Author: Professor Rafael J. Yáñez-Muñoz, Royal Holloway University of London
Mitochondria produce ATP and are generally considered the powerhouses in the cells that make up the human body. They are vital to normal cell function and mitochondrial defects can cause debilitating physical and developmental symptoms particularly affecting body parts with the highest energy demands including the brain, heart, muscles, liver and kidney.
Mitochondria are unique in that they carry their own minimal DNA, separate to that found in the nucleus of a cell where chromosomes reside, and each person inherits their mitochondria from the mother and not the father. The frequency of mitochondrial disease is at least 1:10,000 adults, but around 1:200 people carry mitochondrial mutations that can cause disease, and predicting disease in children of affected women can be complicated (Chinnery et al., 2012). General information on mitochondrial disease is available from the NHS (http://www.mitochondrialncg.nhs.uk/) and The Lily Foundation (http://www.thelilyfoundation.org.uk/mitochondrial-disease/).
No treatment is available for mitochondrial disease. Mitochondrial donation is being researched as a possible way to prevent mitochondrial disease in families known to be affected. In mitochondrial donation, a donor egg (from a third party) with healthy mitochondria would be used, and the nuclear DNA from the third party would be removed and replaced with DNA from the father and affected mother. Two possible avenues for mitochondrial donation have been proposed: maternal spindle transfer (involving the use of a donor egg or “oocyte”) and pronuclear transplantation (PNT, involving the use of a fertilised donor egg or “zygote”) as described in http://www.nature.com/news/eggs-jpg-7.2493?article=1.9883. Hyslop et al., (2016) report experimental studies to optimise the PNT procedure of mitochondrial donation before clinical application, which would have to be licensed by the Human Fertilization and Embryology Authority (HFEA). The authors have improved the PNT procedure between normally fertilised zygotes to reduce the chances of technical manipulations and mutations resulting in low-quality or diseased embryos. PNT involves the transfer of the pronuclei (the still separate maternal and parental nuclear DNA from the fertilised egg) from the affected zygote to the donor zygote. Technology is not perfect so carryover of some mitochondria from the affected egg alongside mother and father DNA is currently unavoidable.
The authors demonstrate that a number of technical manipulations improve the efficiency and outcome of the PNT procedure, which can be assessed by the quality of the blastocyst, the ball of cells resulting from the initial development of the zygote. This quality is assessed in clinical IVF procedures. The timing of pronuclei transfer (better early after fertilisation, about 8 hours after insemination, referred to as “ePNT”), changes to the manipulation medium (removing calcium and magnesium, and reducing the amount of the protein that mediates the fusion event), and the use of a one-step medium in which the embryos remained for the duration of the manipulation, all were beneficial. Under these conditions, blastocyst formation was the same in unmanipulated and control samples (zygotes that received their own pronuclei back). The quality of the resulting blastocysts did not seem particularly affected in transfers involving zygotes from a fresh and a preserved oocyte, as would be done in the clinic, but the frequency of blastocyst formation was somewhat reduced when involving a zygote from a preserved oocyte.
The authors went on to study the resulting blastocysts for aneuploidy (frequency of abnormal chromosomes), and pattern of gene expression (which genes are active and at what level). The conclusion was that good quality ePNT blastocysts could not be distinguished from controls.
The authors finally studied mitochondrial carryover. Removal of the nuclei in the absence of sucrose helped. Either donor or affected mother oocytes have to be preserved during the process, and the authors found that it was better to use preserved donor oocytes and fresh affected oocytes. Combination of these two improvements led to mitochondrial carryover levels below 2% in the majority of blastocysts, and below 5% in all of them. 5% mitochondrial carryover is considered a relatively stringent level below which disease development or transmission are low (Samuels et al., 2013). When the studies of mitochondrial carryover were done in embryonic stem cells derived from ePNT blastocysts (to allow for more cell divisions during which instability could manifest), some stem cell lines displayed instability and an increase in mitochondrial DNA carryover with time. It should be said that stem cell lines are quite different from normal embryonic development and these results should be interpreted with caution. In any case, embryos from ePNT could be screened for increased levels of mutant mitochondria.
Overall the authors present an encouraging set of experiments suggesting that mitochondrial donation techniques are approaching the quality likely to be required for clinical application.
References
Chinnery, P.F., Elliot, H.R. et al., (2012). Epigenetics, epidemiology and mitochondrial DNA diseases. Int J Epidemiol. 41, 177-187.
Hyslop, L.A., Blakeley, P. et al. (2016). Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature. http://dx.doi.org/10.1038/nature18303
Samuels, D. C., Wonnapinij, P. et al. (2013). Preventing the transmission of pathogenic mitochondrial DNA mutations: can we achieve long-term benefits from germ-line gene transfer? Hum. Reprod. 28, 554–559.