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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 analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).
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Figure 5: Methylation analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).

Mentions: The methylation status was determined by two methods: cloning and sequencing of PCR products from amplification of bisulfite mutated genomic DNA of individual blastocysts and direct sequencing of this same PCR product. To validate the direct sequencing approach, ten clones from each blastocyst were sequenced as well as the PCR product, in both orientations. Overall, one hundred clones were analyzed for a total of ten blastocysts from the different experimental groups (Figure 5). For each CpG position analyzed, the allele specific methylation status observed by cloning-sequencing analysis appeared always as a single nucleotide polymorphism (C/T), also visible after direct sequencing (Figure 5). Alternatively, the unmethylated status of a given CpG obtained by cloning and sequencing appeared as a thymine by direct sequencing. By contrast, a methylated status observed by cloning/sequencing appears as a cytosine by direct sequencing. Therefore, we concluded that the direct sequencing analysis was clearly representative of the blastocyst methylation status. The expected allele-specific methylation status was easily discriminated from absence of methylation as shown in Figure 6. After this validation step, direct sequencing was systematically used in further experiments.


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 analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Methylation analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).
Mentions: The methylation status was determined by two methods: cloning and sequencing of PCR products from amplification of bisulfite mutated genomic DNA of individual blastocysts and direct sequencing of this same PCR product. To validate the direct sequencing approach, ten clones from each blastocyst were sequenced as well as the PCR product, in both orientations. Overall, one hundred clones were analyzed for a total of ten blastocysts from the different experimental groups (Figure 5). For each CpG position analyzed, the allele specific methylation status observed by cloning-sequencing analysis appeared always as a single nucleotide polymorphism (C/T), also visible after direct sequencing (Figure 5). Alternatively, the unmethylated status of a given CpG obtained by cloning and sequencing appeared as a thymine by direct sequencing. By contrast, a methylated status observed by cloning/sequencing appears as a cytosine by direct sequencing. Therefore, we concluded that the direct sequencing analysis was clearly representative of the blastocyst methylation status. The expected allele-specific methylation status was easily discriminated from absence of methylation as shown in Figure 6. After this validation step, direct sequencing was systematically used in further experiments.

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