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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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Embryo cleavage kinetics according to culture media and fertilization method. Daily observations for all cultured zygotes and color code classification according to the cell number are depicted. At day 0, two pronuclei were classically observed (2 PN) and defined the zygote stage. Zygotes obtained after in vitro fertilization (Figure 4A group D) or after in vivo fertilization (Figure 4B group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs.
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Figure 4: Embryo cleavage kinetics according to culture media and fertilization method. Daily observations for all cultured zygotes and color code classification according to the cell number are depicted. At day 0, two pronuclei were classically observed (2 PN) and defined the zygote stage. Zygotes obtained after in vitro fertilization (Figure 4A group D) or after in vivo fertilization (Figure 4B group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs.

Mentions: To analyze the effects of different culture media on preimplantation embryo development, a detailed analysis of cleavage kinetics was done each day for embryos from in vivo (group C) and in vitro fertilization (group D), cultured in both media from one cell stage to blastocyst stage (Figure 4). Differences of cleavage kinetics between both media were observed as early as day 2 and maintained until day 4 of culture, with a faster cell cycle in the sequential G1.2/G2.2 medium than in M16 medium in both groups C and D.


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)

Embryo cleavage kinetics according to culture media and fertilization method. Daily observations for all cultured zygotes and color code classification according to the cell number are depicted. At day 0, two pronuclei were classically observed (2 PN) and defined the zygote stage. Zygotes obtained after in vitro fertilization (Figure 4A group D) or after in vivo fertilization (Figure 4B group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Embryo cleavage kinetics according to culture media and fertilization method. Daily observations for all cultured zygotes and color code classification according to the cell number are depicted. At day 0, two pronuclei were classically observed (2 PN) and defined the zygote stage. Zygotes obtained after in vitro fertilization (Figure 4A group D) or after in vivo fertilization (Figure 4B group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs.
Mentions: To analyze the effects of different culture media on preimplantation embryo development, a detailed analysis of cleavage kinetics was done each day for embryos from in vivo (group C) and in vitro fertilization (group D), cultured in both media from one cell stage to blastocyst stage (Figure 4). Differences of cleavage kinetics between both media were observed as early as day 2 and maintained until day 4 of culture, with a faster cell cycle in the sequential G1.2/G2.2 medium than in M16 medium in both groups C and D.

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