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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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Direct sequencing analysis of proximal part of H19 promoter in individual blastocysts. Examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of H19 promoter in individual blastocysts without (Figure 8A) or with methylation defects (Figure 8B) are shown.
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Figure 8: Direct sequencing analysis of proximal part of H19 promoter in individual blastocysts. Examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of H19 promoter in individual blastocysts without (Figure 8A) or with methylation defects (Figure 8B) are shown.

Mentions: In order to identify possible epigenetic defects affecting the H19 promoter, normally differentially methylated, we also assessed its methylation status for 8 CpG positions (Figure 8) on 17 individual blastocysts. For blastocysts fertilized and developed in vivo, the expected allele specific methylation was found (6 individual blastocysts). Methylation defects were observed for 5 blastocysts out of 6 from group D M16 and 4 out of 5 from group D G1.2/G2.2.


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)

Direct sequencing analysis of proximal part of H19 promoter in individual blastocysts. Examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of H19 promoter in individual blastocysts without (Figure 8A) or with methylation defects (Figure 8B) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Direct sequencing analysis of proximal part of H19 promoter in individual blastocysts. Examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of H19 promoter in individual blastocysts without (Figure 8A) or with methylation defects (Figure 8B) are shown.
Mentions: In order to identify possible epigenetic defects affecting the H19 promoter, normally differentially methylated, we also assessed its methylation status for 8 CpG positions (Figure 8) on 17 individual blastocysts. For blastocysts fertilized and developed in vivo, the expected allele specific methylation was found (6 individual blastocysts). Methylation defects were observed for 5 blastocysts out of 6 from group D M16 and 4 out of 5 from group D G1.2/G2.2.

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