June 8, 2016

expert reaction to preclinical application of pronuclear transfer to prevent mitochondrial DNA disease

Faulty mitochondria contribute to a range of diseases and a research group working towards the use of mitochondrial donation techniques for women at risk of passing on those diseases to their children have described their preclinical work in the journal Nature. They report that embryos which underwent the donation techniques were largely similar to normal ones, and that they were able to minimise ‘carryover’ of the original faulty mitochondria to the new embryo. These comments accompanied a briefing.

All our previous output on this subject can be seen here.

Professor Sian Harding, Member of the Nuffield Council on Bioethics working party on the ethical review “Novel Techniques for the Prevention of Mitochondrial DNA Disorders” and Director of the British Heart Foundation Cardiovascular Regenerative Medicine Centre, Imperial College London, said:

“This paper takes us a number of steps forward to the clinical translation of IVF techniques to prevent or reduce the risk of transmission of mitochondrial mutations. Of note, the researchers concentrate on the more ethically challenging pronuclear transfer (PNT) which uses fertilised eggs, rather than maternal spindle transfer (MST) where the egg is unfertilised. Although PNT technically represents the destruction of an embryo, recent studies have shown that this is the safer option. The key goals were to produce good quality blastocysts, similar to those shown to produce successful live births after IVF, and to reduce the carryover of mutant mitochondria to levels  which strongly reduce the likelihood of developing (<18%) or transmitting (<5%) the disease.

“For this study normally fertilised human zygotes were used, unlike previous studies which were on abnormally fertilised ones, and the researchers found it necessary to alter the conditions previously established. Changes in the manipulation medium, as well as a move from late to early stages after first appearance of pronuclei, produced blastocysts which were statistically indistinguishable from those produced in normal IVF by measures of morphology, ploidy and gene expression. A further modification to the transfer medium reduced mitochondrial carryover from >5% in 28% of samples to <5% in all samples and <2% in 79%. However, one line with 4% carryover showed a wide drift when converted into an embryonic stem cell (hES) line, with some clones eventually having around 50% of the original mitochondrial type. This must cause concern, though it has been questioned before whether the hES line is a good model for the developing embryo. The authors suggest that lines with >2% heteroplasmy were associated with identifiable technical problems during transfer, and that this could be used to exclude them from selection for clinical implantation:  however this remains to be established.

“Overall, the methods described here are likely to reduce dramatically the future risks for the fetus of developing mitochondrial disease, though not to eliminate them completely. An important further recommendation was that the patient oocytes, rather than those from the donor, should be frozen. This buys time for those mothers who are now waiting for this technology to be safe enough for licencing but who might have deteriorating oocyte production with age. ”

Dr Rafael J. Yáñez-Muñoz, Reader in Advanced Therapy, Royal Holloway, University of London said:

“This paper reports experimental studies to optimize the procedure of mitochondrial donation before clinical application, which would have to be licensed by the Human Fertilization and Embryology Authority (HFEA). The authors have optimized the procedure of pronuclear transplantation (PNT) between normally fertilized 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 nuclei from the fertilized zygote) from the affected zygote to the donor zygote, after removal of the donor’s own pronuclei. Technology is not perfect so carryover of some mitochondria alongside pronuclei 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 fertilization, 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 vitrified (preserved) oocyte, as would be done in the clinic, but the frequency of blastocyst formation was somewhat reduced when involving a zygote from a vitrified 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 and use of zygotes from vitrified patient rather than donor oocytes were beneficial, and 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. 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.”

Professor Robin Lovell-Badge, Group Leader at The Francis Crick Institute, said:

“This is a detailed investigation of the pronuclear transfer (PNT) technique as a way to avoid transmission of mitochondrial disease, where the authors have addressed questions posed by the HFEA’s Scientific Panel in their last Report in 2014. With respect to the possibility of using PNT clinically, it was essential that they explore the methods in normally fertilised eggs, which they have now done. Indeed, they needed to modify the previous techniques that had been used with abnormally fertilised eggs, in order to have good survival to blastocyst stages and to reduce carryover of mitochondria along with the pronuclei. The latter must to be low to reduce the chances of any child born having mitochondrial disease, or, if a girl, for her children to be at risk.

“I expect that all those interested in the application of the methods, including the scientists and clinicians involved, and prospective patients, will be encouraged by the results, where a thorough analysis of the embryos generated by PNT suggests that they are indistinguishable from control embryos. Analysis of embryonic stem (ES) cells derived from the embryos after PNT was also mostly consistent with expectations. The exception was one cell line, out of 5, where the mitochondrial DNA type associated with the transferred pronuclei increased over time to much higher levels than present in the embryo after PNT.  This result was similar to recently published data from the Egli lab in New York, which used an alternative technique, that of maternal spindle transfer or MST in eggs prior to fertilization, where they also had one aberrant ES cell line where this occurred (out of 8). It is not obvious why this happened in either case or whether it is relevant to clinical use of the methods, given that maintenance of ES cells in culture over long periods has little to do with normal embryo development.

“When reconstituted, the HFEA’s Scientific Panel will need to assess this work along with other relevant studies that have been carried out since their last Report about two years ago.”

Dr Marita Pohlschmidt, Director of Research for Muscular Dystrophy UK, said:

“This is an encouraging and thorough study into the safety and efficacy of mitochondrial donation IVF. The researchers have significantly refined the procedure, identifying the optimum timings for the best possible chance of success. They have also confirmed the need to keep transfer of mutated mitochondrial DNA at an absolute minimum to reduce the risk of mitochondrial disease being passed on.

“While mitochondrial donation IVF significantly reduces the risk of a child born developing mitochondrial disease, it cannot offer a complete guarantee. The clarification of risk that is offered by this study is therefore a very important step forward. It is vital to helping parents who are considering the technique make informed choices. For people whose lives have been blighted by the shadow of mitochondrial disease, mitochondrial donation IVF could offer an invaluable choice.

“We hope the HFEA’s review of these findings opens the door to the first licensed clinics.”

Dr Dusko Ilic, Reader in stem cell science, King’s College London, said:

“The paper is a meticulous dissection of all technical steps involved in the procedure of nuclear DNA transfer for clinical purposes of preventing mitochondrial DNA (mtDNA) disease inheritance. The authors demonstrated that an early pronuclear transfer (ePNT) yields better blastocyst survival in comparison with late pronuclear transfer. Pronuclear stage is a stage after fertilization when maternal and paternal haploid genomes are still packaged separately.

“The authors also demonstrated that the ePNT procedure does not lead to increased incidence of chromosomal abnormalities in comparison with normal non-manipulated blastocysts and that their gene expression pattern indistinguishable from each other.

“They looked into expression of mtDNA encoded genes and found no difference between ePNT and control blastocysts.

“It has been shown earlier that reducing mtDNA carryover to <5% has the potential to prevent transmission of disease to subsequent generations. Using pyrosequencing to determine mtDNA carryover they found that using frozen rather than fresh eggs leads to higher carryover. Regardless, the majority of blastocysts had <2% heteroplasmy (carryover) and none higher than 5%. The majority of the oocytes with 2-5% carryover encountered technical issues during the procedure.

“Finally, to assess the potential fate of mtDNA under condition when the cells can replicate extensively (longer than allowed by UK law), they derived human embryonic stem cell (hESC) lines from five ePNT blastocysts. Four out of five lines showed low level of heteroplasmy, whereas one derived from a blastocyst with around 4% mtDNA carryover showed an upward drift of heteroplasmy and variation among hESC colonies at the passage 12.

“The take home message is that the Newcastle group has clearly demonstrated that they took the highest possible level of precautions and that they are ready for clinical application”

Prof. Bert Smeets, Professor in Clinical Genomics with a focus on mitochondrial disease, Maastricht UMC, Maastricht, said:

“This is an exciting paper, truly setting the stage for the first clinical application of pronuclear transfer in mtDNA disease. The amount and quality of the data is impressive and the added value of being able to use normally fertilized went far beyond my expectations. The data shows that studies on poor-quality embryos have a limited value for establishing a clinical procedure, which is an important message as such. The only correct way forward is using normally fertilized zygotes and embryos. The paper describes a treatment, optimized for  vitrification, manipulation medium and  developmental stage, which generates normal embryos with a  low carry-over of less than 2% in the vast majority. This procedure seams ready for a clinical setting”

“In one ES-cell line an enrichment of the carry-over mtDNA can be observed. This indicates that in a situation of mixed mtDNA templates, a replication or selection advantage, in addition to genetic drift, may occur. From our experience with Preimplantation Genetic Diagnosis (PGD) we saw that a low mutation load at the blastomere stage reflects the mutation load in the child when it is born. Replicative differences are not an issue in PGD, as long as it is not related to the mutation itself, as apart from the mutation the mtDNA is completely identical. However, we do encounter negative and positive selection, in addition to genetic drift, at the level of the mutation, usually at high mutation loads. Keeping the mutation load in the selected PGD embryo far below the level of expression, prohibits that by genetic drift and/or selection the mutation load can reach pathogenic levels. For pronuclear transfer and the additional replicative difference between the mtDNA species, which by the way could go either way, it seems preferable to go as low as possible and stay below the 2%, which fortunately is achieved for almost 80% of the embryos. Further insight in the mtDNA variants affecting replication, would enable selection of mtDNA  combinations in which the mutant will not out-replicate the wild-type mtDNA. One should also not forget that a mixture of different wild-type mtDNAs has been tested and that the situation for the mutated mtDNA might be less favourable.”

“Using a variety of approaches the authors show that blastocysts generated by nuclear transfer do not differ from control IVF blastocysts. This is reassuring, the interventions and manipulations do not cause developmental differences. Still, it is far more difficult to  demonstrate that a blastocyst is normal, than that it is abnormal, and also for controls we do not fully understand what the optimal blastocyst should look like. Normal individual variation between women, but also between blastocysts of the same woman may occur, without necessarily being negative. The RNA-Seq data at least demonstrate that any variation, which might be there is not stronger than differences in quality or in cell lineage. Part of the normal variation, which is equally present between blastocysts of both groups, can be observed in the gene expression of levels mtDNA encoded OXPHOS proteins. As we know that mtDNA copy number can vary largely between oocytes, also from the same woman, I would not be surprised if these gene expression differences reflect normal mtDNA copy number variation at a stage, when at least in the trophectoderm, mtDNA replication starts.”

‘Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease’ by name of Hyslop et al. will be published in Nature at 18:00 UK time on Wednesday 8th June, which is also when the embargo will lift. 

Declared interests

Professor Sian Harding: No conflict of interest apart from membership of the NCOB working party.

Robin Lovell-Badge: Robin Lovell-Badge has no financial conflicts of interest to declare. He works at the same Institute as Kathy Niakan and her group, who carried out some of the studies described in the paper, and who are listed as co-authors. Nevertheless, he has not been involved in any aspects of this research, nor does he have any management role over Dr Niakan. He has been, however, a member of the HFEA’s Scientific Panel looking into ways to avoid mitochondrial disease since its inception.

Dr Marita Pohlschmidt: Dr Marita Pohlschmidt: The Muscular Dystrophy UK does not have a financial interest in mitochondrial research.  We have been a long-term funder of research into mitochondrial disease, conducted by Professor Doug Turnbull and his team at Newcastle University.  Professor Turnbull was appointed a Vice President of the Muscular Dystrophy UK in 2013, an honorary position which carries no payment or reward.

No others received.