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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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Blastocyst maturity. At day 4, each blastocyst was observed and classified into four different categories according to the maturity characteristics (size of embryonic cavity and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χ2 test, P < 0.05)
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Figure 3: Blastocyst maturity. At day 4, each blastocyst was observed and classified into four different categories according to the maturity characteristics (size of embryonic cavity and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χ2 test, P < 0.05)

Mentions: In order to determine the effect of different environmental manipulations, we analyzed the morphology of blastocysts at day 4 after fertilization (Figure 3). When fertilization and embryo development occurred in vivo, there was no difference of blastocyst maturity between superovulated and non-superovulated mice (groups A and B). After in vivo fertilization and in vitro development (group C), blastocysts were more mature on day 4 than blastocysts which developed in vivo (groups A + B). The proportion of fully expanded and hatching blastocysts was 64.4% in group C versus 25.4% in groups A + B (P < 0.001, χ2 test). The fertilization method did not influence the in vitro embryo development since the percentage of fully expanded and hatching blastocyst was 60.7% after IVF (group D). The drastic effects of in vitro development on blastocyst maturity were modulated by the culture medium used. There was a greater proportion of hatching blastocysts when embryos from groups C and D were cultured in G1.2/G2.2 medium (48.3% and 53.6%) compared to M16 medium (23.3% and 17.3%; P < 0.05, χ2 test). Furthermore, the morphometric analysis at day 4 showed equal blastocyst perimeter and area means in groups A and B. The perimeter and area means tended to increase in groups C and D when blastocysts were cultured in G1.2/G2.2 medium as compared to those in M16 medium. Blastocysts from group C were significantly larger than blastocysts from group B (p < 0.05, t test- data not shown).


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)

Blastocyst maturity. At day 4, each blastocyst was observed and classified into four different categories according to the maturity characteristics (size of embryonic cavity and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χ2 test, P < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Blastocyst maturity. At day 4, each blastocyst was observed and classified into four different categories according to the maturity characteristics (size of embryonic cavity and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χ2 test, P < 0.05)
Mentions: In order to determine the effect of different environmental manipulations, we analyzed the morphology of blastocysts at day 4 after fertilization (Figure 3). When fertilization and embryo development occurred in vivo, there was no difference of blastocyst maturity between superovulated and non-superovulated mice (groups A and B). After in vivo fertilization and in vitro development (group C), blastocysts were more mature on day 4 than blastocysts which developed in vivo (groups A + B). The proportion of fully expanded and hatching blastocysts was 64.4% in group C versus 25.4% in groups A + B (P < 0.001, χ2 test). The fertilization method did not influence the in vitro embryo development since the percentage of fully expanded and hatching blastocyst was 60.7% after IVF (group D). The drastic effects of in vitro development on blastocyst maturity were modulated by the culture medium used. There was a greater proportion of hatching blastocysts when embryos from groups C and D were cultured in G1.2/G2.2 medium (48.3% and 53.6%) compared to M16 medium (23.3% and 17.3%; P < 0.05, χ2 test). Furthermore, the morphometric analysis at day 4 showed equal blastocyst perimeter and area means in groups A and B. The perimeter and area means tended to increase in groups C and D when blastocysts were cultured in G1.2/G2.2 medium as compared to those in M16 medium. Blastocysts from group C were significantly larger than blastocysts from group B (p < 0.05, t test- data not shown).

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