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Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes.

ABSTRACT: ART is suspected to generate increased imprinting errors in the lineage. Following an intra cytoplasmic sperm injection (ICSI) procedure, a certain number of embryos fail to develop normally and imprinting disorders may be associated to the developmental failure. To evaluate this hypothesis, we analysed the methylation profile of H19DMR, a paternally imprinting control region, in high-graded blastocysts, in embryos showing developmental anomalies, in the matching sperm and in oocytes of the concerned couples when they were available. Significant hypomethylation of the paternal allele was observed in half of the embryos, independently of the stage at which they were arrested (morula, compacted morula, pre blastocyst or BC-graded blastocysts). Conversely, some embryos showed significant methylation on the maternal allele, whereas few others showed both hypomethylation of the paternal allele and abnormal methylation of the maternal allele. The matching sperm at the origin of the embryos exhibited normal methylated H19 patterns. Thus, hypomethylation of the paternal allele in the embryos does not seem inherited from the sperm but likely reflects instability of the imprint during the demethylating process, which occurred in the early embryo. Analysis of a few oocytes suggests that the defect in erasure of the paternal imprint in the maternal germ line may be responsible for the residual methylation of the maternal allele in some embryos. None of these imprinting alterations could be related to a particular stage of developmental arrest; compared with high-grade blastocysts, embryos with developmental failure are more likely to have abnormal imprinting at H19 (P<0.05).

SUBMITTER: Ibala-Romdhane S

PROVIDER: S-EPMC3198154 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes.

Ibala-Romdhane Samira S Al-Khtib Mohamed M Khoueiry Rita R Blachère Thierry T Guérin Jean-François JF Lefèvre Annick A

European journal of human genetics : EJHG 20110608 11

ART is suspected to generate increased imprinting errors in the lineage. Following an intra cytoplasmic sperm injection (ICSI) procedure, a certain number of embryos fail to develop normally and imprinting disorders may be associated to the developmental failure. To evaluate this hypothesis, we analysed the methylation profile of H19DMR, a paternally imprinting control region, in high-graded blastocysts, in embryos showing developmental anomalies, in the matching sperm and in oocytes of the conc ...[more]

PMID: 21654730

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Project description:BACKGROUND: In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. RESULTS: We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. CONCLUSION: Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

Project description:During mammalian pre-implantation embryonic development, dramatic and orchestrated changes occur in gene transcription. Pregnancy rates were low when yak females were crossbred with cattle breeds, but few studies exist to describe the unique molecular network regulation behind the pre-implantation development of these embryos. We determined the transcriptomes of crossbred embryos derived from yak oocytes in vitro fertilized with Jersey sperm using Illumina RNA-seq for the first time in this study. Embryos were sampled at the 2-, 4-, and 8-cell, morula and blastocyst stages. The results showed that in total, 291.9 million short reads were generated from the five libraries of 2-, 4-, and 8-cell, morula and blastocyst stages, with 276.2 million high-quality reads selected for further analysis. Eighty to 91% of the clean reads were aligned against the yak reference genome. A total of 19,072 transcripts were identified in five libraries, of which 7,785 transcripts were co-expressed in each stage and 2,013 transcripts were stage-specific. When a |log2 ratio| ≥1 and q-value ≤ 0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 3,690 to 10,298 DEGs between any two consecutive stages. Based on the results of GO and KEGG enrichment, some of these DEGs potentially play an important role in regulating pre-implantation development, but they are most likely stage-specific. There were 2,960, 7,287, 6,420, 7,724 and 10,417 DEGs in 2-, 4-, 8-cell, morula and blastocyst stages between the crossbred embryos and purebred embryos of the yak, respectively, leading to a large difference in GO terms and pathways. In conclusion, we sequenced transcriptomes of in vitro-produced crossbred embryos of yak and cattle during pre-implantation and provided comprehensive examinations of gene activities. These will be helpful for development of assisted reproductive technology and better understanding the early maternal-fetal or maternal-embryonic dialog in inter-species crossbreeding.

Project description:The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.

Project description:DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ) and asthenozoospermia (AZ) usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control), with only complete methylation and mild hypomethylation(<50% unmethylated CpGs) identified, and there was no significant difference in the occurrence of these two methylation patterns between AZ and NZ (P>0.05). However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs ) and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05%) and AZ (0.58±1.77%) (P<0.001).Additional, our data indicated the occurrence of >20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05). In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

Dataset Information

Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes.

Publications

Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes.

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