38 EFFECTS OF 5-AZACYTIDINE ON DNA HYPERMETHYLATION STATUS OF CLONED MINIATURE PIG EMBRYO

2009 ◽  
Vol 21 (1) ◽  
pp. 119
Author(s):  
H. S. Kim ◽  
Y. I. Jeong ◽  
J. H. Kim ◽  
J. Choi ◽  
Y. W. Jeong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Level ◽  
Methylation Status ◽  
Cell Mass ◽  
Somatic Cells ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Donor Cells

During somatic cell nuclear transfer, somatic cells should undergo epigenetic reprogramming in order to attain successful totipotency. Although there are many reasons of low efficiency of cloning, the aberrant DNA methylation status in donor cells is thought to reduce efficiency and increase abnormalities in cloned embryos. The methylation level of cloned embryos is higher than that of in vivo-produced or in vitro-fertilized embryos before occurring de novo methylation. This hyper DNA methylation status has been considered as a reason for the abnormal development of cloned embryos and the related decrease in term number. The aim of this research is to validate the DNA methylation pattern in cloned porcine embryos and to confirm whether reduction of hyper DNA methylation levels results in an improvement in cloning efficiency. First, immunostaining was used to evaluate the stage-specific changing pattern of DNA methylation of cloned embryos. In this result, we demonstrated that the 1-cell stage embryos and blastocyst had the highest level of methylation compared to 2- or 4-cell stage embryos. Second, this methylation level was higher in SCNT-derived embryos compared to parthenogenic embryos, suggesting that the methylation level was associated with the nucleus of donor cells. Third, cloned embryos were treated with various concentration of 5-azacytidine, which is a demethylating agent, to find adequate concentration. In results, 500 nm of 5-azacytidin group from 0 to 24 h after activation produced significantly more blastocysts compared to control group (control; 5.4 ± 2.3, 500 nm/0 24-h treatment; 12.7 ± 2.1, P < 0.05) and also showed low DNA methylation level of inner cell mass different from that of control group. In conclusion, our results suggest that cloned porcine embryos show typical demethylation. But, they are generally on a hyper level of DNA methylation. This high DNA methylation level is associated with somatic cells and affects on blastocyst development. We also suggest that 5-azacytidine could improve blastocyst development rate of cloned porcine embryos by aiding weak and abnormal demethylation process.

scholarly journals Combination Analysis of NR3C1, MTHFR and IGFBP3 Gene Polymorphisms and DNA Methylation With Steroid-induced Osteonecrosis of the Femoral Head Risk in Chinese Han Population 

2020 ◽  
Author(s):  
Ronglan Huang ◽  
Qinghao Zhan ◽  
Wenbin Hu ◽  
Renmin Yang ◽  
Nan Cheng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Femoral Head ◽  
Methylation Level ◽  
Gene Polymorphisms ◽  
Methylation Status ◽  
Chinese Han Population ◽  
Control Group ◽  
Chinese Han ◽  
Han Population ◽  
Wide Range

Abstract Background: Although the precise etiology of osteonecrosis of the femoral head (ONFH) has yet to be fully elucidated, it is known that nuclear receptor subfamily3, group C, member 1 (NR3C1), 5, 10-methylenetetrahydrofolate reductase (MTHFR) and insulin-like growth factor-binding protein 3 (IGFBP3) are related to the pathophysiology of steroid-induced osteonecrosis of the femoral head (SONFH). The expression of NR3C1, MTHFR and IGFBP3 are regulated by epigenetics and genetic profiles. Objective: The primary objective of this study was to investigated the association between NR3C1, MTHFR and IGFBP3 gene polymorphisms and DNA methylation status and SONFH.Methods: This case-control study included 79 patients with SONFH and 114 patients who took steroids but did not develop SONFH. We evaluated 5 single-nucleotide polymorphisms (SNPs) out of 3 genes in Chinese Han population. These SNPs were genotyped by improved multiplex ligation detection reaction (iMLDR). Methyltarget was used to test the methylation level of positive sites, the interaction between SNPs and DNA methylation level was analyzed using eQTLD technique.Results: We identified rs3110697 in the IGFBP3 gene that was potentially associated with a reduced risk of SONFH in the genotype (P=0.008; odds ratio [OR]: 0.741; 95% confidence intervals [CI]: 0.456–1.205) and in the recessive model (P=0.003; OR: NA; 95% CI: NA–NA). Furthermore, CpG sites with significant differences in methylation levels were screened as follows: IGFBP3_2-143, MTHFR_1-36, MTHFR_1-77, MTHFR_1-139, MTHFR_2-42, NR3C1_2-163, NR3C1_4-47, and the differences were statistically significant compared with the control group (p<0.05). A total of 10 pairs of linear regression tests of SNP and methylation sites were statistically significant (p<0.05).Conclusions: SONFH is a polygenic disorder in which a wide range of interactions between SNPs and DNA methylation levels may dominate the course of the disease.

42 DEVELOPMENTAL POTENTIAL AND REPROGRAMMING EFFICIENCY OF BOVINE EMBRYOS CLONED WITH ADULT FIBROBLASTS TREATED BY A DEMETHYLATING AGENT, S-ADENOSYL-HOMOCYSTEINE

2006 ◽  
Vol 18 (2) ◽  
pp. 130 ◽  
Author(s):  
B.-G. Jeon ◽  
S. D. Perrault ◽  
G.-J. Rho ◽  
D. H. Betts ◽  
W. A. King
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Somatic Cells ◽  
Telomerase Activity ◽  
Dna Demethylation ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Reprogramming Efficiency ◽  
Low Efficiency

Animal cloning by somatic cell nuclear transfer (SCNT) has been successfully applied to several species although with low efficiency and often associated with severe abnormalities. These poor outcomes are thought to be a consequence of aberrant DNA methylation patterns that result from incomplete epigenetic reprogramming of the transplanted nucleus into recipient oocytes. Telomerase, an enzyme not expressed in most somatic cells, should be expressed in cloned embryos. Therefore its activity has been used as an index of reprogramming in SCNT embryos. The objective of this study was to investigate the DNA methylation status of donor fibroblasts treated with a non-cytotoxic transmethylation inhibitor, S-adenosyl homocysteine (SAH), and to assess the relative telomerase activity (RTA) and developmental potential of SCNT embryos derived from such cells. Adult ear skin fibroblasts were cultured in DMEM supplemented with 0, 0.5, 1.0, or 2.0 mM SAH for 144 h by daily media change prior to nuclear transfer. The SAH-treated fibroblasts were immunostained with a fluorescein isothiocyanate (FITC) conjugated 5-methylcytosine antibody and the relative fluorescence intensity (RFI) was analyzed using a fluorescence microscope equipped with an Openlab" program (Improvision, Coventry, UK). RTA was measured in Day 8 SCNT blastocysts using the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP). Fibroblasts treated with 0.5, 1.0, and 2.0 mM SAH showed lower levels of DNA methylation compared to nontreated controls, and the values did not differ among the treatment groups. Cleavage rates did not differ between the SCNT embryos derived from 0.5 mM SAH-treated cells and nontreated control cells (92.3% vs. 91.3%, respectively). However, the rates of blastocyst development and hatching were significantly (P < 0.05) higher in SCNT embryos derived from 0.5 mM SAH treated donor cells compared to controls (60.0 and 40.0% vs. 34.3 and 26.4%, respectively). Moreover, RTA of the 0.5 mM SAH SCNT embryos was significantly (P < 0.05) increased (1.5-fold) in relation to controls. S-adenosyl homocysteine treatment induces global DNA demethylation in donor fibroblasts and enhances the blastocyst frequencies for bovine SCNT embryos that also exhibit greater telomerase activity levels. These results suggest that use of hypomethylated donor somatic cells increases the developmental potential for SCNT embryos by enhancing the nuclear reprogramming efficiency. This work was funded by NSERC, OMAF, OCAG, and CRC.

29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

2021 ◽  
Vol 33 (2) ◽  
pp. 122
Author(s):  
T. Somfai ◽  
N. T. Hiep ◽  
K. Kikuchi ◽  
Y. Hirao
Keyword(s):  
Dna Methylation ◽  
Embryo Development ◽  
Polar Body ◽  
Immature Stage ◽  
Negative Control ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference ◽  
Polar Body Extrusion

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P&lt;0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.

scholarly journals DNA methylation of DISC1 gene in schizophrenia using methylight Taqman assay

2016 ◽  
Vol 15 (1) ◽  
Author(s):  
Nour El Huda Abd Rahim ◽  
Mohd Nabil Fikri Rahim ◽  
Norsidah Ku Zaifah ◽  
Hanisah Mohd Noor ◽  
Kartini Abdullah ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Level ◽  
Clinical Symptoms ◽  
Smoking Status ◽  
Methylation Status ◽  
Genetic Defect ◽  
Taqman Assay ◽  
Control Group ◽  
Antipsychotic Treatment ◽  
Significant Difference

Introduction: Significant evidences from functional studies have shown that DISC1 gene has a role in the pathogenesis of schizophrenia, although the basis of the genetic defect has yet to be established. There has been a shift of emphasis from DISC1 gene variations to other types of genetic defects namely copy number and epigenetic, both of which have not been well investigated. Thus, the aim of this study is to examine the DNA methylation status of DISC1 gene in patients with schizophrenia. Methods: In this study, 239 subjects were included, 117 schizophrenia patients and 122 healthy controls. Genomic DNA was derived from peripheral blood and bisulfite converted. The DNA methylation level was quantitatively measured by Methylight Taqman analysis. Sociodemographic and the clinical parameters were noted. The severity of the clinical symptoms was assessed using Positive and Negative Syndrome Scale (PANSS). Results: The mean age and gender distribution between the study groups were similar. There was no significant difference in the methylation level of DISC1 between the patients and control group. When patients were compared by age, duration of illness, age at diagnosis, body mass index, smoking status, PANSS score and types of antipsychotic treatment, the DNA methylation level of DISC1, did not show any significant difference (p>0.05). Conclusions: This study found no significant difference in methylation level of DISC1 gene between schizophrenia patients and healthy control. Therefore, it is suggested that aberrant DNA methylation of DISC1 most probably does not contribute to the pathogenesis of schizophrenia.

scholarly journals Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Mai Mahmoud Shaker ◽  
Taghreed Abdelmoniem shalabi ◽  
Khalda said Amr
Keyword(s):  
Dna Methylation ◽  
Dna Sequences ◽  
Promoter Region ◽  
Pregnancy Loss ◽  
Recurrent Pregnancy Loss ◽  
Methylation Status ◽  
Control Group ◽  
Case Group ◽  
Methyl Radical ◽  
Fertile Women

Abstract Background DNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. Results The median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). Conclusion Findings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

scholarly journals ANALYSIS OF THE ASSOCIATION OF THE METHYLATION LEVELS OF MIR10B AND MIR21 GENES IN BLOOD LEUKOCYTES WITH ADVANCED CAROTID ATHEROSCLEROSIS

2018 ◽  
Vol 33 (2) ◽  
pp. 77-82
Author(s):  
Iu. A. Koroleva ◽  
A. A. Zarubin ◽  
A. V. Markov ◽  
A. N. Kazancev ◽  
O. L. Barbarash ◽  
...  
Keyword(s):  
Risk Factors ◽  
Dna Methylation ◽  
Methylation Level ◽  
Carotid Atherosclerosis ◽  
Gene Promoter ◽  
Control Group ◽  
Coding Region ◽  
Blood Leukocytes ◽  
Atherosclerosis Risk Factors

Complications of atherosclerosis remain the leading cause of morbidity and mortality worldwide. MiRNAs are short regulatory molecules that are involved in all processes of pathogenesis. Expression of miRNAs is regulated by DNA methylation. Methylation and/or expression of MIR10B and MIR21 genes are known to vary in atherosclerotic tissues of the arteries, but there is no data about the changes in the methylation levels of these genes in blood leukocytes and their association with atherosclerosis risk factors.Objective.To evaluate the association of methylation levels of MIR10B and MIR21 genes in the blood leukocytes with risk factors and pathogenetically significant traits of carotid atherosclerosis.Material and Methods. DNA for the study was extracted from the samples of blood leukocytes of 122 patients with advanced carotid atherosclerosis as well as from blood leukocytes of 135 individuals in the control group. The DNA methylation level was analyzed by bisulfite pyrosequencing.Results.The methylation level of the MIR10B and MIR21 genes in leukocytes of patients with atherosclerosis is higher than in the leukocytes of the control group. In leukocytes of patients with carotid atherosclerosis the methylation level of the MIR21 gene promoter was correlated with type 2 diabetes and serum cholesterol level, and the methylation level of the coding region of the MIR10B gene was correlated with smoking.Conclusions.The level of DNA methylation in the regions of MIR10B and MIR21 genes in blood leukocytes is associated with the risk of advanced atherosclerosis of the carotid arteries. 

Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 601-615
Author(s):  
M. H. L. Snow
Keyword(s):  
Specific Activity ◽  
Inner Cell Mass ◽  
Cell Damage ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Abnormal Development ◽  
Tritium Concentration ◽  
Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P &lt; 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

Regulation of adenosine A2Areceptor gene expression in a model of binge eating in the amygdaloid complex of female rats

2019 ◽  
Vol 33 (12) ◽  
pp. 1550-1561 ◽  
Author(s):  
Maria Vittoria Micioni Di Bonaventura ◽  
Mariangela Pucci ◽  
Maria Elena Giusepponi ◽  
Adele Romano ◽  
Catia Lambertucci ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Binge Eating ◽  
Epigenetic Regulation ◽  
Methylation Status ◽  
Amygdaloid Complex ◽  
Female Rats ◽  
Control Group ◽  
Compensatory Mechanism ◽  
Mrna Levels

Background:Pharmacological treatment approaches for eating disorders, such as binge eating disorder and bulimia nervosa, are currently limited.Methods and aims:Using a well-characterized animal model of binge eating, we investigated the epigenetic regulation of the A2AAdenosine Receptor (A2AAR) and dopaminergic D2 receptor (D2R) genes.Results:Gene expression analysis revealed a selective increase of both receptor mRNAs in the amygdaloid complex of stressed and restricted rats, which exhibited binge-like eating, when compared to non-stressed and non-restricted rats. Consistently, pyrosequencing analysis revealed a significant reduction of the percentage of DNA methylation but only at the A2AAR promoter region in rats showing binge-like behaviour compared to the control animals. Focusing thus on A2AAR agonist (VT 7) administration (which inhibited the episode of binge systemically at 0.1 mg/kg or intra-central amygdala (CeA) injection at 900 ng/side) induced a significant increase of A2AAR mRNA levels in restricted and stressed rats when compared to the control group. In addition, we observed a significant decrease in A2AAR mRNA levels in rats treated with the A2AAR antagonist (ANR 94) at 1 mg/kg. Consistent changes in the DNA methylation status of the A2AAR promoter were found in restricted and stressed rats after administration of VT 7 or ANR 94.Conclusion:We confirm the role of A2AAR in binge eating behaviours, and we underline the importance of epigenetic regulation of the A2AAR gene, possibly due to a compensatory mechanism to counteract the effect of binge eating. We suggest that A2AAR activation, inducing receptor gene up-regulation, could be relevant to reduction of food consumption.

scholarly journals DNA methylation of insulin-like growth factor 2 and H19 cluster in cord blood and prenatal air pollution exposure to fine particulate matter

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Congrong Wang ◽  
Michelle Plusquin ◽  
Akram Ghantous ◽  
Zdenko Herceg ◽  
Rossella Alfano ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Particulate Matter ◽  
Growth Factor ◽  
Cord Blood ◽  
Gene Cluster ◽  
Methylation Level ◽  
Fine Particulate Matter ◽  
Methylation Status ◽  
Insulin Like Growth Factor ◽  
Fine Particulate

Abstract Background The IGF2 (insulin-like growth factor 2) and H19 gene cluster plays an important role during pregnancy as it promotes both foetal and placental growth. We investigated the association between cord blood DNA methylation status of the IGF2/H19 gene cluster and maternal fine particulate matter exposure during fetal life. To the best of our knowledge, this is the first study investigating the association between prenatal PM2.5 exposure and newborn DNA methylation of the IGF2/H19. Methods Cord blood DNA methylation status of IGF2/H19 cluster was measured in 189 mother-newborn pairs from the ENVIRONAGE birth cohort (Flanders, Belgium). We assessed the sex-specific association between residential PM2.5 exposure during pregnancy and the methylation level of CpG loci mapping to the IGF2/H19 cluster, and identified prenatal vulnerability by investigating susceptible time windows of exposure. We also addressed the biological functionality of DNA methylation level in the gene cluster. Results Prenatal PM2.5 exposure was found to have genetic region-specific significant association with IGF2 and H19 during specific gestational weeks. The association was found to be sex-specific in both gene regions. Functionality of the DNA methylation was annotated by the association to fetal growth and cellular pathways. Conclusions The results of our study provided evidence that prenatal PM2.5 exposure is associated with DNA methylation in newborns’ IGF2/H19. The consequences within the context of fetal development of future phenotyping should be addressed.

scholarly journals Repeats as global DNA methylation marker in bovine preimplantation embryos

2017 ◽  
Vol 62 (No. 2) ◽  
pp. 43-50 ◽  
Author(s):  
W. Li ◽  
A. Van Soom ◽  
L. Peelman
Keyword(s):  
Dna Methylation ◽  
Mutation Rate ◽  
Methylation Level ◽  
Methylation Status ◽  
Repetitive Sequences ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Repeat Sequence ◽  
Preimplantation Embryos ◽  
Global Dna Methylation

DNA methylation undergoes dynamic changes and is a crucial part of the epigenetic regulation during mammalian early development. To determine the DNA methylation levels in bovine embryos, we applied a bisulfite sequencing based method aimed at repetitive sequences including three retrotransposons (L1_BT, BovB, and ERV1-1-I_BT) and Satellite I. A more accurate estimate of the global DNA methylation level compared to previous methods using only one repeat sequence, like Alu, could be made by calculation of the weighted arithmetic mean of multiple repetitive sequences, considering the copy number of each repetitive sequence. Satellite I and L1_BT showed significant methylation reduction at the blastocyst stage, while BovB and ERV1-1-I_BT showed no difference. The mean methylation level of the repetitive sequences during preimplantation development was the lowest at the blastocyst stage. No methylation difference was found between embryos cultured in 5% and 20% O<sub>2</sub>. Because mutations of CpGs negatively influence the calculation accuracy, we checked the mutation rate of the sequenced CpG sites. Satellite I and L1_BT showed a relatively low mutation rate (1.92 and 3.72% respectively) while that of ERV1-1-I_BT and BovB was higher (11.95 and 24% respectively). Therefore we suggest using a combination of repeats with low mutation rate, taking into account the proportion of each sequence, as a relatively quick marker for the global DNA methylation status of preimplantation stages and possibly also for other cell types.

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