Quantitative Methylation Analysis Of PEG1 In Chorionic Villus Obtained From ART Or Spontaneous Pregnancy

Assisted reproductive technologies (ART) are important medical treatments for infertile people at reproductive age and are assumed to account for 1-3% of the total annual births in developed countries. Although ART and related procedures have been considered one of the safest medical treatments, few people born with the assistance of ART have yet reached adulthood and the assessment of long-term outcomes of ART pregnancies is lacking. Recently, some human and animal studies have suggested a possible link between ART and certain birth defects related to epigenetic abnormalities. But the absolute risk of imprinting disorder post-ART is too small to make a definitive statement. ART may affect epigenetic regulation, particularly the expression of imprinted genes, thus affecting the embryo implantation, placentation, organ formation and fetal growth. The prevalence of miscarriage has been estimated asl5-20% of all clinically recognized pregnancies. To some extent, the occurrence of early miscarriage reduced the birth effects. Some studies suggests that genomic imprinting defects affecting the maintenance of early pregnancy. Our study used two different methods to obtain the PEG1 methylation values in four groups of chorionic villus. The goal of present study is designed to investigate the possible correlationship between ART and imprinting diseases. PartⅠDetermination of quantitative DNA methylation of PEG1 by COBRAOBJECTIVETo evaluate the correlation between methylation of PEG1 and early pregnancy. The methylation status of PEG1 was assessed using combined bisulfite restriction analysis (COBRA). Four groups of chorionic villus have been analyzed.MATERIALS AND METHODSChorionic villus come from multi-fetal reduction (12 samples) and spontaneous abortion of ART(31 samples), early pregnancy (24 samples) and spontaneous abortion (29 smaples) of nature pregnancy were collected between May 2008 and December 2009 at Nanfang hospital. The duration of pregnancy ranged from 6 to 10 weeks. There was no anatomic and genetic abnormalities existed in spontaneous abortion patients. The early spontaneous abortion inclusion criteria included:no fetal heart beat, empty gestational sac and gestational sac less than gestational weeks.All chorionic villus were collected under the sterile condition. Villi genomic DNA was extracted by Wizard Genomic DNA Purification Kit (Promega, America). DNA of villus was bisulfite converted with the MethylDetector Bisulfite Modification Kit (Active Motif, America). The PEG1 was amplified by PCR of the bisulfite converted DNA. The primers were (forward primer F) 5'-TYGTTGTTGG TTAGTTTTGTYGGTT-3', and (reverse primer R) 5'-CCCAAAAACAACCCCAA CTC-3'. To determine the overall methylation pattern, COBRA was performed with the Taq I restriction enzyme, which recognizes a single site in the bisulfite converted PEG1 sequence and cuts only methylated DNA. The purified PCR product was digested with 5 U Taq I restriction enzyme (Promega, Madison, WI) at 65℃for 3-3.5 hours in a total volume of 20 mL and separated by 4% agarose electrophoresis. Gel images were saved as TIFF files. Band densities were determined using Labworks image acquisition and analysis software (Ultra-violet Products, Ltd., Cambridge, UK). Percent methylation was calculated as the density ratio of the methylated bands to the two total bands.Data were analyzed using SPSS version 13 software (SPSS Inc., Chicago, IL), all tests were two-tailed, and P<0.05 was considered statistically significant. Continuous variables were presented as mean±SD and were tested by one-way ANOVA analysis. Count data were presented as percentage (%).RESULTSThere was no extremely high methylation (100%) or low methylation (0%) existed in the tested specimens. The fluctuations of methylation values were 39.3-83.7% in early abortion of ART,38.9-75.4% in early abortion of nature pregnancy, and 42.1-58.3% in normal pregnancy,423.4-56% in normal pregnancy of ART. Methylation in PEG1 showed no significant difference among the four groups. Defined the mean±2SD methylation values of nature pregnancy as normal methylation range (39.0-59.0%), the methylation in two abortion groups were compared. Three were both seven specimens were outlined the range in ART abortion No significant difference existed between the two groups (χ2= 0.020, P= 0.887).CONCLUSIONSNeither full methylation nor unmethylation values have been detected in our study. We speculated that extreme methylation abnormalities may restrict the early development of embryos and prevent the occurrence of implantation. There was no significant difference between the two abortion groups in methylation values of PEG1. Because only one CpG site was studyed in this part, further quantitative analysis needed to make a more definitive conclusion.PartⅡDetermination of quantitative DNA methylation of PEG1 by PyrosequencingOBJECTIVEFour groups of chorionic villus as described in partⅠwere analyzed by pyrosequencing. The effective of Bisulfite conversion and DNA methylation of four CpG sites have been evaluated in this part.MATERIALS AND METHODSThe collection and grouping of chorionic villus were as same as in the first part of present study.Four CpG sites of the PEG1 were selected for analysis by pyrosequencing. This region was amplified by PCR from bisulfite-treated genomic DNA isolated from four groups of chorionic villus. The following primers were used (GenBank AC144863, nucleotide positions 17954-18263):forward5'-TYGTTGTTGGTAGTTTGTAYGGT T- 3', and reverse Biotin-5'-CCCAAAAACAACCCCAACTC-3'. Pyrosequencing reactions were conducted by PyroMark 96 (Bitoage, America) using the following pyrosequencing primer:5'-GAGGGGGTGTGGTTG-3'.The average level of the PEG1 methylation was calculated used the four CpG sites methylation values. Bisulfite conversion efficient was been evaluated by two T/C sites. Several samples of sperm were pysequenced as standard criteria of unmethylation.RESULTS1. The theoretical methylation valures corresponds perfectly to the sequence found by pyrosequencing. The quantitative methylation values vary over a range of up to 5.3%, and bisulfite conversion efficiency were 95.6%-97.5%. This is also the range of the standard deviation obtained by the pyrosequencing system.2. In present study, we found that the fluctuations in the bisulfite-treated efficiency ranged from 89.9% to 100%. Methylation values have been adjusted as following:exact methylation values=metylation measured by pyrosequencing-(95%- conversion). Methylation levels in all chorionic villus appeared to vary up and down from a low of 42% to a high of 85.3%. Extremely high or low methylation did not exist in chorionic villus. The mean methylation values showed significant among the four groups. This effect was due to a reduction of methylation in the normal pregnancy of nature fertilization. Methylation levels in normal pregnancy of ART also showed the same trend, because of the small sample studied, no statistical significant was obtained.3. There was no significant methylation difference existed between IVF and ICSI.4. To evaluate the relationship between methylation levels and early pregnancy loss, all chorioic villus were regrouped as abortion group and normal group. A ROC carve was carried out. The under carve area was 0.684 (95%CI: 0.577～0.791) (SD=0.055,P=0.003). We speculated that the higher the methylation level, the possibility of early miscarriage occurs also greater.CONCLUSIONSAssisted reproductive technology did not affect the imprint genes. ART as a treatment for infertility was general safe. The deviation of methylation levels in early miscarriage groups were significant greater than those in normal pregnancy groups. The ROV carve also suggested that miscarriage happened more frequency in the high methylation level group. High methylation level of PEG1 may induce the occurrence of early miscarriage. Further study was needed to derive a definite conclusion.Pyrosequencing was more effective and suitable for methylation quantitative analysis comparing with COBRA. Although the two methods got the similar conclusion in the population level, pyrosequencing was more suitable than COBRA in the quantitative analysis of individual sample.PartⅢKaryotype of chorionic villus and methylation of PEG1 in relation to early miscarriageOBJECTIVEThe purpose of this part was to explore the relationship between methylation values of PEG1 and the frequency of chromosomal abnormalities in chorionic villus obtained from patients with early miscarriages. MATERIALS AND METHODS Chorionic villus and peripheral blood from patients with early miscarriage were collected in Nanfang Hospital between May 2008 and December 2009. Chromosomal analysis of villus and blood was all conducted by Department of Cell Biology and Medical Genetics. The methylation values of chorionic villus were examined by pyrosequencing.Data were analyzed using SPSS version 13 software (SPSS Inc., Chicago, IL), all tests were two-tailed, and P<0.05 was considered statistically significant. Continuous variables were presented as mean±SD and were tested by Welch's test.RESULTSThere was no abnormal karyotype existed in maternal peripheral blood. One villi sample was failed to culture due to the contamination of blood. The karyotypes were ascertained with use of a standard G-banding technique. Among the 25 chorionic villus,15 (60%) had abnormal chromosome. The abnormal karyotypes included tetrasomy, trisomy, triploidy, mosaic and inversion. The results showed that methylaion values in abnormal karyotype group was 65%, and 59.5% in normal karyotype group. There was no statistical difference in methylation level between the normal and abnormal karyotype groups (F=2.505, P=0.128), because of the small simple recruited in study.CONCLUSIONSThe increased metylation level in abortion chorionic villus may induced by abnormal karyotype. The definite conclusion need further study.