| Background and ObjectiveSpontaneous abortion (SA) is a common pathological pregnancy. The incidence is high both at home and abroad, with the rate at10%to15%of all pregnancies in domestic, and more than80%of spontaneous abortion occurred in early pregnancy (pregnancy of12weeks or less). There are various possible etiological factors associated with SA, involving genetic factors, immune factors, endocrine factors, reproductive tract anatomic abnormalities, infection factors, trauma as well as environment and other unknown reasons. It is still unable to find the certain causes for60%to70%of SA by the existing medical technology and inspection means, which have greatly improved. Tested by the traditional cytogenetics observation (karyotype analysis), the incidence of chromosomal abnormalities is more than50%happened in less than15weeks of pregnancy. The research results of our laboratory also indicated that chromosomal abnormalities accounted for53.3%(88/165) of the early SA, but46.7%(77/165) normal karyotype can not find the causes. Therefore, it is still necessary to make further exploration in the etiology and pathogenesis of spontaneous abortion. Epigenetics can be defined as a branch subject of genetic referring to alterations in DNA function without alterations in DNA sequence. DNA methylation is the major form of epigenetic information. In mammals, genomic DNA methylation refers to transferring the methyl from s-adenosylmethionine (SAM) to5-Position of carbon atoms of Cytosine and generating5-methyl cytosine (5-dmC) with the catalytic action of DNA methyltransferase (Dnmt). In the DNA molecule, the amount of5methyl cytosine represent the level of DNA methylation. In mammals, the Normal patterns of DNA methylation are very important for the growth and development. The specific methylation patterns are established during the development of embryo through gene imprinting, gene silencing, female X chromosome inactivation et al, and transmit stably in the process of development and proliferation of embryo. The methylation patterns play an important role in the whole process of growth and development. Thus, it speculated in theory that aberrant DNA methylation may be associated with spontaneous abortion.However, the study on DNA methylation level is mostly used in various tumors, such as stomach cancer, colon cancer, etc. It is rare for the study on the relationship between DNA methylation level and spontaneous abortion. So far, there are only2pieces of paper available. Cezar, who studied the relationship between the whole genome methylation level and cloned embryo development ability in1983, involved in the entire genome methylation level of9cases of spontaneous abortion cattle fetus,7cases of fetus survival of cattle, and11cases of healthy adult cattle. And, Li-Jun Y compared the DNA methylation level with the expression of Dnmtl, Dnmt3a among the16cases of spontaneous abortion villus and16cases of induced abortion villus for the analysis of the role of DNA methylation level in spontaneous abortion. The results showed that DNA methylation level may be associated with spontaneous abortion. Nevertheless, the results above are not strong enough to sustain the conclusion about the relationship between DNA methylation level and early abortion due to the limited research reports and the small sample size.The activity of DNA methyltransferase, abnormal histone methylation, RNA interference and diet environment factors are likely to affect the level of DNA methylation according to the previous studies. DNA methylation can only be achieved under the catalysis of enzymes. Being the key enzyme of DNA methylation, DNA methylation transferase plays an critical role in the process. Hitherto, three main DNA methyltransferases with methyl transferase activity, including Dnmtl, Dnmt3a and Dnmt3b, have been identified. As one of the main mammals DNA methyltransferase, Dnmtl, which expresses in the proliferation of cells in the replicated state, and preferentially catalyze half a double-stranded DNA methylation after replication, can make the low methylation substring fully methylation. Dnmtl plays an important role in the maintenance of DNA methylation by the determination in the normal replication patterns of cellular DNA methylation. Dnmt3a and Dnmt3b, which act on the double-stranded DNA methylation unmethylated, exert their main function by establishment of methylation patterns in gamete formation and early embryo. Moreover, these two DNA methyltransferases can complement each other in the catalytic function, with Dnmt3a primarily making the outside of nucleosome naked part of DNA methylated, while Dnmt3b making the core methylated.So far, there are many reports on the DNA methyltransferases and the degree of DNA methylation at home and abroad focusing on the study of tumor cells. However, the studies on the association between DNA methylation and SA are rare with inconsistent results. The researches carried by Li-Jun, Liu Y Q, Robert, Suzuki and others show that Dnmtl plays a dominant role in DNA methylation process, while Rheel and Leu show that, in the process of DNA methylation, Dnmtl and Dnmt3b work together. And the two experiments operated by Ting illustrate that the degree of dependence on Dnmtl is different among various cells, namely Dnmtl does not play a leading role in the process of methylation in some cells. However, there is only one paper on the effect of DNA methyltransferases to DNA methylation. Li-Jun Y et al determined and compared the DNA methylation level and the expression of Dnmtl, Dnmt3a proteins of the16cases of spontaneous abortion villus and16cases of induced abortion villus. The results show:Compared with the induced abortion villus, the presence of DNA methylation levels decrease in spontaneous abortion. As for the deficient expression of Dnmtl protein, there appears to be a correlation between Dnmtl deficiency and the reduced DNA methylation level of chorionic villus of spontaneous abortion. However, The questions, of both what is the role of DNA methyltransferase playing on the promotion and maintenance process of DNA methylation in spontaneous abortion villus, and what is the relationship between them, still remain unsettled due to such few researches above.Based on the analysis above, the aim of this study is to explore the relationship between the level of DNA methylation and unexplained early spontaneous abortion, and the role of DNA methyltransferase Dnmtl, Dnmt3aand Dnmt3b playing on the promotion and maintenance of DNA methylation in the villus of early spontaneous abortion. This experiment includes three parts as followed:In the first part:The establishment of the detection method of global DNA methylation level by high performance liquid chromatograph (HPLC). The chromatographic conditions:Agilent1100high performance liquid chromatography (Agilnt G1311A pump, AgilntG1316A UV detector, Agilnt G1313A autosampler), Thermo C18column (6mm×250mm,5μm);0.1%formic acid and methanol(volume ratio99:1) serve as the mobile phase; the flow rate is1ml/min, the injection volume was20μ1, the UV detection wavelength is280nm, the column temperature is30℃.(1) The optimization of ultraviolet absorption wavelength. Chromatographic scanning in240-300nm is carried out with the two standard mixture in order to choose the best Ultraviolet absorption wave length.(2) Establishment of standard formulas.5pipes of standard solution were prepared. The concentration of deoxycytidine (dc) were1,5,10,50,100μmol/L and5-dmc concentrations were0.05,0.25,0.5,2.5,5μmol/L, respectively. Then inject the samples in turn. Draw standard curve of the dC and5-dmC with the concentration (μmol/L) and corresponding peak area.(3) Precision experiment. Inject the5pipes of two standard mixed solution continuously5times, then calculate the standard deviation and relative standard deviation (RSD) of the peak area and retention time to reflect the precision of the instrument.(4) Stability experiment. Inject the5pipes of two standard mixed solution5times every hour, then calculate the standard deviation and relative standard deviation (RSD) of the peak area and retention time to reflect the stability of the samples and the instrument.(5) Specificity experiment. Pure water is used instead of the enzyme hydrolysis of samples and DNA solution as the negative and blank controls, the standard and the sample as well as the blank control. And inject the negative control continuously. The retention time and peak shape are compared to determine the specificity of the method for the detection of dC and5-dmc.In the second part:Study on relationship between DNA methylation level and unexplained early spontaneous abortion. This part of the experiment contains four groups:the spontaneous abortion mother group, spontaneous abortion villus group, induced abortion mother group and induced abortion villus group. The dC and5dmC peak area of each sample are measured by the HPLC method eatablished in the first part. The ratio of5-dmc/(dc+5-dmc) ratio was determined according to the area of dc and5-dmc, which represents the overall level of DNA methylation,The level of DNA methylation (%)=5-dmC/(5-dmC+dC)×100%In the third part:Research of the effect of Dnmtl, Dnmt3a and Dnmt3b on early spontaneous abortion villus genome DNA methylation in the light of their expression. This part of the experiment contains two groups:spontaneous abortion villus group and induced abortion villus group. The expression of Dnmtl, Dnmt3a, Dnmt3b mRNA was measured by qRT-PCR in villi tissues, respectively.The results were as followed:In the first part:The establishment of the detection method of global DNA methylation level by high performance liquid chromatograph (HPLC)(1) The optimization of ultraviolet absorption wavelength.The result of Chromatographic scanning in240-300nm with the two standard mixture showed that the absorbance of dC were similar between280-295nm, with the maximum absorption of5-dmC at280nm, which was chosen as the optimization wavelength ultraviolet absorption.(2) Establishing of standard formulas. The standard formulas of the dC and5-dmC were drawn with the concentration (μmol/L) and corresponding peak area. The standard formulas for dC is:Y=0.17X+0.0004, r=0.999; The standard formulas for5-dmC is:Y=0.18X+1.711， r=1. These results showed there was a good linear relationship between concentration and peak area.(3) Precision experiment. The retention time for dC is:5.98±0.09, RSD=1.5%. The peak area for dC is:5540.3±59.13, RSD=1.1%. The retention time for5-dmC is:11.38±0.17, RSD=1.5%. The peak area for5-dmC is:259.2±3.38, RSD=1.3%. The results show that the precision of the instrument is fine.(4) Stability experiment. The retention time for dC is:5.94±0.83, RSD=1.3%. The peak area for dC is:5536.1±61.43, RSD=1.1. The retention time for5-dmC is: 11.45±0.11, RSD=0.9%. The peak area for5-dmC is:258.06±2.33, RSD=0.9%.The results show that the stability of the instrument is fine.(5) Specificity experiment. The peak pattern of the blank control, the negative control and the sample were compared with the standard. There was no target peak type in the blank control and the negative control. There was target peak type and other impurity peak in the sample, while the impurity peak cannot cause interference to the target peak. The results show that the Specificity of the method is fine.In the second part:Study on relationship between DNA methylation level and unexplained early spontaneous abortion.(1) Comparison of the mothers’age between spontaneous abortion and induced abortion group mother group.In the34cases of spontaneous abortion, the average age of mother was30.85±5.28(years). In the33cases of induced abortion, the average age of mother was29.52±4.95(years). No significant difference of age was found by statistic analysis (P=0.289).(2) Comparison of DNA methylation level between spontaneous abortion and induced abortion group mother group.In the34cases of spontaneous abortion, the average DNA methylation level of mother was4.41±0.65. In the33cases of induced abortion, the average DNA methylation level of mother was4.15±0.63. No significant difference of DNA methylation level was found by statistic analysis (P=0.103).(3) Comparison of DNA methylation level between spontaneous abortion and induced abortion group villus group.In the45cases of spontaneous abortion, the average DNA methylation level of mother was2.29±0.37.In the44cases of induced abortion, the average DNA methylation level of mother was4.56±1.02. Significant difference of DNA methylation level was found by statistic analysis (P=0.000).In the third part:Research of the effect of Dnmtl, Dnmt3a and Dnmt3b on early spontaneous abortion villus genome DNA methylation by their expression.(1) Comparison of Dnmtl mRNAlevel between spontaneous abortion villus group and induced abortion villus group.In the30cases of spontaneous abortion, the average Dnmtl mRNA level of villus was0.033±0.007.In the33cases of induced abortion, the average Dnmtl mRNA level of villus was0.071±0.0172. Significant difference of Dnmtl mRNA level was found by statistic analysis (P=0.000).(2) Comparison of Dnmt3a mRNA level between spontaneous abortion villus group and induced abortion villus group.In the30cases of spontaneous abortion, the average Dnmt3a mRNA level of villus was0.072±0.013. In the33cases of induced abortion, the average Dnmt3a mRNA level of villus was0.184±0.049. Significant difference of Dnmt3a mRNA level was found by statistic analysis (P=0.000).(3) Comparison of Dnmt3b mRNA level between spontaneous abortion villus group and induced abortion villus group.In the30cases of spontaneous abortion, the average Dnmt3b mRNA level of villus was0.008±0.003. In the33cases of induced abortion, the average Dnmt3bmRNA level of villus was0.005±0.001. Significant difference of Dnmt3b mRNA level was found by statistic analysis (P=0.071).Conclusions(1)The high performance liquid chromatography method established by the experiment can detect genome DNA methylation level accurately and sensitively. The precision and stability of instrument is fine, and the specificity of the method for the detection of dC and5-dmc is good. (2) There exists lowing level of genomic DNA methylation in spontaneous abortion villus tissues. Insufficient maintenance of DNA methylation is likely to be a spontaneous abortion risk factor independent of chromosomal abnormalities.(3) Dnmtl and Dnmt3a, but not Dnmt3b might play a major role in the drop of genomic DNA methylation level in villus. |
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