IVF based approaches towards the treatment and prevention of mitochondrial disease

i Acknowledgements . iii Presentations . iv Publications . iv Abbreviations . v List of Figures and Tables . viii Chapter 1: Introduction . 2 1.1 Mitochondrial Biology . 2 1.1.1 Basic Mitochondrial Biology . 2 1.1.2 Mitochondrial origins . 2 1.1.3 Mitochondrial Structure . 5 1.1.4 The mitochondrial respiratory chain . 8 1.1.4.1 Complex I (NADH-coenzyme Q oxidoreductase) . 8 1.1.4.2 Complex II (Succinate-Q oxioreductase) . 8 1.1.4.3 Complex III (Q-cytochrome c oxidoreductase) . 9 1.1.4.4 Complex IV (Cytochrome c oxidase) . 10 1.1.4.5 FoF1ATP synthase . 10 1.2 Mitochondrial DNA . 12 1.2.1 Mitochondrial genome . 12 1.2.2 Mitochondrial genome organisation . 14 1.2.3 MtDNA Replication . 14 1.2.3.1 Strand displacement model . 14 1.2.3.2 Strand coupled model . 15 1.3 Mitochondrial DNA Inheritance . 18 1.3.1 Maternal inheritance . 18 1.3.2 Mitochondrial DNA mutations . 21 1.3.2.1 MtDNA point mutations . 21 1.3.2.2 MtDNA deletions . 21 1.3. 3 Homoplasmy and Heteroplasmy . 23 1.3. 4 Threshold Effect . 25 1.3. 5 The mtDNA Bottleneck . 27 1.4 Management and diagnosis of mtDNA disease . 31 1.4.1 Diagnosing mtDNA disease . 31 1.4.2 Management of mitochondrial disease . 33 1.4.2.1 Pharmacological treatments . 33 1.4.2.2 Dietary supplementation . 35 1.4.2.3 Exercise studies . 35 1.4.2.4 Personalised gene therapies . 35 1.5 Female Gametogenesis and Early Preimplantation Development . 37 1.5.1 Oogenesis . 37 1.5.2 Mammalian Meiosis . 37 1.5.3 Fertilisation . 39 1.5.4 Abnormally fertilised human embryos . 39 1.5. 5 Pre-implantation development . 40 1.5. 6 Blastocyst development . 43 1.5. 6.1 Lineage specification . 43 1.6 Cryopreservation strategies used to preserve fertility . 46 1.6.1 Controlled rate freezing . 46 1.6. 2 Vitrification . 47 1.7 Current and future approaches for the treatment and prevention of mtDNA disease . 50 1.7. 1 Genetic counselling . 50 1.7. 2 Oocyte donation . 50 1.7. 3 Prenatal genetic diagnosis . 51 1.7. 4 Preimplantation genetic diagnosis . 54 1.7. 5 Currently explored IVF strategies to prevent the transmission of mitochondrial disease . 57 1.7. 5.1 Germinal vesicle transfer . 57 1.7. 5.2 Metaphase II spindle transfer . 57 1.7. 5.3 Pronuclear transfer . 58 1.7. 5.4 Comparison between metaphase II spindle transfer and pronuclear transfer . 59 1.8 Ethical issues associated with the IVF based reproductive techniques. 63 1.8. 1 Ethical issues associated with Preimplantation genetic diagnosis (PGD) . 63 1.8. 1.1 Ethical issues associated with the use of PGD for mtDNA inherited disorders . 64 1.8. 2 Ethical issues surrounding the nuclear transfer technique pronuclear transfer (PNT) . 65 1.8. 3 Are PNT and other nuclear transfer techniques being explored for their ability to prevent the transmission of mtDNA disease, forms of cloning? 65 1.9 Aims . 67 Chapter 2 Materials and Methods . 69 2.1 Collection of oocytes and embryos . 69 2.1.1 Human Oocytes . 69 2.1.2 Abnormally Fertilised Human Embryos . 69 2.1.2.1 Blastomere biopsy . 70 2.1.2.2 Whole embryo disaggregation to obtain individual blastomeres . 70 2.2 Molecular Biology Techniques . 70 2.2.1 DNA isolation . 70 2.2.1.1 DNA extraction from single cells . 70 2.2.1.2 Follicular fluid DNA extraction . 71 2.2.2 PCR . 71 2.2.3 Gel electrophoresis, visualization and analysis . 71 Chapter 3 Analysis of embryos for pre-implantation genetic diagnosis for mitochondrial DNA point mutations . 74 3.