Is three-parent IVF the answer to preventing mitochondrial defects?

Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases

Study question Can pronuclear and spindle transfer rescue embryo development and pregnancy outcomes in patients with diminished ovarian reserve (DOR) who consistently yield no euploid blastocysts? Summary answer Mitochondrial replacement therapy led to high fertilization, euploid blastocyst development, pregnancy and live birth rates, allowing DOR patients to maintain genetic similarity with their offspring. What is known already Women with DOR and advanced maternal age must often turn to oocyte donation due to the significantly reduced quality and quantity of autologous oocytes. As mitochondrial dysfunction leads to oocyte aneuploidy and failed IVF outcomes, mitochondrial replacement techniques (MRT) are being explored to prevent transmission of mutations in maternally inherited mitochondrial DNA (mtDNA). Maternal spindle transfer into young donor oocytes improves morphokinetic development and reduced aneuploidy rates while pronuclear transfer at the zygote stage enhances blastocyst development. Due to ethical restrictions, there are few clinical studies on MRT and none that investigated pregnancy outcomes in AMA patients. Study design, size, duration In this prospective cohort study, pronuclear transfer was performed on 323 mature oocytes retrieved following short antagonist controlled ovarian stimulation between June 2022 and January 2024.Main outcomes were the clinical pregnancy, ongoing pregnancy and live birth rates following frozen embryo transfer (FET). Secondary outcomes included the fertilization rate, embryo development and euploidy status determined by preimplantation genetic testing for aneuploidy (PGT-A). Additionally maternity test used for DNA matching. Participants/materials, setting, methods Oocytes were derived from 66 DOR patients (≤46 years of age) who had ≥5 IVF attempts without yielding blastocysts. Karyoplast-containing meiotic spindles from the patients‘ oocytes were transferred into enucleated donor oocytes before fertilization. For pronuclear transfer, the pronuclei of patient and donor zygotes were fused into the HVJ-E virus while the cytoplasmic fragments were neutralized. Embryos were cultured in single-step culture. Statistical differences were evaluated using the Shapiro-Wilk test followed by the Student’s t-test. Main results and the role of chance Participants had a mean age of 39 years and produced, on average, 9.6 mature oocytes . Following spindle or pronuclear transfer, 88.4% of oocytes were successfully fertilized. 97.2% of the embryos reached the 8-cell stage on day 3 on pronuclear transfer and 94.2% for meiotic spindle transfer, 55.7% reached the blastocyst stage (AA, AB, BA, BB quality on day 5/6) for pronuclear transfer and 52.5% reached blastocyst stage as following morphological qualities for meiotic spindle transfer.(p < 0.05 in all cases). PGT-A revealed a 60.3% euploidy rate for meiotic spindle transfer and 56.9% for pro nuclear transfer. Remarkably, 59.6% of these patients achieved clinical pregnancy from pro nuclear transfer and 52.2% of patients achieved clinical pregnancy from meiotic spindle transfer for following FET, 60% had an ongoing pregnancy for pro nuclear transfer and 55% for meiotic spindle transfer. 44% achieved a live birth after 37 weeks’ gestation for pro nuclear transfer and 40% achieved a live birth after 37 weeks‘ gestation for meiotic spindle transfer. All cases confirmed with matched nuclear DNA with source of nuclear DNA from recipiend and mitochondrial DNA match rate found avarage 89,51% with donor-recipient match. Heteroplasmy rate was avarage 9,21% for all cases. Limitations, reasons for caution While pronuclear transfer has minimal risks of micromanipulation compared to spindle transfer, neonatal testing is recommended to screen for any potential genetic and morphologic abnormalities. Larger prospective studies are needed to validate these findings, explore epigenetic alterations, and confirm long-term safety. Wider implications of the findings This study underscores the potential of spindle and pronuclear transfer to achieve competent euploid embryos and live births in DOR patients who exploited conventional IVF options. DOR patients will be able to maintain genetic bonds and physical characteristics with their children, avoiding social stigmas and potential legal issues. Trial registration number Yes

Preventing the transmission of mitochondrial DNA disorders: selecting the good guys or kicking out the bad guys

Women can produce eggs with a high load of abnormal mtDNA. Those who know they have a disease caused by anomalies in the mtDNA can opt for pre-implantation genetic diagnosis and select for the implantation the embryos without mutations in mtDNA or with the lowest proportion of abnormal mtDNA. Another option is to have a baby using donated eggs. The UK House of Commons recently approved two methods for replacing abnormal mtDNA: maternal spindle transfer and pro-nuclear transfer. In maternal spindle transfer (MST), the genetic material in the nucleus of an egg is removed from the intended mother’s egg and transferred into an egg from a donor that has had its maternal spindle removed. The reconstituted egg would then be fertilised by the intended father’s sperm and the newly formed embryo implanted into the intended mother. In pronuclei transfer (PNT), the pronuclei from the donor embryo are transferred into the donor embryo, which has had its pronuclei removed. The essay will discuss the main moral objections against the various types of nuclear transfer to prevent mitochondial DNA (mtDNA) disorders: the risks for the baby and for the future generations, the problem related to changing of the germ line and the use of similar techniques for other purposes. Our conclusion is that the two main developed methods for replacing abnormal mtDNA are morally approvable because they allow the child who will be born to have a better quality of the life.

This paper examines whether there are moral differences between the mitochondrial replacement techniques that have been recently developed in order to help women afflicted by mitochondrial DNA diseases to have genetically related children absent such conditions: maternal spindle transfer (MST) and pronuclear transfer (PNT). Firstly, it examines whether there is a moral difference between MST and PNT in terms of the divide between somatic interventions and germline interventions. Secondly, it considers whether PNT and MST are morally distinct under a therapy/creation optic. Finally, it investigates whether there is a moral difference between MST and PNT from a human embryo destruction point of view. I conclude, contra recent arguments, that regarding the first two points there is no moral differences between PNT and MST; and that regarding the third one MST is morally preferable to PNT, but only if we hold a gradualist account of the moral value of human embryos where zygotes have slight moral value.

Mitochondrial donation and human reproduction: Clinical, ethical and legal aspects.

New IVF techniques put mitochondrial diseases in focus

This article argues that two forms of mitochondrial replacement therapy, maternal spindle transfer (MST) and pro-nuclear transfer (PNT), are not therapies at all because they do not treat children who are coming into existence. Rather, these technologies merely create healthy children where none was inevitable. Even if creating healthy lives has some value, it is not to be confused with the medical value of a cure or therapy. The article addresses a recent Bioethics article, 'Mitochondrial Replacement: Ethics and Identity,' by Wrigley, Wilkinson, and Appleby, who argue that PNT is morally favorable to MST due to the Non-Identity Problem. Wrigley et al. claim that PNT, since it occurs post-conception, preserves the identity of the resulting child, whereas MST, since it occurs pre-conception, is an identity-altering technique. As such, a child born with mitochondrial disease could complain that her parents failed to use PNT, but not MST. The present article argues that the authors are mistaken: both MST and PNT are identity-affecting techniques. But this is of little matter, for we should be cautious in drawing any moral conclusions from the application of the Non-Identity Problem to cases. The article then argues that the authors are mistaken in inferring that PNT is a type of embryonic cure or therapy for children with mitochondrial disease. The article cautions against the mistaken life-saving rhetoric that is common in bioethics discussions of MRTs.

Study question Can pronuclear transfer (PNT) or maternal spindle transfer (ST) be applied to overcome poor embryo development associated with advanced maternal age or early embryo arrest in a mouse model? Summary answer Both PNT and ST may have the potential to restore embryonic developmental potential in a mouse model of reproductive ageing and embryonic developmental arrest. What is known already Germline nuclear transfer (NT) techniques, such as PNT and ST, are currently being applied in humans to prevent the transmission of mitochondrial diseases. Yet, there is also growing interest in the translational use of NT for treating infertility and improving IVF outcomes. Nevertheless, direct scientific evidence to support such applications is currently lacking. Moreover, it remains unclear which infertility indications may benefit from these novel assisted reproductive technologies. Study design, size, duration We applied two mouse models to investigate the potential of germline NT for overcoming infertility. Firstly, we used a model of female reproductive ageing (B6D2F1 mice, n = 155), with ages ranging from 6 to 8 weeks (young), 56 (aged) to 70 weeks (very-aged), corresponding to a maternal age of Participants/materials, setting, methods Ovarian reserve was assessed by histological analysis in the reproductive-aged mice. Mitochondrial membrane potential (△Ψm) was measured by JC-1 staining in MII oocytes, while spindle-chromosomal morphology was examined by confocal microscopy. Reciprocal ST and PNT were performed by transferring the meiotic spindle or pronuclei (PN) from unfertilised or fertilised oocytes (after ICSI) to enucleated oocytes or zygotes between aged or very-aged and young mice. Similarly, NT was also conducted between NZB/OlaHsd (embryo arrest) and B6D2F1 (non-arrest control) mice. Finally, the effect of cytoplasmic transfer (CT) was examined by injecting a small volume (∼5%) of cytoplasm from the oocytes/zygotes of young (B6D2F1) mice to the oocytes/zygotes of aged or very-aged mice or embryo-arrest mice. Overall, embryonic developmental rates of the reconstituted PNT (n = 572), ST (n = 633) and CT (n = 336) embryos were assessed to evaluate the efficiency of these techniques. Finally, chromosomal profiles of individual NT-generated blastocysts were evaluated using next generation sequencing. Main results and the role of chance Compared to young mice, the ovarian reserve in aged and very-aged mice was severely diminished, reflected by a lower number of ovarian follicles and a reduced number of ovulated oocytes (P 0.05). In the second series of experiments, we primarily confirmed that the majority (61.8%) of in vivo zygotes obtained from NZB/OlaHsd mice displayed two-cell block during in vitro culture, coinciding with a significantly reduced blastocyst formation rate compared to the B6D2F1 mice (13.5% vs. 90.7%; P 0.05). Surprisingly, chromosomal analysis revealed that euploidy rates in PNT and ST blastocysts generated following the transfer of very-aged PN to young cytoplasts and very-aged spindles to young cytoplasts were comparable to ICSI controls (with young mouse oocytes). A high euploidy rate was also observed in the blastocysts obtained from either PNT or ST between young mice. Conversely, the transfer of young PN and young spindles into very-aged cytoplasts led to a higher rate of chromosomal abnormalities in both PNT and ST blastocysts. Large scale data N/A. Limitations, reasons for caution The limited number of blastocysts analysed warrants careful interpretation. Furthermore, our observations should be cautiously extrapolated to humans given the inherent differences between mice and women in regards to various biological processes, including centrosome inheritance. The findings suggest that ST or PNT procedures may be able to avoid aneuploidies generated during embryo development, but they are not likely to correct aneuploidies already present in some aged MII oocytes. Wider implications of the findings To our knowledge, this is the first study to evaluate the potential of PNT and ST in the context of advanced maternal age and embryonic developmental arrest in a mouse model. Our data suggest that PNT, and to a lesser extent ST, may represent a novel reproductive strategy to restore embryo development for these indications. Study funding/competing interest(s) M.T. is supported by grants from the China Scholarship Council (CSC) (Grant no. 201506160059) and the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) (Grant no. 01SC2916 and no. 01SC9518). This research is also supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N, G051516N and G1507816N). The authors declare no competing interests. Trial registration number N/A.

In the discussions leading up to the enactment of the UK Human Fertilisation and Embryology (Mitochondrial Donation) Regulations 2015, it was repeatedly emphasised, by many commentators, that maternal spindle transfer (MST) and pronuclear transfer (PNT) did not give rise to children who could be considered as having three or more parents. This was because it was argued that only the genetic material found in the chromosomes should be considered as the determining factor for the formation of parent–child relationships and the resulting kinship identities. In this present study, however, this assertion will be questioned in the light of different kinds and different understandings of kinship identities. It will also be suggested that any person who is partly responsible for the very existence of a child, through any means, may qualify as a causal parent — a parent whom the resulting child may want to identify. As a result, a positive response should be given to a request from a person born from MST and PNT concerning identifying information for all the individuals responsible for bringing him or her into existence. In the light of this, the article will conclude that it is regrettable that the UK government enacted binding legislation making sure that children, born through MST and PNT, will never be able to contact the egg donors and, in the case of PNT, the sperm donors. This reflects a very limited understanding of who parents really are and may give rise to serious long-term psychological distress in the prospective children.

First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility

Mitochondrial Replacement Therapy (MRT) is a development in the area of reproductive health, as it provides new possibilities for women who are at risk of transmitting mitochondrial diseases to their offspring. A cell’s powerhouse, known as mitochondria, contains inherited Deoxyribonucleic Acid (DNA) called mitochondrial DNA (mtDNA) and is inherited only through the mother. Changes in specific areas of the mtDNA can lead to severe mitochondrial disorders affecting major bodily processes and might involve metabolic or neurological problems. New In-Vitro Fertilisation (IVF) procedures, incorporated into Pronuclear Transfer (PNT) and Maternal Spindle Transfer (MST), use MRT to transplant mtDNA along with a donor’s mitochondria, thereby reducing the chances of passing on mitochondrial diseases. The advantage of MRT is the ability to minimise mitochondrial diseases, which is achievable through key techniques. However, MRT poses specific technical issues and raises ethical concerns, particularly concerning germline modifications. The following paper examines the clinical, ethical and technical aspects of MRT with regard to IVF compatibility, success rates and resulting health impacts.

Abstract: Mitochondrial DNA (mtDNA) mutation is associated with serious human disorders and affects multiple organs and tissues with high-energy requirements. Since the transmission of mtDNA is complex and is not fully understood, an accurate estimation of mtDNA disease transmission by preimplantation genetic diagnosis (PGD) or by prenatal diagnosis (PND) remains challenging. Recently, nuclear transfer techniques, including maternal spindle transfer (MST), pronuclear transfer (PNT) and polar body transfer (PBT), have shown the promising results. These methods avoid the transmission of mutated mtDNA from mother to offspring, and are collectively known as the mitochondrial replacement therapy (MRT). Further, the United Kingdom Parliament approved the Human Fertilisation and Embryology Authority (HFEA) to grant licenses for experimental use of MST and PNT in humans in 2015. Thus, a new era of assisted reproductive technology (ART), in which cures can be provided at the gamete or early zygote stages, is reali...

Mitochondrial replacement techniques (MRTs) have the potential to allow prospective parents who are at risk of passing on debilitating or even life‐threatening mitochondrial disorders to have healthy children to whom they are genetically related. Ethical concerns have however been raised about these techniques. This article focuses on one aspect of the ethical debate, the question of whether there is any moral difference between the two types of MRT proposed: Pronuclear Transfer (PNT) and Maternal Spindle Transfer (MST). It examines how questions of identity impact on the ethical evaluation of each technique and argues that there is an important difference between the two. PNT, it is argued, is a form of therapy based on embryo modification while MST is, instead, an instance of selective reproduction. The article's main ethical conclusion is that, in some circumstances, there is a stronger obligation to use PNT than MST.

The question whether maternal spindle transfer (mst) and pronuclear transfer (pnt) can be prohibited under eu legislation was examined by the non-governmental organisation European Bioethics Research (ebr). It did so by submitting an official complaint to the eu Commission proposing that the uk Human Fertilisation and Embryology (Mitochondrial Donation) Regulations 2015 breached the prohibition on the modification of a person’s germ line genetic identity of the eu Clinical Trials Directive 2001/20/EC and the new Regulation eu 536/2014. A discussion then took place, during 2016, between ebr and the eu Commission whether mst and pnt principally involved a ‘medicinal product’ in which case the eu Clinical Trials Directive 2001/20/EC and Regulation eu 536/2014 would be applicable or whether the procedures just involved a medical procedure in which case the Tissue and Cells Directive 2004/23/EC was applicable which did not include any prohibition on the intentional modification of a person’s germline.