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Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT, and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates among PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregate (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In October 2015 the UK enacted legislation to permit the clinical use of two cutting edge germline-altering, IVF-based embryonic techniques: pronuclear transfer and maternal spindle transfer (PNT and MST). The aim is to use these techniques to prevent the maternal transmission of serious mitochondrial diseases. Major claims have been made about the quality of the debates that preceded this legislation and the significance of those debates for UK decision-making on other biotechnologies, as well as for other countries considering similar legislation. In this article we conduct a systematic analysis of those UK debates and suggest that claims about their quality are over-stated. We identify, and analyse in detail, ten areas where greater clarity, depth and nuance would have produced sharper understandings of the contributions, limitations and wider social impacts of these mitochondrial interventions. We explore the implications of these additional considerations for (i) the protection of all parties involved, should the techniques transfer to clinical applications; (ii) the legitimacy of focussing on short-term gains for individuals over public health considerations, and (iii) the maintenance and improvement of public trust in medical biotechnologies. We conclude that a more measured evaluation of the content and quality of the UK debates is important and timely: such a critique provides a clearer understanding of the possible, but specific, contributions of these interventions, both in the UK and elsewhere; also, these additional insights can now inform the emerging processes of implementation, regulation and practice of mitochondrial interventions.

Background: The use of nuclear transfer (NT) has been proposed as a novel reproductive treatment to overcome the transmission of maternally-inherited mitochondrial DNA (mtDNA) mutations. Pathogenic mutations in mtDNA can cause a wide-spectrum of life-limiting disorders, collectively known as mtDNA disease, for which there are currently few effective treatments and no known cures. The many unique features of mtDNA make genetic counselling challenging for women harbouring pathogenic mtDNA mutations but reproductive options that involve medical intervention are available that will minimize the risk of mtDNA disease in their offspring. This includes PGD, which is currently offered as a clinical treatment but will not be suitable for all. The potential for NT to reduce transmission of mtDNA mutations has been demonstrated in both animal and human models, and has recently been clinically applied not only to prevent mtDNA disease but also for some infertility cases. In this review, we will interrogate the different NT techniques, including a discussion on the available safety and efficacy data of these technologies for mtDNA disease prevention. In addition, we appraise the evidence for the translational use of NT technologies in infertility., Objective and Rationale: We propose to review the current scientific evidence regarding the clinical use of NT to prevent mitochondrial disease., Search Methods: The scientific literature was investigated by searching PubMed database until Jan 2017. Relevant documents from Human Fertilisation and Embryology Authority as well as reports from both the scientific and popular media were also implemented. The above searches were based on the following key words: 'mitochondria', 'mitochondrial DNA'; 'mitochondrial DNA disease', 'fertility'; 'preimplantation genetic diagnosis', 'nuclear transfer', 'mitochondrial replacement' and 'mitochondrial donation'., Outcomes: While NT techniques have been shown to effectively reduce the transmission of heteroplasmic mtDNA variants in animal models, and increasing evidence supports their use to prevent the transmission of human mtDNA disease, the need for robust, long-term evaluation is still warranted. Moreover, prenatal screening would still be strongly advocated in combination with the use of these IVF-based technologies. Scientific evidence to support the use of NT and other novel reproductive techniques for infertility is currently lacking., Wider Implications: It is mandatory that any new ART treatments are first adequately assessed in both animal and human models before the cautious implementation of these new therapeutic approaches is clinically undertaken. There is growing evidence to suggest that the translation of these innovative technologies into clinical practice should be cautiously adopted only in highly selected patients. Indeed, given the limited safety and efficacy data, close monitoring of any offspring remains paramount., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

The recent birth from a mitochondrial DNA mutation carrier of a child, conceived after transfer in a donor oocyte of the meiotic spindle, taken from the maternal oocyte, revived the debate on the safety of these procedures. The doubts concern mainly the possibility of genetic reversion, the uncertainties about potential disturbances of the dialogue between nuclear and mitochondrial genomes and the side effects of a heteroplasmic state induced by these techniques. The possibility to expand nuclear transfer applications to patients experiencing in vitro fertilization failure, urges us to answer these questions rapidly., (© 2017 médecine/sciences – Inserm.)

In this article, I argue that there is no compelling therapeutic 'need' for human nuclear genome transfer (so-called mitochondrial replacement) to prevent mitochondrial diseases caused by mtDNA mutations. At most there is a strong interest in (i.e. 'want' for) this technology on the part of some women and couples at risk of having children with mitochondrial disease, and perhaps also a 'want' on the part of some researchers who see the technology as a useful precedent - one that provides them with 'a quiet way station' in which to refine the micromanipulations techniques essential for other human germline interventions and human cloning. In advance of this argument, I review basic information about mitochondrial disease and novel genetic strategies to prevent the transmission of mutated mitochondria. Next, I address common features of contemporary debates and discussions about so-called mitochondrial replacement. First, I contest the cliché that science-and-(bio)technology is fast outpacing ethics. Second, I dispute the accuracy of the term 'mitochondrial replacement'. Third, I provide a sustained critique of the purported 'need' for genetically-related children. In closing, I call into question the mainly liberal defense of human nuclear genome transfer. I suggest an alternative frame of reference that pays particular attention to issues of social justice. I conclude that our limited resources (time, talent, human eggs, and money) should be carefully expended in pursuit of the common good, which does not include pandering to acquired desires (i.e., wants)., (© 2017 John Wiley & Sons Ltd.)

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention., Competing Interests: The authors declare no competing financial interests.

To evaluate and compare mitochondrial DNA (mtDNA) carry-over and embryonic development potential between different nuclear transfer techniques we performed germinal vesicle nuclear transfer (GV NT), metaphase-II spindle-chromosome-complex (MII-SCC) transfer and pronuclear transfer (PNT) in mice. No detectable mtDNA carry-over was seen in most of the reconstructed oocytes and embryos. No significant differences were seen in mtDNA carry-over rate between GV NT (n=20), MII-SCC transfer (0.29 ± 0.63; n=21) and PNT (0.29 ± 0.75; n=25). Blastocyst formation was not compromised after either PNT (88%; n=18) or MII-SCC transfer (86%; n=27). Further analysis of blastomeres from cleaving embryos (n=8) demonstrated undetectable mtDNA carry-over in all but one blastomere. We show that NT in the germ line is potent to prevent transmission of heritable mtDNA disorders with the applicability for patients attempting reproduction., (Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial DNA diseases are relatively common, sometimes devastating, and transmitted exclusively through the egg to children of carrier mothers. A study in Cell by Wang et al. (2014) adds the exciting possibility of a new therapy for preventing mitochondrial disease transmission predicated on the use of polar body genomes in mice., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inherited mtDNA diseases transmit maternally and cause severe phenotypes. Currently, there is no effective therapy or genetic screens for these diseases; however, nuclear genome transfer between patients' and healthy eggs to replace mutant mtDNAs holds promises. Considering that a polar body contains few mitochondria and shares the same genomic material as an oocyte, we perform polar body transfer to prevent the transmission of mtDNA variants. We compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Reconstructed embryos support normal fertilization and produce live offspring. Importantly, genetic analysis confirms that the F1 generation from polar body transfer possesses minimal donor mtDNA carryover compared to the F1 generation from other procedures. Moreover, the mtDNA genotype remains stable in F2 progeny after polar body transfer. Our preclinical model demonstrates polar body transfer has great potential to prevent inherited mtDNA diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inherited diseases caused by mitochondrial gene (mtDNA) mutations affect at least 1 in 5000-10,000 children and are associated with severe clinical symptoms. Novel reproductive techniques designed to replace mutated mtDNA in oocytes or early embryos have been proposed to prevent transmission of disease from parents to their children. Here we review the efficacy and safety of these approaches and their associated ethical and regulatory issues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

Unlabelled: BACKGROUND; Mitochondrial DNA (mtDNA) disorders are an important cause of human diseases. In view of the limitations of prenatal diagnosis and preimplantation genetic diagnoses, alternatives such as ooplasmic transfer (OT) and nuclear transfer (NT) have been proposed to prevent the transmission of mtDNA mutations. Both OT and NT are radical in the sense that they do not entail genetic selection, but genetic intervention to correct the genetic cause of the disease., Methods: After interviews with experts in the field, the relevant literature was searched and analyzed. Bioethical issues were divided into conceptual and normative points., Results: OT is the transfer of normal mitochondria to a carrier's oocyte containing mutant mtDNA. In case of NT, a donated oocyte is enucleated and replaced with the nuclear DNA from a woman carrying a mtDNA mutation. NT can be performed both before and after in vitro fertilization, respectively, with the nucleus of an unfertilized oocyte, with the pronuclei of the zygote, or with the nucleus of a blastomere of an embryo. Conceptual questions regard whether these techniques amount to germ-line modification and human cloning. Normative questions concern, among others, the significance of intervening in the mtDNA, the implications of having 'three genetic parents', the ethics of oocyte donation and the health and safety risks for children conceived as a result of one of these techniques., Conclusions: Further interdisciplinary debate and research is needed to determine whether a clinical application of OT and NT can be morally justified, and if so, under which conditions.