Title: Development of assisted reproductive technologies to prevent the transmission of mitochondrial DNA disease.
Funder: Health Education England

Abstract: The over-arching aim of my research proposal is to optimise the clinical translation of IVF-based techniques to prevent the transmission of mitochondrial DNA (mtDNA) disease from mother to child. Pathogenic mtDNA mutations have been shown to cause a wide variety of diseases with variable severity and age of onset. After more than 5 years of preclinical research in our clinic into the technique called pronuclear transfer (PNT) we have received the necessary approval and the first patients will be treated later this year. PNT involves transplantation of the nuclear genome after fertilization of the oocyte when the haploid maternal and paternal genomes are separately packaged in clearly visible pronuclei. The pronuclei in a fertilized oocyte from an affected woman are transplanted to that of a donor fertilized oocyte which has had its pronuclei removed. Our findings indicate that while PNT provides a promising approach, further improvements are required to maximise the chance of a successful pregnancy. Embryos develop into viable blastocysts following the PNT procedure but the proportion that develop is reduced compared to unmanipulated controls. Reduced blastocyst development is associated with a reduced pregnancy rate. One hypothesis is that asynchrony between the fertilized oocytes when they undergo the PNT procedure may contribute to the reduced blastocyst development. We found there was no detectable effect on blastocyst development when the pronuclei were removed but fused to reconstitute the fertilized oocyte. The reduced blastocyst development was detected following transplantation of the pronuclei between fertilized oocytes from different egg donors. The first aim of this research proposal is to use a time-lapse system which has been purchased in preparation for the introduction of this clinical service to optimize the timings of the PNT procedure. Embryo development will be monitored and compared to unmanipulated controls. The potential benefit to patients is an improvement in embryo development to the blastocyst stage and chance of a successful pregnancy.Our preclinical research has focused on the PNT procedure however there is an alternative procedure called maternal spindle transfer (MST) which has shown promise to prevent transmission of mtDNA disease. MST is performed before fertilisation when oocytes are arrested at metaphase of meiosis II (MII) awaiting fertilization by a sperm. There is a lack of robust published evidence on the safety and efficacy of MST. The second aim of this research proposal is to compare MST to PNT and depending upon the outcome of the comparison publish either 1) the body of evidence that would be required to obtain regulatory approval to translate the research into the clinical service or 2) why MST should not be translated into clinical service. The benefit to patients is our clinic will offer the procedure which reduces the risk of transmission of mitochondrial DNA (mtDNA) disease but maximizes the chance of a successful pregnancy.

Lay abstract: Mitochondria are small structures within cells which are responsible for producing energy for cells to function. A relatively small amount of DNA is located within the mitochondria and is known as mitochondrial DNA (mtDNA). The mtDNA contains information that allows the mitochondria to make some of the components it needs to produce energy. Mistakes (mutations) in the mtDNA can cause mitochondria to stop working properly and leads to mtDNA disease. The organs with a high energy requirement such as heart and muscles are worst affected. Severe mitochondrial DNA disease is often fatal before adulthood. DNA that is not located within the mitochondria is known as nuclear DNA. Children inherit a copy of nuclear DNA from both parents. Inheritance of mtDNA is different because children inherit all their mtDNA from just their mother. If a woman has a mtDNA mutation all her children are at risk of developing the same mtDNA disease. The risk can be reduced with a range of reproductive options. Mitochondrial donation is a treatment option for women that are high risk of passing on severe mtDNA disease. During the procedure the majority of the faulty mtDNA in a mother's egg is replaced with healthy mtDNA from a donor's egg. A child born following this procedure will inherit DNA from the mother, father and egg donor so have been called three-person or three-parent babies by the press. We have received approval to perform the mitochondrial donation procedure called pronuclear transfer (PNT) and the first patients will be treated in the near future. PNT is performed after fertilisation of the egg by the sperm when the nuclear DNA is packaged inside visible structures called pronuclei. Fertilised eggs at the pronuclear stage contain two pronuclei. One pronucleus contains the nuclear DNA from the sperm and the other the nuclear DNA from the egg. The pronuclei are removed from the fertilised egg of the mother and transferred to a healthy donor's fertilised egg that has had its pronuclei removed. Prior to receiving approval to treat patients our research has shown that embryos can develop after the PNT procedure but a reduced number develop compared to fertilised eggs that haven't undergone PNT. Patients were concerned this could reduce their chance of pregnancy therefore optimising the procedure was identified as a priority area for research by patients at the Reproductive options focus group. A reason why PNT embryos stop developing could be because the procedure was performed between fertilised eggs at slightly different stages of development. The first aim of the research is to use a camera to record pictures of the fertilised eggs as they develop to determine when the pronuclei appear. The PNT procedure will be performed with the mother's and donor's fertilised eggs matched for the time of appearance of the pronuclei. The subsequent embryo development will be compared to fertilised eggs that have not undergone PNT to determine if the proportion of embryos that develop is improved by optimising the timings. Approval to perform the mitochondrial donation procedure called maternal spindle transfer (MST) cannot currently be sought because there is a lack of robust published research data to provide an indication of safety and efficacy of the procedure. MST is performed before fertilization when the eggs are naturally synchronized and waiting to be fertilized by a sperm. Having a naturally synchronized stage to perform the mitochondrial donation procedure could circumvent the timing related issues with PNT. The second aim of the research is to perform the MST procedure and compare with all our data on the PNT procedure. There are two potential outcomes for patients following this comparison. 1. If the proportion of good quality embryos developing after the procedure is higher with MST compared to PNT our research on the MST procedure will be used to apply for the necessary approval in order to be able to offer MST as a treatment option for patients. 2. If the timings of the PNT procedure are optimised and the proportion of good quality embryos developing after the procedure is better than MST the clinic will continue to offer PNT as a treatment option. The results will be published in peer reviewed journals and presented at academic and patient conferences. Public and patients will be involved in designing the presentation to ensure the research is presented in a clear and easy to understand manner for different audiences. Patients will be involved in updating the patient information documents with the research findings and help direct and develop further research questions arising.

Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: REPRODUCTIVE HEALTH AND CHILDBIRTH