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Project
Investigation of epigenetic stability and safety of assisted reproduction technologies (FWOAL568)
Introduction
The epigenome is less stable than the genome (Bennett-Baker et al., 2003). Epimutations (i.e. modifications of the chromatin configuration or the DNA methylation pattern, resulting in altered gene expression) occur mostly in a stochastic manner and their incidence is dependent on genetic and environmental influences. Genomic imprinting is an epigenetic mechanism characterised by parent-specific marks at several chromosomal loci. The imprint marks are reset during gametogenesis along with genome-wide epigenetic reprogramming, but after fertilisation they escape the second wave of epigenetic reprogramming. The mechanisms of epigenetic reprogramming have predominantly been investigated in mice and less so in human tissue. Previous work from our lab has focused on human imprint reprogramming of three genes and revealed aspects on germ-line imprints, time of resetting and imprint maintenance in the embryo (De Rycke et al. 2002; De Rycke, 2003; Geuns et al., 2003; Geuns et al., 2007a; Geuns et al., 2007b).
Although the majority of children born after assisted reproductive technology (ART) are healthy, it cannot be denied that ART bypasses a number of biological selection filters, and that it subjects gametes and preimplantation embryos to hormonal stimulation, culture media and physical stress in time windows with major epigenetic reprogramming events. Recent years have seen the publication of several papers that report on the association of ART and imprinting disorders (Cox et al., 2002, Maher et al., 2003, Halliday et al., 2004, Ludwig et al., 2005). More recently there has been a follow-up study reporting on an association between IVF and increased blood pressure and fasting glucose levels (Ceelen et al., 2007, 2008). It is not clear whether this association is related to the genetic predisposition of subfertile couples or/and to the interference of specific ART aspects with epigenetic reprogramming during gametogenesis and early embryonic development. Previous evidence that ART can influence the epigenetic make-up of embryos has been delivered in animal studies (Young et al., 2001, Shi and Haaf, 2002).
The consequences of epigenetic abnormalities can become obvious at different levels; firstly, aberrant chromatin organisation and defective genome-wide DNA methylation reprogramming after fertilisation can have a devastating impact on the developmental ability of embryos. In mouse, a good correlation was found between aberrant genome-wide DNA methylaton patterns and abnormal embryonic development and preimplantation embryonic loss (Shi and Haaf, 2002). In human, a significant proportion of ART embryos do not reach the blastocyst stage. One possible cause is the high incidence of postzygotic chromosomal abnormalities. Key elements for proper chromosome segregation are the centromeres whose function is determined by chromatin organisation with highly methylated heterochromatin (Probst et al., 2008). During preimplantation development these centromeric heterochromatin domains are reorganised from ring structures around nucleoli to chromocenters. Mouse embryos that fail this reorganisation arrest development. Secondly, epigenetic abnormalities may deregulate genomic regions without impact on implantation, but affect development far beyond this stage and contribute to a number of more subtle problems, such as low birth weight (Schieve et al., 2002), late-onset metabolic and cardiovascular diseases (Maher et al., 2003), oncological disorders (Klip et al., 2001) and alterations of the long-term expression pattern of several genes (Gluckman and Hanson, 2004).
Aim
The aim of the proposal is to gain a more detailed insight into the human basic epigenetic reprogramming process and to investigate the influence of ART on the epigenome.
Objectives
Part 1: investigation of epigenetic reprogramming in human gametes and preimplantation embryos through immunohistochemical techniques and time-lapse observations.
A/ Analysis of genome-wide DNA methylation patterns and chromatin organisation.
B/ Analysis of the expression and intracellular trafficking of DNA methyltransferases.
This part should elucidate details of epigenetic reprogramming and its machinery in human, reveal if aberrant reprogramming is a mechanism contributing to the high incidence of preimplantation developmental failure in ART and whether hormonal stimulation or culture media influence reprogramming.
Part 2: investigation to see whether the epigenetic profile (DNA methylation and expression) of selected genes is influenced in ART-pregnancies as compared to spontaneous pregnancies
The epigenome is less stable than the genome (Bennett-Baker et al., 2003). Epimutations (i.e. modifications of the chromatin configuration or the DNA methylation pattern, resulting in altered gene expression) occur mostly in a stochastic manner and their incidence is dependent on genetic and environmental influences. Genomic imprinting is an epigenetic mechanism characterised by parent-specific marks at several chromosomal loci. The imprint marks are reset during gametogenesis along with genome-wide epigenetic reprogramming, but after fertilisation they escape the second wave of epigenetic reprogramming. The mechanisms of epigenetic reprogramming have predominantly been investigated in mice and less so in human tissue. Previous work from our lab has focused on human imprint reprogramming of three genes and revealed aspects on germ-line imprints, time of resetting and imprint maintenance in the embryo (De Rycke et al. 2002; De Rycke, 2003; Geuns et al., 2003; Geuns et al., 2007a; Geuns et al., 2007b).
Although the majority of children born after assisted reproductive technology (ART) are healthy, it cannot be denied that ART bypasses a number of biological selection filters, and that it subjects gametes and preimplantation embryos to hormonal stimulation, culture media and physical stress in time windows with major epigenetic reprogramming events. Recent years have seen the publication of several papers that report on the association of ART and imprinting disorders (Cox et al., 2002, Maher et al., 2003, Halliday et al., 2004, Ludwig et al., 2005). More recently there has been a follow-up study reporting on an association between IVF and increased blood pressure and fasting glucose levels (Ceelen et al., 2007, 2008). It is not clear whether this association is related to the genetic predisposition of subfertile couples or/and to the interference of specific ART aspects with epigenetic reprogramming during gametogenesis and early embryonic development. Previous evidence that ART can influence the epigenetic make-up of embryos has been delivered in animal studies (Young et al., 2001, Shi and Haaf, 2002).
The consequences of epigenetic abnormalities can become obvious at different levels; firstly, aberrant chromatin organisation and defective genome-wide DNA methylation reprogramming after fertilisation can have a devastating impact on the developmental ability of embryos. In mouse, a good correlation was found between aberrant genome-wide DNA methylaton patterns and abnormal embryonic development and preimplantation embryonic loss (Shi and Haaf, 2002). In human, a significant proportion of ART embryos do not reach the blastocyst stage. One possible cause is the high incidence of postzygotic chromosomal abnormalities. Key elements for proper chromosome segregation are the centromeres whose function is determined by chromatin organisation with highly methylated heterochromatin (Probst et al., 2008). During preimplantation development these centromeric heterochromatin domains are reorganised from ring structures around nucleoli to chromocenters. Mouse embryos that fail this reorganisation arrest development. Secondly, epigenetic abnormalities may deregulate genomic regions without impact on implantation, but affect development far beyond this stage and contribute to a number of more subtle problems, such as low birth weight (Schieve et al., 2002), late-onset metabolic and cardiovascular diseases (Maher et al., 2003), oncological disorders (Klip et al., 2001) and alterations of the long-term expression pattern of several genes (Gluckman and Hanson, 2004).
Aim
The aim of the proposal is to gain a more detailed insight into the human basic epigenetic reprogramming process and to investigate the influence of ART on the epigenome.
Objectives
Part 1: investigation of epigenetic reprogramming in human gametes and preimplantation embryos through immunohistochemical techniques and time-lapse observations.
A/ Analysis of genome-wide DNA methylation patterns and chromatin organisation.
B/ Analysis of the expression and intracellular trafficking of DNA methyltransferases.
This part should elucidate details of epigenetic reprogramming and its machinery in human, reveal if aberrant reprogramming is a mechanism contributing to the high incidence of preimplantation developmental failure in ART and whether hormonal stimulation or culture media influence reprogramming.
Part 2: investigation to see whether the epigenetic profile (DNA methylation and expression) of selected genes is influenced in ART-pregnancies as compared to spontaneous pregnancies
Date:1 Jan 2010 → 31 Dec 2013
Keywords:reproductive genetics, andrology, clinical genetics, embryology, assisted reproductive technology
Disciplines:Basic sciences, Biological sciences