DNA Methylation And Regulation Of Asthenozoospermia Associated Genes

About15%of couples do not achieve pregnancy within1year; almost50%of them do so spontaneously in the second year of unprotected intercourse, and another14%in the third year. According to the definition of World Health Organization（WHO）,"Infertility is the inability of a sexually active contraception couple to achieve pregnancy in one year". A male contribution to infertility is found in45～50%of the remaining cases, low sperm motility is one of the most important causes in male infertility. One study indicated that among1,085sperm samples analyzed from infertile males,81%were defective in motility, being approximately20%isolated asthenozoospermia.Abnormal gene expression is decreased sperm motility etiology of the hot spots, especially pathogenesis for idiopathic asthenospermia, in related research, the development of idiopathic weak spermatogenesis and sperm abnormal gene expression has obvious correlation.Sperm movement is mainly related to the sperm tail, the sperm through the the tail flagellum swing forward momentum. Adenosine triphosphate （ATP） to start the next sperm flagellar axoneme dual microtube A microtubule two rows arm with the adjacent double micro tube B microtubules binding sites constantly detachment and attachment, in order The longitudinal force generated by the sliding between the doublets. Longitudinal force generated by the arm further formed by various subsidiary structures related to the double microtubule sliding resistance into lateral bending force, and power steering and a wavy spread. The main energy source of sperm motility from with a large number of mitochondria in the sperm, the mitochondria produce ATP is an important factor for activation of sperm flagellar system. The main factors involved in the regulation of sperm motility following aspects:（1） sperm calcium levels.（2） kinase phosphorylation System:Protein phosphorylation is an important mechanism for the regulation of sperm motility, tyrosine-specific protein phosphorylation and sperm motility of the sperm tail tyrosine and serine-threonine kinase system are closely related.（3） reactive oxygen species （Reactive Oxygen Species）:close to the reactive oxygen species produced by white blood cells in the sperm and semen and sperm motility, reactive oxygen species induced cyclic AMP-protein kinase signaling pathway at low concentrations which increase sperm motility.（4） immune factors:male semen overdose of anti-sperm antibodies can be induced the sperm agglutination thereby affect sperm motility.（5） seminal plasma osmotic pressure:the sperm in the seminal plasma of low osmolality activate the osmotic pressure-sensitive calcium channels induce calcium influx, thereby improving sperm motility.Dihydrolipoamide dehydrogenase （DLD）, Corydalis hydratase （FH） and cytochrome oxidase （COX-6） and other genes ATP production the DLD, FH and COX-6will be an exception, will affect sperm motility, thus affecting the pregnancy. Was found by comparing normal subjects and patients with weak sperm infertility semen asthenozoospermia group DLD-2, FH, COX-6expression abnormalities.DNA methylation, as one of the epigenetics is an important mechanism for the regulation of transcription, control of gene expression and imprinting. Existing research data show that methylation abnormalities and male infertility sperm abnormalities, the occurrence of specific tumors, nervous system disease, Rett syndrome, and so on. Therefore, DNA methylation abnormalities as a highly sensitive biomarker, become a hotspot in the development of research in a variety of human diseases. In related research, indicate that some abnormal DNA methylation of certain sites will lead to inadequate sperm motility, causing Nanzaibuyu with sperm development-related genes （such as EST, KCNQ1, LIT1, SNRPN and CREM, etc.）. Detected some sperm development related gene methylation status, important for early diagnosis and prediction of sperm motility, specific CpG island methylation patterns at the same time from a whole new perspective to clarify the molecular the the asthenospermia development mechanism. The gene methylation spectrum, and for early diagnosis so that patients receive personalized treatment and improve efficacy find asthenospermia.The present study also shows that the decline in abnormal sperm motility of sperm development related gene upstream regulatory gene also has a significant correlation. Increased or decreased expression of the upstream gene, will lead to its downstream gene abnormalities, the occurrence of sperm cell development is disturbed affect sperm motility.It has been the surface, HSPA-2involved in sperm cell production process, it was found that the knockout the HSPA-2mice, will affect the spermatogonia of meiosis, leading to apoptosis of spermatogonia. Studies have shown that there are significant differences in normal subjects and patients with asthenospermia HSPA-2expression. The HSPA-2upstream gene heat shock protein Factor-1（HSF-1） is related to abnormal expression of heat shock protein Factor-2（HSF-2） is the development of abnormal sperm. The study found that HSF-1to HSPA resistance induced apoptosis in male germ cells, research by transcriptional regulation of HSF-1and HSF-2knockout HSF-1transgenic mice proved an important role in sperm production and development.The experimental detected by the MassArray technology platform quantitative HSPA in the normal group,28cases and31cases of idiopathic asthenozoospermia patients semen-2, DLD-2, FH and COX-6, CpG island methylation level; fluorescence quantitative PCR detection of the normal group of43cases and40cases of idiopathic asthenozoospermia patients semen HSPA-2, HSF-1and HSF-2mRNA expression levels to explore the the idiopathic asthenospermia related gene expression in the pathogenesis of abnormal mechanism.Materials and Methods1. Idiopathic weak sperm with normal sperm motility and sample selectionDiagnostic standard （WHO1999fourth edition）, select at least3consecutive semen examinations were diagnosed patients with inadequate sperm motility, and the exclusion of semen liquefaction abnormal urogenital infections, epididymitis or orchitis, systemic disease, fine cause of varicocele, cryptorchidism, unhealthy living habits, specialist medical examinations were normal. The control group for any a routine examination of semen for sperm motility normal volunteers. All samples included in the study are to inform patients and obtain their informed consent to collect adult male sperm and semen samples with normal sperm motility,50%1layer Percoll separation, purification sperm, to explore what kind of separation method is more suitable for the study of this subject.2. Sperm DNA and RNA extraction and detectionCollect sperm samples removed from the refrigerator at-80℃,37℃water For box thaw DNA kit Biotake solution extracted sperm DNA and RNA extracted sperm DNA, Sperm RNA extracted using Trizol; spectrophotometer detected, the determination of the concentration and purity of the DNA and RNA.3. DNA methylation level detection Application of genome-wide genomic DNA extraction kit extracted tissues and cell lines treated with sulfites application MassArray@EpiTYPER technology platform （Sequenom） is detected in the normal group of28cases and31cases of idiopathic asthenozoospermia patients semen HSPA-2, DLD-2, FH and COX-6, CpG island methylation levels.4. mRNA expression levels of detectionIn order to study the normal group of43cases and40cases of idiopathic asthenozoospermia patients semen HSPA-2, HSF-1and HSF-2mRNA expression levels, we used real-time quantitative PCR （Real-Time PCR） detection of gene expression level. GAPDH as reference gene mutation of the RNA content than positive, each sample three independent repeat experiments, serial dilutions of standard templates to make the standard curve.5. The level of methylation analysis in the differences between the groupWilcox rank sum test analysis purposes CpG island methylation level differences in asthenozoospermia group and normal group. The tests were two sided probability test, select the test level a=0.05, data SPSS13.0software analysis.6.The target gene mRNA expression differences in the correlation analysisOne-way the anova analysis purposes gene expression in normal group and the idiopathic asthenospermia group differences using the Spearman test analysis of the normal group with weak azoospermia group of target gene expression.Result1. Study included71patients with low sperm motility,71cases with normal sperm motility.2. The case group asthenospermia patients fast before the sperm （A）, forward sperm （A+B） ratio and density with the control group significantly reduced, the difference was significant. And no significant difference in age, abstinence time and other indicators, the two sets of parameters.3. According to the results of time-of-flight mass spectrometry detection, the methylation level of weak azoospermia group in HSPA-CpG₁9and DLD CpG₂DNA CpG island were significantly different compared with the normal group.4. The expression of HSPA-2and its regulatory gene HSF-1and HSF-2in the asthenospermia group were significantly different from the normal group, and these different expression of HSPA-2, HSF-1and HSF-2are of a significant correlation.Conclusion1. The abnormal expression of HSPA-2may be affected by the abnormal methylation of the CpG₁9sites and the abnormal regulatory gene.2. DLD abnormal expression may be affected by the CpG₂sites aberrant methylation.3. The DNA methylation level of FH and COX-6B and normal controls have no effect in the FH and COX-6B abnormal expression, there may be some other transcriptional regulation mechanisms during the expressoion process of FH and COX-6B.