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

BMC Developmental Biology Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos Patricia Fauque 1 2 Pierre Jouannet 2 Corinne Lesaffre 1 Marie- Anne Ripoche 1 Luisa Dandolo 1 Daniel Vaiman 0 1 Hlne Jammes 1 3 0 Genetics Department, INRA , Jouy en Josas , France 1 Genetique et Developpement, INSERM Institut Cochin U567, CNRS (UMR 8104), Universite Paris Descartes , Paris , France 2 Biologie de la Reproduction, Hopital Cochin, AP-HP, Universite Paris Descartes , Paris , France 3 PHASE Department, INRA , Jouy en Josas , France 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. 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. - Background Many studies have focused on the effects of Assisted Reproductive Technology (ART) on children's health. ART babies now account for approximately 2.2% and 0.6% of all births in France and in the United States, respectively [1]. Singleton pregnancies obtained after ART are at a higher risk for adverse perinatal outcomes than natural pregnancies. These risks include perinatal mortality, preterm delivery, and low birth weight [2-4]. Since low birth weight has been associated to increased rates of cardiovascular and metabolic diseases in adulthood, ART is at least a partial cause of such long-term consequences [5]. Different studies have also reported rare congenital malformations [6-8], chromosomal abnormalities [9], and alterations of cognitive and motor development [10,11]. However it has been difficult to assess the real interaction effects between the mode of conception and the incidence of these anomalies. Recently, the concept of epigenetic risk has associated disturbances of embryonic development with aberrant genomic imprinting. Some reports suggest that ART increases the risk of diseases such as the Beckwith-Wiedemann syndrome (BWS), Prader Willi syndrome (PWS), Angelman syndrome (AS) and Silver-Russel syndrome (SRS) [12-18]. Genomic imprinting leads to a parent-of-origin specific gene expression. Imprinted genes are known to play important roles in regulating embryonic growth, placental functions [19], postnatal metabolic pathways and behavior associated with the control of resources [20]. Moreover, oncogenesis may also be associated with altered epigenetic regulations [21]. Imprinted genes [22] are generally located in clusters, epigenetically marked by DNA methylation on key regulatory sequences (Differentially Methylated Regions, DMRs), by histone modifications (acetylation/deacetylation and methylation) and often associated with antisense RNAs [23,24]. The allele specific methylation of DMRs occurs in germ cells and provides a heritable "memory" that must be maintained throughout fertilization and embryo development. The differential methylation at DMRs is preserved during preimplantation development, in spite of genome-wide changes in global DNA methylation occurring at these early stages [25]. During this period of dynamic epigenetic changes, environmental manipulations, such as hormone-induced superovulation, in vitro fertilization (IVF) and embryo culture, could modify genomic imprints and have deleterious effects on later fetal and postnatal stages. Few studies have reported on the imprinted gene expression in human preimplantation embryos, due to major limitations, such as the scarcity of embryos available for research and the associated ethical restrictions. A monoallelic paternal expression has been shown for both SNRPN [26] and IGF2 [27,28]. More recently, DNA methylation analysis of control regions of the SNRPN gene and DLK1/ GTL2 locus on human early embryo has been reported [29,30]. Most studies made use of mouse models in order to evaluate the impact of in vitro fertilization and/or embryo culture using various culture media. Using preimplantation embryos (from the two cell- to the blastocyst stages), an aberrant imprinting of H19 gene has been previously found under different culture conditions [31,32]. After implantation, embryonic tissues preserved correct genomic imprints although aberrant H19 imprinting was maintained in some placentae [33]. However, alteration of allele-specific methylation of H19 gene was shown in fetuses obtained from embryos cultured in medium supplemented with fetal calf serum [34]. Finally studies on the long-term effects of in vitro culture on mouse embryos have shown by analyzing development and behavioral parameters in relation to imprinting, that the postnatal development could be affected by embryo culture during the preimplantation period [35,36]. Visual observation of blastocysts suggests a great individual variability of development after IVF and embryo culture. Knowledge about the molecular grounds of such variation is still scarce, but could be of interest for optimizing culture and IVF conditio (...truncated)