Following fertilization, dramatic epigenetic remodeling occurs on both the maternal and paternal chromosomes, which is critical to the establishment of totipotency,
the ability of an individual embryonic cell to generate all cell types in an organism (Edwards and Beard, 1997) (Figure 1.4). Immediately after fertilization, remodeling of the sperm chromatin begins and consists of the replacement of protamines by acetylated histones, and active, genome-wide demethylation (Oswald et al., 2000). On the other hand, the maternally inherited genome is passively demethylated over the course of the next several rounds of cell division, which is thought to be due to a lack of maintenance methylation (Carlson et al., 1992). During this early stage of preimplantation development, methylation is lost from all areas of the genome except imprinted genes and retroviral sequences (Lucifero et al., 2004).
Maintenance of DNA methylation at imprinted loci relies on DNMT1, which recognized and methylates hemi-methylated DNA (Fatemi et al., 2001). A number of isoforms of DNMT1 have been identified (Pradhan et al., 1997). The longer isoform, DNMT1s is most predominant in somatic cells (Hermann et al., 2004), while the shorter DNMT1o is present in growing oocytes and during preimplantation development (Howell et al., 2001). The majority of the time, DNMT1s is localized within the nucleus, associated with the DNA replication machinery at replication foci during S-phase (Szyf, 2001). During preimplantation development, DNMT1s is excluded from the nucleus, allowing for passive demethylation of the maternal genome (Carlson et al., 1992). The oocyte-specific isoform localizes to the nucleus at the 8-cell stage, and along with DNMT1s activity, is thought to be responsible for maintaining methylation at imprinted loci throughout preimplantation development (Ding and Chaillet, 2002). In addition, disruption of number of maternal-effect genes, that are transcribed and stored in the
developing oocyte and are required for preimplantation development, have been shown to result in loss of methylation at a number of imprinted loci including Snrpn, Peg3, Peg1/ Mest and H19 (Nakamura et al., 2007; .Li et al., 2008).
As preimplantation development proceeds, different cell lineages begin to emerge. As such, de novo methylation begins around the time of implantation to allow for differentiation of embryonic and extraembryonic lineages, and further differentiation into the numerous tissue types of the adult organism (Monk et al., 1987).
1.3 - Assisted Reproductive Technologies 1.3.1 Prevalence of ARTs and Their Sequelae
Since the first reported birth through the use of assisted reproductive technologies (ART) in 1978, the use of these technologies has dramatically increased. It is estimated that 1-3% of total births in developed countries result from some form of ART (Klemetti et al., 2002; Wright et al., 2008). The field of assisted reproduction is broad and consists of a variety of techniques, from non-invasive procedures such as ovarian hyperstimulation, to highly invasive interventions such as intracytoplasmic sperm injection (ICSI) of retrieved oocytes. However, all involve the manipulation of human gametes and preimplantation embryos, and many involve embryo culture during preimplantation development. As described above, germ cell and preimplantation development are critical periods in the erasure and maintenance of proper imprinted methylation patterns (Santos and Dean, 2004). As such, the timing of ARTs during these
critical periods provides a mechanism for the disruption of imprinting establishment and maintenance through the environmental insult caused by the use of these procedures.
In addition to epigenetic consequences of ARTs, a number of other sequelae have been observed. Couples who undergo ART carry intrinsic subfertility, which itself is a risk factor for early pregnancy loss (Gray and Wu, 2000), and are on average 5 years older than those who conceive naturally (Katalinic et al., 2004). In addition, ART carries a higher risk of multiple births, which itself is associated with higher rates of prematurity, low birth weight, neonatal mortality, congenital malformations and disability (Koivisto et al., 1975; Fauser et al., 2005). However, all of the risk associated with ARTs cannot be attributed to intrinsic subfertility of the couples and risk of multiple births. Singleton pregnancies occurring through the use of ARTs have an increased risk of prematurity, low birth weight, neonatal mortality, and neonatal intensive care unit admission (Helmerhorst et al., 2004; Jackson et al., 2004; McDonald et al., 2005), as well as an increased risk of congenital malformations (Lancaster, 1985; Rimm et al., 2004; Bonduelle et al., 2005; Hansen et al., 2005; Klemetti et al., 2005; Olson et al., 2005), and cerebral palsy (Ericson et al., 2002; Lidegaard et al., 2005; Hvidtjorn et al., 2006) and epilepsy (Ericson et al., 2002; Sun et al., 2007). Most important for the studies contained in this thesis is the increase in the incidence of the human imprinting disorders Angelman Syndrome (AS) (Cox et al., 2002; Orstavik et al., 2003) and Beckwith-Wiedemann Syndrome (BWS) (DeBaun et al., 2003; Gicquel et al., 2003; Maher et al., 2003) with the use of ARTs.
The incidence of AS in the general population is approximately one case per 16,000 births, with only 5% of these cases related to imprinting abnormalities (Cox et al.,
2002; Williams, 2007). As the prevalence of AS is low, large-scale studies containing sufficient numbers of patients have been difficult to achieve. However, seven cases of AS following the use of ARTs have been reported to date, 5 of which displayed imprinting abnormalities (71%) (Cox et al., 2002; Orstavik et al., 2003; Ludwig et al., 2005; Sutcliffe et al., 2006). This is a significantly higher proportion than in the non-ART population.
Beckwith-Wiedemann Syndrome is a second imprinting disorder that is associated with ARTs and is estimated to affect 1 in 13 700 children (Shuman et al., 1993). As with AS, in a number of studies, parents of children with BWS were more likely to have undergone fertility treatments than the general population (Chang et al., 2005; Doornbos et al., 2007) and a higher incidence of BWS was seen in ART children than in the general population (Gicquel et al., 2003; Arnaud and Feil, 2005). The link between BWS and ARTs has been strongly established, and the relative risk of ART use is 4-9 times greater for BWS patients. Silver-Russell Syndrome has also been associated with the use of ARTs (Hitchins et al., 2001; Svensson et al., 2005; Bliek et al., 2006; Kagami et al., 2007; Galli-Tsinopoulou et al., 2008; Chopra et al., 2010). Taken all together, ARTs may impose inherent risk for normal development.
Attributing any of these risks to specific forms of ART has proven difficult, and as procedures vary from clinic to clinic, and protocols vary between patients, most studies simply group the observed effects under the umbrella of “ARTs”. The remainder of this work will focus specifically on the effects of superovulation and embryo culture on genomic imprinting.