Biological foundation for epigenetic investigations of ART derived human oocytes and embryos

A growing body of evidence suggests that some of the technologies used to treat infertility in humans and/or the infertility itself may compromise the function of key genes which are associated with imprinting. The imprint is a unique mark found on a small number of genes. The imprint is established during the growth of eggs and sperm and it is vital for the development of healthy embryos before they implant to produce a pregnancy. Importantly, the imprinted mark can, in some cases, be changed erroneously during the laboratory procedures used to treat infertility. Errors in how the gene imprints are established and read may predispose an individual to diseases such as cancer and/or specific disorders associated with imprinted gene disruption including rare mental retardation syndromes such as Angelman syndrome, and Beckwith-Wiedemann syndrome. This is a major cause of concern as over a million babies have been born worldwide following assisted conception. Furthermore, it is possible that the more invasive assisted reproductive technologies (ART) which are increasingly being developed and used to treat infertile patients may predispose human embryos and the children born subsequently to imprinting errors. It is therefore of paramount importance to understand how imprinted genes work in healthy human eggs, sperm and embryos and to investigate how their programming can go wrong. This project will predominantly use highly sensitive and sophisticated molecular biology tools called microarrays and follow-up analyses to simultaneously study multiple imprinted genes and their regulators throughout the development of human eggs and embryos. The information generated by these unique studies will be used to confirm the safety of a new treatment for the in vitro growth of eggs- a technique which involves an extended period of culture in the laboratory during which time the eggs are ripened before they are fertilised. It is anticipated that status of the imprinted genes and their regulators can be used to test the safety of new ARTs, such as the in vitro maturation of oocytes, before they are introduced into routine clinical practise. The proposed work will therefore help us to understand and hopefully prevent imprinting errors and diseases being passed onto the next generation of children born following assisted conception.

Imprinted genes are expressed predominantly or exclusively from one parental allele. This mode of regulation is uniquely sensitive to disruption, and therefore imprinted genes themselves are cellular biomarkers of epigenetic disruption, cellular stress and cancer. In assisted reproductive technology (ART) in humans, gametes and embryos are manipulated in vitro at the exact time when the complex and delicate processes of epigenetic reprogramming occur. Recent evidence suggests a range of epigenetic mutations leading to diseases such as Angelman syndrome and Beckwith-Wiedemann syndrome have arisen in children born following assisted conception. Although some of these of imprinting defects may be caused by infertility itself, experimental evidence also indicates that ARTs can disrupt epigenetic mechanisms and alter the regulation of imprinted genes. The need to directly quantify the epigenetic consequences of ART and infertility on the health of the human preimplantation embryos is therefore becoming increasingly important. Due to the limited availability of human gametes and embryos for research and the requirement for analytical methods which are sensitive at the single oocyte/embryo level, the epigenetic processes that regulate early human development remain largely unknown. Despite these limitations we established a programme to investigate early epigenetic processes in humans and have developed appropriate tools and techniques to permit studies of oogenesis and embryogenesis. This project therefore aims to conduct a robust series of investigations of the imprinted genes in human oocytes and preimplantation embryos and to relate these observations to the epigenetic health of embryos derived following ART. This application will test the hypothesis that disruption of imprinting attributable to in vitro manipulations of human gametes and embryos will result in aberrant expression of imprinted genes in the preimplantation embryos so produced. Specifically this application will: 1) establish whether the imprinted genes and epigenetic regulators serve as biomarkers of epigenetic disruption in human blastocysts derived by conventional ART including IVF, ICSI and the in vitro maturation (IVM) of oocytes; 2) monitor allelic expression of imprinted gene transcripts in blastocysts to determine whether expression is monoallelic or otherwise; 3) use bisulphite genomic sequencing to analyse methylation imprints in human ART-derived oocytes and embryos; and 4) conduct a detailed analysis of imprinted gene expression and methylation in a cohort of IVM blastocysts. The data generated will significantly advance our knowledge of the mechanism(s) behind ART-induced imprinting defects in human oocytes and embryos and will help us understand how to alleviate these problems in the future.