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Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.

Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, Jammes H - BMC Dev. Biol. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biologie de Reproduction, Hôpital Cochin, AP-HP, Université Paris Descartes, Paris, France. patricia.fauque@cch.aphp.fr

ABSTRACT

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.

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Methylation status of H19 ICR (A: ICRCTCF 1–2 region analysis and B: ICRCTCF 3–4 region analysis) for individual blastocysts determined by bisulfite/sequencing analysis. For each group, methylation status of individual blastocysts was analyzed by direct sequencing and is represented by the black and white lollipops. The CTCF binding sites are shaded in grey. Only examples are shown for clarity.
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Figure 7: Methylation status of H19 ICR (A: ICRCTCF 1–2 region analysis and B: ICRCTCF 3–4 region analysis) for individual blastocysts determined by bisulfite/sequencing analysis. For each group, methylation status of individual blastocysts was analyzed by direct sequencing and is represented by the black and white lollipops. The CTCF binding sites are shaded in grey. Only examples are shown for clarity.

Mentions: The methylation status of the H19 ICRCTCF 1–2 region was assessed H19 for 19 CpG positions on 85 individual blastocysts. A preserved differential methylation of H19 ICRCTCF 1–2 was found for each analyzed CpG of embryos fertilized in vivo as measured in 8 blastocysts of group A, 26 of group B, 8 of group C cultured in M16 medium and 7 of group C in G1.2/G2.2 medium (Figure 7). Methylation defects were often observed after in vitro fertilization in blastocysts of group D and in both culture media (n = 36). In this group, the proportion of individual blastocysts with unmethylated alleles and the number of non-methylated CpGs were more important when cultured in M16 compared to sequential G1.2/G2.2 medium (18 out of 19 in M16 and 13 out of 17 in G1.2/G2.2 medium – Figure 7). Only one blastocyst out of 19 from group D M16 and 4 out of 17 from group D G1.2/G2.2 had a correct differential methylation status at all the CpG positions. The unmethylated CpGs appeared preferentially outside of the CTCF binding sites, thereby possibly reducing the deleterious consequences of the anomaly.


Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.

Fauque P, Jouannet P, Lesaffre C, Ripoche MA, Dandolo L, Vaiman D, Jammes H - BMC Dev. Biol. (2007)

Methylation status of H19 ICR (A: ICRCTCF 1–2 region analysis and B: ICRCTCF 3–4 region analysis) for individual blastocysts determined by bisulfite/sequencing analysis. For each group, methylation status of individual blastocysts was analyzed by direct sequencing and is represented by the black and white lollipops. The CTCF binding sites are shaded in grey. Only examples are shown for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169233&req=5

Figure 7: Methylation status of H19 ICR (A: ICRCTCF 1–2 region analysis and B: ICRCTCF 3–4 region analysis) for individual blastocysts determined by bisulfite/sequencing analysis. For each group, methylation status of individual blastocysts was analyzed by direct sequencing and is represented by the black and white lollipops. The CTCF binding sites are shaded in grey. Only examples are shown for clarity.
Mentions: The methylation status of the H19 ICRCTCF 1–2 region was assessed H19 for 19 CpG positions on 85 individual blastocysts. A preserved differential methylation of H19 ICRCTCF 1–2 was found for each analyzed CpG of embryos fertilized in vivo as measured in 8 blastocysts of group A, 26 of group B, 8 of group C cultured in M16 medium and 7 of group C in G1.2/G2.2 medium (Figure 7). Methylation defects were often observed after in vitro fertilization in blastocysts of group D and in both culture media (n = 36). In this group, the proportion of individual blastocysts with unmethylated alleles and the number of non-methylated CpGs were more important when cultured in M16 compared to sequential G1.2/G2.2 medium (18 out of 19 in M16 and 13 out of 17 in G1.2/G2.2 medium – Figure 7). Only one blastocyst out of 19 from group D M16 and 4 out of 17 from group D G1.2/G2.2 had a correct differential methylation status at all the CpG positions. The unmethylated CpGs appeared preferentially outside of the CTCF binding sites, thereby possibly reducing the deleterious consequences of the anomaly.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biologie de Reproduction, Hôpital Cochin, AP-HP, Université Paris Descartes, Paris, France. patricia.fauque@cch.aphp.fr

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus