Mitochondrial Replacement Therapy Research Articles

‘Recombining’ biological motherhoods. Towards two ‘complete’ biological mothers

Within feminist literature from the early 1970s to this day, assisted reproductive technologies have been largely known to divide, replace or eliminate biological motherhood. For example, while in the past...

Implications of Law’s Response to Mitochondrial Donation

Changes to Australian law in 2022 made Australia the second country to expressly permit the clinical use of mitochondrial donation (MD), a technology that makes heritable changes to the human genome. This paper considers these changes in the context of Australia’s broader controls on human embryo use to provide insights into future regulatory responses to other emerging genetic technologies, which could be used in reproduction.

Abstract B085: Carcinoma-associated mesenchymal stem cells promote high-grade serous ovarian cancer metastasis and drive tumor heterogeneity at the metastatic site through direct mitochondrial transfer

Abstract High-grade serous ovarian cancer (HGSC) is the most common and lethal form of ovarian cancer, accounting for over 70% of ovarian cancer cases. HGSC mortality is largely driven by early and diffuse transcoelomic spread from the primary site throughout the peritoneal cavity, resulting in roughly three-quarters of cases presenting with advanced stage disease at time of diagnosis. Following detachment from the primary tumor, disseminated ovarian cancer cells are spread by the peritoneal fluid and ascites where they must adapt to their new microenvironment to survive and successfully form metastases. Crucial to this spread is the formation of multicellular aggregates containing both cancer and non-malignant stromal cells, which enhance cancer cell survival and are associated with poor clinical outcome. While most studies on HGSC multicellular aggregates have focused on ovarian cancer cells alone, the interactions between ovarian cancer cells and other non-malignant cells have yet to be fully elucidated. Prior work by our group have shown that stromal-progenitor cells, termed carcinoma-associated mesenchymal stem/stromal cells (CA-MSCs), enhance HGSC metastasis through direct cellular interaction and formation of heterocellular CA-MSC:cancer cell complexes. Here we demonstrate CA-MSCs enhance metastasis by preferentially donating their mitochondria to cancer cells with the least amount of endogenous mitochondria content (‘mito poor’ cancer cells), increasing their ability to survive and driving tumor heterogeneity at the metastatic site in vivo. Mito poor cancer cells were shown to have decreased proliferative capacity, increased sensitivity to platinum-based chemotherapy and display decreased oxidative phosphorylation (OXPHOS) compared to ‘mito rich’ cancer cells. Primary patient-derived CA-MSCs rescue this vulnerable phenotype by increasing proliferation, chemoresistance and OXPHOS in mito poor cancer cells. These findings were validated in a fully autologous system using CA-MSCs and cancer cells derived from the same patient to prevent confounding effects of cellular response to foreign organelle/DNA. Through lentiviral knockdown of the mitochondrial motor protein MIRO1 we further demonstrate mitochondrial transfer is necessary for CA-MSC-mediated rescue of mito poor cancer cells. Further, we developed a haplotype-specific quantification of mitochondrial DNA to differentiate CA-MSC from endogenous cancer cell mitochondria which was used to quantify the amount of CA-MSC mitochondrial donation and demonstrate donated mitochondria persist in cancer cells up to 14 days. Importantly, CA-MSC mitochondrial donation occurred in vivo and was associated with decreased survival in an orthotopic ovarian cancer murine model. Genomic barcoding was used to quantify tumor cell clonal heterogeneity; using this system we demonstrate CA-MSC mitochondrial donation significantly enhance tumor cell heterogeneity at the metastatic site in vivo. Collectively, we report CA-MSC mitochondrial transfer as a driver of HGSC progression, heterogeneity and metastasis. Citation Format: Leonard G. Frisbie, Catherine A. Pressimone, Roja Baruwal, Geyon L. Garcia, Zanib Javed, Claudette St. Croix, Simon Watkins, Michael Calderone, Huda I. Atiya, Nadine Hempel, Alexander Pearson, Lan G. Coffman. Carcinoma-associated mesenchymal stem cells promote high-grade serous ovarian cancer metastasis and drive tumor heterogeneity at the metastatic site through direct mitochondrial transfer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B085.

What importance do donors and recipients attribute to the nuclear DNA-related genetic heritage of oocyte donation?

How do oocyte donors and recipients perceive the genetic link related to the transfer of nuclear DNA between donors and offspring? Whether they are donors or recipients, individuals attach great importance to the transmission of their genetic heritage, since 94.5% would opt for the pronuclear transfer method to preserve this genetic link in the context of oocyte donation. Since 1983, the use of oocyte donation has increased worldwide. Performed in France since the late 1980s and initially offered to women with premature ovarian insufficiency, its indications have progressively expanded and now it is proposed in many indications to prevent the transmission of genetically inherited diseases. This has resulted in an increase in the waiting time for access to oocyte donation due to the difficulty in recruiting oocyte donors in French ART centres. Several articles have discussed how to fairly distribute donor oocytes to couples, but few have interviewed women in the general population to record their feelings about oocyte donation, as either the donor or recipient and the importance given to the genetic link between the oocyte donors and the children born. Mitochondrial replacement therapy (MRT) is a technique originally developed for women at risk of transmitting a mitochondrial DNA mutation. Recently, MRT has been considered for embryo arrest and oocyte rejuvenation as it could help females to reproduce with their own genetic material through the transfer of their oocyte nucleus into a healthy donor oocyte cytoplasm. We conducted an opinion survey from January 2021 to December 2021, during which 1956 women completed the questionnaire. Thirteen participants were excluded from the analysis due to incomplete responses to all the questions. Consequently, 1943 women were included in the study. We specifically developed a questionnaire for this study, which was created and distributed using the Drag'n Survey® software. The questionnaire consisted of 21 items presented alongside a video created with whiteboard animation software. The aim was to analyse whether certain factors, such as age, education level, marital status, number of children, use of ART for pregnancy, video viewing, and knowledge about oocyte donation, were associated with feelings towards oocyte donation, by using a univariate conditional logistic regression model. This statistical method was also used to assess whether women would be more inclined to consider oocyte donation with the pronuclear transfer technique rather than the whole oocyte donation. All parameters found to be statistically significant in the univariate analysis were subsequently tested in a multivariate model using logistic regression. Most women were concerned about the biological genetic contribution of the donated oocyte (94.8%). The most common reason for a women's reluctance to donate their oocytes was their unwillingness to pass on their genetic material (33.3%). Nearly 70% of women who were initially hesitant to donate their oocytes indicated that they would reconsider their decision if the oocyte donation was conducted using donated cytoplasm and the pronuclear transfer technique. Concomitantly, >75% of the respondents mentioned that it would be easier to receive a cytoplasm donation. The largest proportion of the population surveyed (94.5%) expressed their support for its legalization. In this study, a substantial portion of the responses came from individuals with medical or paramedical backgrounds, potentially introducing a recruitment bias among potential donors. The rate of missing responses to the question regarding the desire to become an oocyte donor was 13.6%, while the question about becoming an oocyte cytoplasm donor had a missing response rate of 23%. These missing responses may introduce a bias in the interpretation of the data. This study was the first to demonstrate that, for the French population studied, the combination of oocyte cytoplasm donation with pronuclear transfer could offer a promising approach to enhance the acceptance of oocyte donation for both the donor and the recipient. No external funding was used for this study. The authors have no conflicts of interest. N/A.

Angiogenic activity of mitochondria; beyond the sole bioenergetic organelle.

Angiogenesis is a complex process that involves the expansion of the pre-existing vascular plexus to enhance oxygen and nutrient delivery and is stimulated by various factors, including hypoxia. Since the process of angiogenesis requires a lot of energy, mitochondria play an important role in regulating and promoting this phenomenon. Besides their roles as an oxidative metabolism base, mitochondria are potential bioenergetics organelles to maintain cellular homeostasis via sensing alteration in oxygen levels. Under hypoxic conditions, mitochondria can regulate angiogenesis through different factors. It has been indicated that unidirectional and bidirectional exchange of mitochondria or their related byproducts between the cells is orchestrated via different intercellular mechanisms such as tunneling nanotubes, extracellular vesicles, and gap junctions to maintain the cell homeostasis. Even though, the transfer of mitochondria is one possible mechanism by which cells can promote and regulate the process of angiogenesis under reperfusion/ischemia injury. Despite the existence of a close relationship between mitochondrial donation and angiogenic response in different cell types, the precise molecular mechanisms associated with this phenomenon remain unclear. Here, we aimed to highlight the possible role of mitochondria concerning angiogenesis, especially the role of mitochondrial transport and the possible relation of this transfer with autophagy, the housekeeping phenomenon of cells, and angiogenesis.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes? PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring. In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence. The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups. Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique. Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 μm versus 73.1 μm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl2). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers. N/A. Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells. PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer. A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.

Generation of mitochondrial replacement monkeys by female pronucleus transfer.

Mutations in mitochondrial DNA (mtDNA) are maternally inherited and have the potential to cause severe disorders. Mitochondrial replacement therapies, including spindle, polar body, and pronuclear transfers, are promising strategies for preventing the hereditary transmission of mtDNA diseases. While pronuclear transfer has been used to generate mitochondrial replacement mouse models and human embryos, its application in non-human primates has not been previously reported. In this study, we successfully generated four healthy cynomolgus monkeys ( Macaca fascicularis) via female pronuclear transfer. These individuals all survived for more than two years and exhibited minimal mtDNA carryover (3.8%-6.7%), as well as relatively stable mtDNA heteroplasmy dynamics during development. The successful establishment of this non-human primate model highlights the considerable potential of pronuclear transfer in reducing the risk of inherited mtDNA diseases and provides a valuable preclinical research model for advancing mitochondrial replacement therapies in humans.

Three to one – an ethicolegal outline of mitochondrial donation in the South African context

Mitochondrial donation or mitochondrial transfer enables a woman with mitochondrial disease to have a genetically related child without transmitting the disease to the child. The techniques used for mitochondrial donation or transfer which are maternal spindle transfer or pro-nuclei transfer, require three gametes to ultimately produce a healthy embryo. Both these techniques result in the child inheriting nuclear DNA from the intending parents and mitochondrial DNA from the female donor. Following the legalisation of mitochondrial donation in the UK, after a rigorous process of scientific and ethical review, and the birth of another baby using the technique, coupled with the fact that there is no cure for mitochondrial disease, suggests that it is prudent for us to consider this reproductive intervention and its application in the South African setting. In addition, the 2019 UNESCO Report of the International Bioethics Committee (IBC) on assisted reproductive technologies (ART) and parenthood, encourages debate on changes in models of parenthood influenced by ART including the emergence of new models of families and forms of parenthood that extend beyond the classical rule of mater semper certa est (the mother is always known). ART, in particular mitochondrial donation, challenges this rule. The aim of this paper is to provide an outline of the legal and ethical positions of mitochondrial donation and resume the discussion with specific focus on the South African context.

Mitochondrial diseases: causes and solutions (literature review)

The discovery of mitochondrial desoxyribonucleic acid (DNA) mutations and the association of these mutations with the development of severe, sometimes, fatal diseases led to new forms of reproductive technologies, i.e., mitochondrial replacement therapy. Currently, researchers are investigating three main methods of mitochondrial replacement therapy: pronuclear transfer, maternal spindle transfer, and polar body genome transfer. Thanks to these methods, the transmission of faulty mitochondrial DNA from the mother to the next generation can be prevented, with the main goal of developing a healthy offspring free of genetic disorders and lethal mitochondrial diseases. However, the implementation of these technologies has raised several moral, social, and cultural concerns, as it marks the emergence of a child with genetic material from three parents for the first time. This review presents an objective summary of the causes of mitochondrial disorders and various techniques employed in mitochondrial replacement therapy, along with their outcomes.

Pre-implantation Genetic Testing in Inherited Metabolic Diseases? Stateof- the-art and current challenges.

Inborn errors of metabolism (IEM) are genetic diseases involving congenital disorders of enzyme activities. Most follow Mendelian autosomal recessive inheritance and few follow mitochondrial inheritance. In many cases, after the birth of an affected child parents discover that have been the carriers for the condition and worry about the risk of recurrence in future offspring. Preimplantation genetic testing (PGT) can analyze embryos before their transfer to the uterus and prevent the transmission of hereditary conditions to descendants, however this procedure is of limited value in mtDNA conditions. The list of diseases currently approved for PGT were reviewed. The process for eligibility, was as for the Comissão Nacional Procriação Medicamente Assistida (CNPMA), of Portugal (PT). Review of international practices for Assisted Reproductive Techniques (ART) in IEM was carried out. As of 07.2022, 23 IEM diseases associated with deleterious variants in nDNA were approved for PGT in PT. Couples at risk for conditions not included in the list can solicit an evaluation from an expert committee, after a medical genetics consultation. To qualify for approval, diseases must cause significant suffering and/or premature death. Due to a greater number of solicitations many more IEM conditions have been approved for PGT across the world. ART for mtDNA is not available in PT. International expert centers include PGT for specific well documented variants and mitochondrial donation. PGT is a reliable approach to reduce the risk of transmission of a genetic condition to the offspring. The list of IEM disorders currently accepted for this technique in Portugal are small, but it is expanding, as many more diseases fit the necessary criteria. While appealing in theory, low success rates coupled with limited availability can be discouraging for patients. Genetic counselling is of paramount importance after the diagnosis of IEM diseases. It is important for both clinicians and patients to be made aware of the available reproductive options and their limitations.

Whose (germ) line is it anyway? Reproductive technologies and kinship

AbstractReproductive biotechnologies can separate concepts of parenthood into genetic, gestational and social dimensions, often leading to a fragmentation of heteronormative kinship models and posing a challenge to historical methods of establishing legal and/or moral parenthood. Using fictional cases, this article will demonstrate that the issues surrounding the intersection of current and emerging reproductive biotechnologies with definitions of parenthood are already leading to confusion regarding social and legal family ties for offspring, which is only expected to increase as new technologies develop. Rather than opposing these new technologies to reassert traditional concepts of the family, however, this article will explore the opportunities that these technologies represent for re‐imagining various culturally cherished values of family‐making in a way that is inclusive of diverse genders, sexualities and cultures. It will consider IVF, gametogenesis, mitochondrial donation, surrogacy, artificial gestation, CRISPR‐Cas9 gene editing, foster care and adoption as some of many possible pathways to parenthood, including for members of the LGBTIAUQ+ community.

MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

MitoHOPE: Introduction to the Australian Mitochondrial Donation Project

Mitochondrial Donation provides a means of reducing the risk of transmitting mitochondrial genetic disease. If successful, families who have suffered in many cases across generations, will be freed from the risks of inheriting mitochondrial genetic disease in their future children. In response to mito community advocacy led by the Mito Foundation, Maeve’s Law, was passed in March 2022 to allow the introduction of mitochondrial donation in Australia. The mitochondrial donation procedure involves removing the nuclear DNA from the egg or zygote of from a woman with a mitochondrial DNA (mtDNA) mutation and transferring it to an enucleated egg or zygote from a donor with healthy mtDNA. Although this technique has been used in animal models for many years and has undergone significant preclinical testing in humans, it is use in the clinic remains at an early stage and, to date, only a small number of babies have been born. To introduce mitochondrial donation in Australia, an extensive regulatory and licensing procedure has been established and the MRFF has funded a Mitochondrial Donation Pilot Program, including a clinical trial. The mitoHOPE (Healthy Outcomes Pilot and Evaluation) Program integrates expertise in clinical genetics, oocyte and embryo development, mitochondrial disease, reproductive technologies, ethics, law, and health economics. The program was developed in conjunction with the mito community and this engagement will inform all steps of the implementation. Our partners include Monash IVF, the Mito Foundation and Newcastle University (UK). Progress is now underway to establish our governance arrangements, an oocyte donation program, clinical pathways into the clinical trial and an underpinning research program to ensure the safety and efficacy of mitochondrial donation as it is being introduced into the clinic.

MitoHOPE: Embryological Aspects

Mitochondrial diseases are a group of rare genetic disorders that affect the function of mitochondria, the energy-producing structures within cells. These disorders result from mutations in either nuclear DNA or mitochondrial DNA (mtDNA), disrupting normal energy production process, with vital organs such as the brain, heart, muscles, and nervous system suffering from insufficient energy supply. Symptoms of mitochondrial disease are diverse however commonly include muscle weakness, fatigue, neurological issues, developmental delays, and organ dysfunction. Treatment options for mitochondrial disease are limited and primarily focus on managing symptoms and supporting affected organ systems. Currently, no cure exists, making early detection crucial to better manage the condition’s progression. Research efforts and advances in genetic technology offer hope for improved diagnostic methods and potential therapies in the future and for patients where the disease is caused by mutations in mtDNA, mitochondrial donation technology has also been developed in the hope of providing patients a means of having a family, with a reduced likelihood of passing on mtDNA disease to their offspring. Mitochondrial donation technology, also known as mitochondrial replacement therapy (MRT), is a ground-breaking medical technique designed to prevent the transmission of mitochondrial diseases from mother to child. This cutting-edge procedure involves replacing defective mitochondria in a woman’s egg with healthy mitochondria/cytoplasm from an egg donor using primarily either miotic spindle transfer (MST) or pro-nuclear transfer (PNT). Recently in Australia a MRFF grant has been awarded to facilitate bringing MRT to patients in Australia who have mtDNA disease and offering it as part of a clinical trial (mitoHOPE). As part of this, considerations must be given to how this technology is applied in a clinical IVF lab setting, specifically the equipment used, the technology that is selected (MST vs PNT) and optomised, the training of the clinical embryologists and the management of the oocyte donor program, all of which are crucial to ensure optimal clinical trial and technology provision.

Mitochondrial Donation: The UK Experience

Mutations in mitochondrial DNA (mtDNA) are inherited exclusively from our mothers and can cause a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of the mitochondrial genome from the nuclear DNA genome may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Such technologies involve transplantation of the nuclear DNA from the egg of an affected woman to an enucleated egg from an unaffected donor. Nuclear genome transplantation can be performed either before or after fertilisation. The latter involves transfer of pronuclei, which separately contain the maternal and paternal genomes. In parallel with legal and regulatory reform to permit therapeutic application of so called “mitochondrial donation” techniques in the UK, we have conducted preclinical studies to test the safety and efficacy of pronuclear transplantation (PNT). In 2017 Human Fertilisation and Embryology Authority (HFEA) approved the cautious use of PNT in the UK and granted the first licence for its therapeutic application in 2018. I will discuss the scientific, legal, and regulatory hurdles in the path from proof of concept to translation of mitochondrial donation to clinical treatment.

Genome Editing Tools to Increase the Efficacy of Mitochondrial Donation

Mitochondrial donation (MD) aims to prevent transmission of mtDNA disease by transplanting the nuclear genome from an affected woman’s egg to an enucleated egg from an unaffected donor. The nuclear DNA is transplanted in a karyoplast, which upon fusion with the enucleated donor egg, introduces a small amount of perinuclear mitochondria resulting in heteroplasmy for maternal mtDNA. Under optimal conditions, this accounts for &lt;2% of the mtDNA content of MD embryos. Despite this, [Formula: see text]20% of embryonic stem (ES) cell lines derived from MD embryos exhibit complete reversion to the maternal mitochondrial genome and elevated levels (40–60%) have recently been reported in a baby born following MD treatment for infertility. Thus, currently available MR treatments are regarded as risk reduction, rather than prevention strategies. A major focus of our ongoing research is to bridge this gap by developing techniques to minimise maternal mtDNA in MD embryos. I will present our recent findings from these investigations.

Advancing deliberative reform in a parliamentary system: prospects for recursive representation

AbstractRecent theories of democratic representation push beyond ‘minimalist’ notions that only rely on periodic elections to connect officials and constituents. For example, Jane Mansbridge (2019) calls for ‘recursive representation’, which seeks ongoing, two-way interaction between representatives and their constituents. Given the scale and complexity of modern representative democracies, how can such ambitious proposals be translated into practice? We analyze two Deliberative Town Halls (DTHs) convened with a Federal Member of Australian Parliament in 2020 to discuss a complex issue, mitochondrial donation, ahead of a parliamentary debate and conscience vote on this issue. Drawing on interviews with participants, we argue that democratic innovations such as DTHs can contribute to realizing recursive representation when three criteria are met: authenticity, inclusion, and impact. We discuss the significance of each criterion and the role of DTHs in advancing recursive representation in a parliamentary system.

Making patients political: Narrating, curating, enacting, and navigating the ‘idealised policy patient’

In this article we develop the concept of the ‘idealised policy patient’ to contribute to a better understanding of patient-family activism and the mechanisms through which powerful and persuasive patient narratives are facilitated and mobilised. The context through which we explore the idealised policy patient is the UK debates about the legalisation of mitochondrial donation, which primarily took place between 2011 and 2015. In our example, the idealised policy patient was constructed around a culturally persuasive narrative of patient suffering, where mitochondrial donation was presented as a desirable and ethical solution. We draw on interviews with patient-families and stakeholders, and documentary analysis to identify four dimensions of the idealised policy patient – narrating, curating, enacting and navigating. Narrating describes how the idealised policy patient appears in public and policy spaces, as a culturally available narrative which conveys certain meanings and is designed to invoke an emotional and practical response. Curating identifies the multiple forms of labour and facilitation involved in supporting patient-families in activist activities which strengthen the dominant narrative and its embodiment. Enacting focuses on the work of patient-families themselves in supporting and contributing to the idealised policy patient in a way that enlivens and embodies the specifically curated narrative. Finally, navigating considers how those offering an opposing viewpoint position themselves in relation to the idealised policy patient. To conclude, we argue that medical sociology has often given insufficient scrutiny to how the capacity of patients to leverage their status for political ends is bolstered through alignment with existing powerful groups, particularly in hegemonic campaigns. We encourage future researchers to examine how the idealised policy patient is reproduced and reorientated within different policy contexts.

Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer.

Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases.

Single-cell multi-omics sequencing reveals chromosome copy number inconsistency between trophectoderm and inner cell mass in human reconstituted embryos after spindle transfer.

Is the chromosome copy number of the trophectoderm (TE) of a human reconstituted embryos after spindle transfer (ST) representative of the inner cell mass (ICM)? Single-cell multi-omics sequencing revealed that ST blastocysts have a higher proportion of cell lineages exhibiting intermediate mosaicism than conventional ICSI blastocysts, and that the TE of ST blastocysts does not represent the chromosome copy number of ICM. Preimplantation genetic testing for aneuploidy (PGT-A) assumes that TE biopsies are representative of the ICM, but the TE and ICM originate from different cell lineages, and concordance between TE and ICM is not well-studied, especially in ST embryos. We recruited 30 infertile women who received treatment at our clinic and obtained 45 usable blastocysts (22 from conventional ICSI and 23 reconstituted embryos after ST). We performed single-cell multi-omics sequencing on all blastocysts to predict and verify copy number variations (CNVs) in each cell. We determined the chromosome copy number of each embryo by analysing the proportion of abnormal cells in each blastocyst. We used the Bland-Altman concordance and the Kappa test to evaluate the concordance between TE and ICM in the both groups. The study was conducted at a public tertiary hospital in China, where all the embryo operations, including oocytes retrieval, ST, and ICSI, were performed in the embryo laboratory. We utilized single-cell multi-omics sequencing technology at the Biomedical Pioneering Innovation Center, School of Life Sciences, Peking University, to analyse the blastocysts. Transcriptome sequencing was used to predict the CNV of each cell through bioinformatics analysis, and the results were validated using the DNA methylation library of each cell to confirm chromosomal normalcy. We conducted statistical analysis and graphical plotting using R 4.2.1, SPSS 27, and GraphPad Prism 9.3. Mean age of the volunteers, the blastocyst morphology, and the developmental ratewere similar in ST and ICSI groups. The blastocysts in the ST group had some additional chromosomal types that were prone to variations beyond those enriched in the blastocysts of the ICSI group. Finally, both Bland-Altman concordance test and kappa concordancetest showed good chromosomal concordance between TE and ICM in the ICSI blastocysts (kappa = 0.659, P < 0.05), but not in ST blastocysts (P = 1.000), suggesting that the TE in reconstituted embryos is not representative of ICM. Gene functional annotation (GO and KEGG analyses) suggests that there may be new or additional pathways for CNV generation in ST embryos compared to ICSI embryos. This study was mainly limited by the small sample size and the limitations of single-cell multi-omics sequencing technology. To select eligible single cells, some cells of the embryos were eliminated or not labelled, resulting in a loss of information about them. The findings of this study are innovative and exploratory. A larger sample size of human embryos (especially ST embryos) and more accurate molecular genetics techniques for detecting CNV in single cells are needed to validate our results. Our study justifies the routine clinical use of PGT-A in ICSI blastocysts, as we found that the TE is a good substitute for ICM in predicting chromosomal abnormalities. While PGT-A is not entirely accurate, our data demonstrate good clinical feasibility. This trial was able to provide correct genetic counselling to patients regarding the reliability of PGT-A. Regarding ST blastocysts, the increased mosaicism rate and the inability of the TE to represent the chromosomal copy number of the ICM are both biological characteristics that differentiate them from ICSI blastocysts. Currently, ST is not used clinically on a large scale to produce blastocysts. However, if ST becomes more widely used in the future, our study will be the first to demonstrate that the use of PGT-A in ST blastocysts may not be as accurate as PGT-A for ICSI blastocysts. This study was supported by grants from the National Key R&D Program of China (2018YFA0107601) and the National Key R&D Program of China (2018YFC1003003). The authors declare no conflict of interest. N/A.