There is mounting experimental evidence that the manipulation and extended culture of human and animal gametes and preimplantation embryos in vitro can result in epigenetic disruption. In humans, a number of surveys also suggest that these changes may bring about an increased incidence of imprinting diseases such as Beckwith Wiedemann Syndrome and Angelman Syndrome following the use of Assisted Reproductive Technologies (ART). Similarly, Large Offspring Syndrome caused by the aberrant methylation of the imprinted 19f2r gene is a well characterised risk following in vitro fertilisation (IVF) in domestic animals. This background has led us to hypothesise that the technologies used for assisted reproduction in humans may alter gene expression during early human embryo development in vitro. Initial experiments were conducted to map the expression of key genes responsible for the establishment of methylation patterns in the human germline. Expression patterns of the DNA methyltransferases DNMTI, DNMT2, DNMT3A, DNMT3B and DNM3L and methyl domain binding proteins MBD1, MBD2, MBD3, MBD4 and MeCP2 were analysed in human cDNA libraries generated from oocytes and somatic cells from across all stages of human folliculogenesis and preimplantation development. It was found that the expression pattern of DNMT3L differed significantly from that found in previous murine work as expression only occurred following fertilisation. The expression of three PeG genes with essential functions during preimplantation and gastrulation in the mouse, EZH2, EED and YY1 was found to be expressed in the human at similar stages to their essential murine functions. The expression of further PeG and HOX genes including HP 1 HSα, β and γ and HOX AI, A4, A5, A 7, A10, B4, B7, C6, CB, C9, DB, CDX2, GBXI and HEX within the same tissues was characterised by the use of degenerate primers enabling an expression profile for these genes in the human germline to be established. Finally, a novel approach to analyse imprinted gene networks using a focussed microarray covering all the known human imprinted genes and epigenetic regulators along with appropriate controls was tested. This experimental series used a custom designed array. After careful characterisation, this array elicited varying expression patterns between human inner cell mass (ICM) and trophectoderm samples and the ICM and early passage human embryonic stem cells (hESCs). Human blastocysts following intracytoplasmic sperm injection and IVF were also contrasted. Real time PCR confirmed the array's findings of upregulation of GNAS, KCNQIDN and SLC22Al8 and downregulation of CD81, DNMT3A2, IPW, MAGEL2, PAR5, PAR-SN, UBE3A and ZNF264 with increasing passage number in hESCs. These studies aim to increase our understanding of early human development and eventually contribute to the improvement of the success rates of these ARTs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598020 |
| Date | January 2009 |
| Creators | Hinkins, Matthew |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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