and objective:DNA methylation is essential for remethylation in spermatogenesis and sperm maturation, some abnormal methylated promoters have been found in the testis and spermatozoa of male infertility. We have previously found the presence of high concentration of cell-free seminal DNA (cfsDNA) in human semen. We proposed that some testis and epididymis-specific methylated promoters could be detected in human cfsDNA, and thus hold promise as noninvasive epigenetic biomarkers for male infertility, of which most cases are caused by defects in testicular sperm production or epididymal sperm maturation. Therefore, we identified the testis and epididymis-specific methylated promoters in human seminal plasma.Methods:We screened testis and epididymis-specific promoter methylation with a NimbleGen HG18Refseq promoter array in pooled cfsDNA between6normozoospermic donors and6post-vasectomy men respectively. The10testis and epididymis-specific methylated promoters (CLPB, DAZ1, DNMT3L, GML, HOXA5, MSH4, MORC1, PEG10, PRAME and SNURF) screened by promoter microarray were validated in the pooled cfsDNA by MeDIP-quantitative PCR, the other9testis and epididymis-specific methylated promoters (CHRFAM7A, USP28, NCK2, ERRFI, FBRS, GLTPD1, HSF1, VPS16and ZNF623) were validated in the seminal plasma of4vasectomized men and4normozoospermic donors by MethyLight. Gene Ontology analysis was used to classify the function of testis and epididymis-specific methylated genes.Results:Promoters of367testis and epididymis-specific hypomethylated genes and134hypermethylated genes were identified. The8testis and epididymis-specific hypomethylated promoters validated by MeDIP-quantitative PCR were concordant with the promoter methylation microarray, except for PEG10and CLPB. As for the other9promoters measured by MethyLight, two testis and epididymis-specific hypermethylated promoters (CHRFAM7A and USP28) and6testis and epididymis-specific hypomethylated promoters (ERRFI, FBRS, GLTPD1, HSF1, VPS16and ZNF623) were consistent with the promoter array besides NCK2. Gene Ontology analysis revealed many genes involved in male reproduction and the epididymal functioin of reabsorption, excretion and defense.Conclusion:We detected the testis and epididymis-specific methylated promoters in human cfsDNA, which may be used for noninvasive epigenetic biomarkers for the study and diagnosis of male infertility. Part Ⅳ Quantifying the methylation level of testis-specific methylated promoters in the cfsDNA of NOABackground and objective: DNA methylation play essential role in spermatogenesis. However, the difficulty of obtaining testis limits the research on human. We have previously identified testis and epididymis-specific methylated promoters from human cfsDNA. Given that remethylation occurs gradually from the prospermatogonia stage to spermiogenesis during spermatogenesis, the testis-specific methylated promoters could show the differential methylation level in the testis of NOA with different pathological types, and the difference could be reflexed in cfsDNA. Some differential methylated promoters, which may be found in the cfsDNA of NOA between hypospermatogenesis and the other pathological types, thus have the potential of predicting the retrieval of sperm. In the present study, the hypothesis was certified by comparing the methylation status in the testicular DNA and cfsDNA of NOA with different pathological types.Methods:Fourteen testicular biopsies of NOA [mature arrest (MA, n=5) and hypospermatogenesis (HO, n=9)] and corresponding9semen samples [MA (n=5) and HO (n=4)]were collected, they were used to analyze the correlation of promoter methylation level between testicular DNA and corresponding cfsDNA, and relative amout of testicular mRNA. On the other hand, the92semen samples of NOA [sertoli-cell only (SCO, n=17), MA (n=25) and HO (n=26)] and normozoospermia (Nor, n=24) collected, they were used to detected the differential methylation level of promoters in cfsDNA. The relative methylation level of6testis-specific methylated promoters (ACRBP, CIB1, CCNA1, DMRT1, HSF1and MORC1) was determined in testicular DNA (n=14) and cfsDNA (n=101) by MethyLight and the relative amount of their mRNAs was determined in testicular samples by RT-qPCR. The correlation of promoter methylation level between testicular DNA and corresponding cfsDNA (n=9), and the correlation between the promoter methylation level and mRNA level in testis (n=14) was analyzed. The methylation level of promoters was analyzed among the groups of SCO, MA, HO and Nor. Finally, the methylation level of promoters was compared between the group of HO (n=26) and the other two groups of NOA (SCO and MA, n=42), and the sensitivity and specificity of the differential methylated promoters were calculated using receiver operating characteristic (ROC) analysis.Results:The correlation coefficient about promoter methylation level of ACRBP, CIB1, CCNA1, DMRT1, HSF1and MORC1between testicular DNA and corresponding cfsDNA was0.90,0.94,0.79,0.99,0.90and0.92respectively. The correlation coefficient between promoter methylation level of ACRBP, CIB1, CCNA1, DMRT1, HSF1and MORC1and the relative amount of their mRNA in testis was-0.667、-0.586、-0.609、-0.584、-0.806and-0.774respectively. The promoter methylation level of ACRBP, CTB1, CCNA1, DMRT1, HSF1and MORC1shows the tendency of changing dynamically among the groups of SCO, MA, HO and Nor. Among them, the methylation level of CCNA1and DMRT1promoters in the cfsDNA of HO was higher than the other two types, P value is0.032and0.065respectively. They were used to predict the hypospermatogenesis of NOA, the sensitivity of CCNA1and DMRT1promoters is27.27%and36.36%respectively, and the specificity of each promoter is94.44%.Conclusions:The methylation level of testis-specific methylated promoters in the cfsDNA of NOA is able to reflex the methylation level of promoters in testis, thus can be used as the noninvasive biomarker for the research of NOA on revealing the etiology and pathogenesis, and the prediction of NOA with hypospermatogenesis.