| As result of the social, environmental, economic and many other reasons, infertility has become a global public health problem, therefore, more and more couples are resorting to assisted reproductive technology (ART). ART is a new technology, which refers to get new life through in vitro manipulation of gametes, embryos or genetic material, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), frozen-thawed embryo transfer (FET), and so on. ART has been performed more than30years. Although the developments of ART have intensified the hopes and the wishes of those couples to resolve their infertility, the delivery rate remains disappointedly low. One major reason for this is that most pregnancies ended with spontaneous abortion (SA). SA refers to pregnancy loss at less than20weeks’ gestation in the absence of elective medical or surgical measures to terminate the pregnancy. Spontaneous abortion is a common medical problem. In the last fifty years, numerous studies have been focused on studying the potential etiologies that lead to SA. Multiple reasons have been proposed. Recently, animal studies have found that imprinting disorder may be associated with SA. However research involving human tissue specimen is still rare. ART is performed just at the time of erasure of parental imprints and imprinting establishment. It is likely that ART might affect the process and cause imprinting disorder. In addition, some studies showed that the single nucleotide polymorphism (SNP) of some imprinted genes is related to placental and fetal growth and development, and the methylation status of imprinted genes as well. However, the reports about SNP and human SA are also still rare. In order to investigate the effect of ART on DNA methylation patterns of imprinted genes, as well as SNP, and the relationship between methylation status, SNP and SA, we analyzed the DNA methylation patterns of three imprinted genes including IGF2, GRB10and PEG3in human chrionic villus samples (CVS) from SA after ART and natural conception in the present study. Besides the association between IGF2and GRB10genetic variant and the susceptibility to pregnancyloss was evaluated. Part I Quantitative methylation analysis of imprinted genes IGF2, GRB10and PEG3in human spontaneous abortion after ART and natural conception [Objective] The delivery rate which remains disappointedly low is an issue of concern. One major reason for this is that most pregnancies ended with SA. Animal studies showed that the imprinting disorder may affect the maintenance of pregnancy. ART procedures are performed just at the time of erasure of parental imprints and imprinting establishment. It is likely that ART might affect the process and cause imprinting disorder. In the present study, we analyzed the DNA methylation patterns of three imprinted genes including IGF2, GRBIO and PEG3in human chrionic villus samples (CVS) from SA after ART and natural conception by pyrosequencing and Bisulfite Sequencing PCR (BSP) to investigate the potential relation between ART, DNA methylation patterns and SA.[Materials and Methods](1) The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all the patients. CVS were collected from women who underwent abortion procedures in Department of Gynecology and Obstetrics in Nanfang hospital from May2008to Nov2012.337samples were collected in total. According to source, the samples were divided into four groups:(A) SA after natural pregnancies (n=84).(B) Induced abortion after natural conceptions (n=94). Fetal heartbeat was observed in these pregnancies. These abortions were performed at the patient’s request for personal or social reasons.(C)SA after ART (n=73). In cases of SA, an intrauterine sac without fetal heartbeat was observed.(D) Fetal reduction by transvaginal ultrasound after ART (n=86). Fetal heartbeat was observed in these pregnancies. Gestational weeks ranged from6to18weeks, and the maternal age ranged from17to45years old. These patients had no known anatomic or genetic abnormalities. Gestational weeks was calculated from the last menstrual period or determined by ultrasound.(2) CVS were washed with physiological saline,. Genomic DNA was extracted using a Genomic DNA Purification Kit (Promega, Madison, WI, USA). Bisulfite treatment of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). PCR was performed to amplify the target gene fragments. The DNA methylation patterns of IGF2, GRBIO and PEG3were analyzed by pyrosequencing, which was performed at PyroMark Q96ID System (Qiagen). The pyrosequencing reactions were performed according to the manufacturer’s instructions. The degree of methylation at each CpG site was determined by Allele Quantification (AQ) software. BSP was then performed to confirm the pyrosequencing results.(3) The statistical analysis program SPSS16.0was used for analyses. Data were shown as mean±SD, or percentage when appropriate. Appropriate statistical tests were used for comparison, including Independent samples t test and Chi-square test. When eaqual variances were not assumed, Satterthwaite t test was used. Analysis of covariance (ANCOVA) was used to exclude the potential effects of maternal age and gestational weeks. Boxplot was used to generate outliers and extreme values. Statistical significance was set at P<0.05.[Results](1) The average methylation percentages of IGF2, GRB10, PEG3gene were54.1±6.1(%)(range18.5-79.3%),48.6±11(%)(range0-80.2%),46.0±6.8(%)(range24.6-71.9%), respectively. Except for GRB10which showed completely non-methylation (0%) in two cases, there was no clear hypomethylation or hypermethylation. Boxplot showed that there were37cases were abnormal methylation status, the overall incidence was3.7%(37/1011). The rates of abnormal methylation level of each gene were showed as GRB10> IGF2>PEG3(4.2%(14/337),3.9%(13/337), and3%(10/337), respectively).(2) All the CVS were divided into ART group and natural conception group according to the way of conception (whether or not ART).159patients were included in the ART group, while178patients in the natural conception group. The age of ART group was significantly older than that in the natural conception group (31.6±3.8vs.29.4±5.6, Satterthwaite t test, t=4.393, P<0.001), and the gestational weeks of ART group were significantly smaller than that in the natural conception group (8.2±1.3vs.9.1±2.0, Satterthwaite t test, t=-4.824, P<0.001). The methylation percentages of IGF2, GRB10, PEG3genes were compared between two groups, which showed no significant differences (IGF2:Independent samples t test, t=1.534, P=0A26(eaqual variances assumed, P=0.136); GRB10:Satterthwaite t test, t=l.218, P=0.224(eaqual variances not assumed, P=0.001); PEG3:Satterthwaite t test, t=-0.849, P=0.396(eaqual variances not assumed, P=0.004)). Concerning the potential effects of maternal age and gestational weeks, ANCOVA was used. And similar results were obtained (IGF2:F=3.625, P=0.058; GRB10:F=1.725, P=0.190; PEG3:F=0.434, P=0.510). The ratios of abnormal methylation of three imprinted genes were further compared between two groups, no significant difference was found (Chi-square test, IGF2:3C2=0.771, P=0.380; GRB10:x^O.026, P=0.872; PEG3:x2=0.028, P=0.868). According the ART procedure, the ART group was then divided into IVF group and ICSI group. The maternal age and gestational weeks were compared between these矛two groups, and no significant difference was found (age:Independent samples t test, t=1.848, P=0.066(eaqual variances assumed, P=0.069); gestational weeks: Satterthwaite t test, t=-1.628, P=0.107(eaqual variances not assumed, P=0.028)). There were no significant differences between these two groups in the methylation level of IGF2, GRB10and PEG3(Independent samples t test, IGF2:t=1.617, P=0.108; GRB10:t=-0.268, P=0.789; PEG3:t=-0.243, P=0.808). So were the ratios of abnormal methylation of three imprinted genes (Chi-square test, IGF2:x2=0.564, P=0.453; GRB10:j■*=0.017, P=0.896; PEG3:■=0351, P=0.550).(3) According to whether or not SA, all the CVS were further divided into SA group and non-SA group.157patients were included in the SA group, while180patients in the non-SA group. The age of ART group was comparable between two groups (31.0■4.8vs.30.0■5.0, Independent samples t test, t=-1.605, P=0.109). However, the gestational weeks of SA group were significantly larger than that in the non-SA group (9.4■2.0vs.8.1■1.4, Satterthwaite t test, t=-6.636, P<0.001). The methylation percentages of IGF2, GRB10, PEG3genes were compared between two groups. Except PEG3gene (eaqual variances not assumed, P=0.031; Satterthwaite t test, t=0.654, P=0.513), significant differences were found between two groups in IGF2and GRB10gene. Both gene showed higher methylation levels in SA groups when compared to the non-SA group (Independent samples t test, IGF2:55.0±5.2vs.53.4±6.8, t=-2.446,P=0.015; GRB10:49.9±10.8vs.47.5±11.1, t=-2.042, P=0.042). Concerning the potential effects of maternal age and gestational weeks, ANCOVA was used. And similar results were obtained. The ratios of abnormal methylation of three imprinted genes were further compared between two groups. The ratio of abnormal methylation level of GRB10in SA group was significantly higher than that in the non-SA group (Chi-square test, x2=5.001, P=0.025). And there were no significant differences between two groups in the ratio of abnormal methylation level of IGF2and PEG3(Chi-square test. IGF2:8/157vs.9/180, x2=0.002, P=0.968; PEG3:2/157vs.6/180, P=0.215).[Conclusions](1) ART might not neither affect the methylation status of imprinted genes IGF2, GRB10and PEG3, nor the occurrence of abnormal methylation status. And compared with IVF, ICSI does not increase the risk of epigenetic abnormality.(2) The methylation status of imprinted gene IGF2and GRB10in CVS might be related to SA, and the imprinted gene PEG3may not be involved. This suggested that imprinting errors may be associated with the occurrence of SA.(3) The possibility that this abnormal methylation seen is not the cause but a consequence of the defect which led to SA cannot be excluded. And the research is limited to minority imprinted gene with partial DMRs. Further studies are required to confirm the conclusions above. Part II Quantitative methylation analysis of developmentally important imprinted genes in human stillbirths from ART and spontaneous conceptions[Objective]SNP refers to the DNA sequence polymorphism caused by a single nucleotide mutation, accounting for more than90%of all known polymorphism. SNP is related to many phenotypic differences, the susceptibility to drugs or diseases, and so on. Recently, the screening of SNP was used to detect the susceptibility genes of S A, but most are limited to the genes associated with immunity. Imprinted gene, a special gene, plays an important role in human growth and development. In recent years, studies on SNP of imprinted genes found some imprinted genes SNP might be related to the placental and fetal growth, birth weight, and so on. There were also some studies which showed that SNP may be associated with the status of methylation of imprinted genes. In this part, we hypothesized that there might be a relationship between SNP of imprinted gene and SA. SNP of four loci including IGF2rs3741205, rs3741206, rs3741211and GRB10rs2237457were detected by pyrosequencing. The relationship of SNP loci and SA were detected.[Materials and Methods](1) The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all the patients. CVS were collected as Part I. CVS were washed with physiological saline,. Genomic DNA was extracted using a Genomic DNA Purification Kit (Promega, Madison, WI, USA). PCR was performed to amplify the target gene fragments. The genotypes of IGF2rs3741205, rs3741206, rs3741211and GRB10rs2237457were analyzed by pyrosequencing which was performed at PyroMark Q96ID System (Qiagen). The pyrosequencing reactions were performed according to the manufacturer’s instructions. The genotype was determined by SNP software. Direct sequencing was then performed to confirm the pyrosequencing results.(2) The statistical analysis program SPSS16.0was used for analyses. Data were shown as mean±SD, and percentage when appropriate. Appropriate statistical tests were used for comparison, including Independent samples t test and Chi-square test. Hardy-Weinberg equilibrium was estimated in both groups using Chi-square test based on allele frequencies which were calculated by counting alleles. ShEsis software was used to analyze the linkage disequilibrium (LD) and haplotype. Statistical significance was set at P<0.05.[Results](1)337patients were included in total. Among them,159were in ART group, and the rest in the natural conception group. No significant deviation from the Hardy-Weinberg equilibrium in both groups was observed. There were also no significant differences in the frequencies of wild genotype and mutant genotype of IGF2rs3741205, rs3741206, rs3741211and GRB10rs2237457in CVS between two groups (Chi-square test. rs3741205:x2=0.064, P=0.801; rs3741206:x2=0.625, P=0.429;rs3741211:x2=0.006, P=0.936; rs2237457:x2=0.072, P=0.789).(2) In the natural conception group, according to whether or not SA, two sub-groups were generated:SA group and non-SA group. There were84patients included in the SA group, and94patients included in the non-SA group. No significant deviation from the Hardy-Weinberg equilibrium in both groups was observed. There were also no significant differences in the frequencies of wild genotype and mutant genotype of IGF2rs3741205, rs3741206, rs3741211and GRB10rs2237457in CVS between these two groups (Chi-square test. rs3741205: x2=2.049, P=0.152; rs3741206:x2=0.606, P=0.436; rs3741211:x2=0.089, P=0.765; rs2237457:x2=0.014, P=0.905).(3) In the ART group, two sub-groups were also generate:SA group and non-SA group (fetal reduction) according to whether or not SA.73patients were included in the SA, and86were in the non-SA group. No significant deviation from the Hardy-Weinberg equilibrium in both groups was observed. The frequencies of IGF2rs3741211TC+CC genotype and TT genotype were difference in two groups (Chi-square test,x2=3.973, P=0.046). Compared with TT genotype, the TC+CC genotype had a1.91-fold increased risk of SA after ART (95%CI:1.008-3.629). There were no significant differences in the distribution of the rest loci (Chi-square test, rs3741205:x2=1.887, P=0.170; rs3741206:x2=0.955, P=0.328; rs2237457: x2=0.363, P=0.547). The relationship between the methylation status of IGF2, H19, KvDMR1and rs3741211TC+CC genotype were further investigated. The differences of the methylation status of IGF2, H19, KvDMR1between the group of TC+CC genotype and the group of TT genotype were compared. The methylation level of KvDMRl was significantly higher in the group of TC+CC genotype than that in the group of TT genotype (51.2±11.8vs.47.5±7.3, Satterthwaite t test, t=-2.007, P=0.048). No significant differences were found between two groups in the methylation level of IGF2and PEG3, though higher level was showed in the TC+CC genotype (IGF2:eaqual variances not assumed, P=0.036; Satterthwaite t test, t=-0.549, P=0.584; H19:eaqual variances assumed, P=0.083; Independent samples t test, t=-1.215, P=0.227). SHEsis analysis software were used to detect the LD among IGF2rs3741205, rs3741206and rs3741211. A strong LD was showed. Then haplotype analysis was made. There were eight haplotypes in total, which were:GAT, GGT, TAC, TAT, TGC, TGT, GAC and GGC. Among these haplotypes, only GGT showed different frequency between two groups (SA vs. non-SA:4%vs.0.7%, x2=3.896, P=0.048). However, it is not a risk factors for SA after ART (OR:5.640, 95%CI:0.821-38.761).[Conclusions](1) ART might not affect the distribution of genotype of IGF2rs3741205, rs3741206, rs3741211, and GRB10rs2237457.(2) In natural conception, IGF2rs3741205, rs3741206, rs3741211and GRB10rs2237457in CVS might be unrelated to SA. However, IGF2rs3741211TC+CC genotype in CVS might be related to SA after ART. And this genotype may be associated with the methylation level of KvDMR1gene. The GGT haplotype (rs3741205, rs3741206, rs3741211) may be associated with SA after ART, but due to the small sample size, it is not sure whether it is a risk factor of SA. Other loci were unrelated to SA after ART.(3) Larger sample size was needed to confirm the preliminary conclusions. Further studies related to the SNP functional were also required. |
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