Mitochondrial Replacement Therapy Research Articles

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

Reproductive 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 <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.

Spatiotemporal Distribution and Function of Mitochondria in Oocytes.

Mitochondria are energy provider organelles in eukaryotic cells that contain their own specific genome. This review addresses structural and functional properties of mitochondria, focusing on recent discoveries about the changes in quality and number of mitochondria per cell during oocyte development. We highlight how oocyte mitochondria exhibit stage-specific morphology and characteristics at different stages of development, in sharp contrast to the elongated mitochondria present in somatic cells. We then evaluate the latest transcriptomic data to elucidate the complex functions of mitochondria during oocyte maturation and the impact of mitochondria on oocyte development. Finally, we describe the methodological progress of mitochondrial replacement therapy to rescue oocytes with developmental disorders or mitochondrial diseases, hoping to provide a guiding reference to future clinical applications.

Muscle mitochondrial transplantation can rescue and maintain cellular homeostasis.

Mitochondria control cellular functions through their metabolic role. Recent research that has gained considerable attention is their ability to transfer between cells. This has the potential of improving cellular functions in pathological or energy-deficit conditions, but little is known about the role of mitochondrial transfer in sustaining cellular homeostasis. Few studies have investigated the potential of skeletal muscle as a source of healthy mitochondria that can be transferred to other cell types. Thus, we isolated intermyofibrillar mitochondria from murine skeletal muscle and incubated them with host cells. We observed dose- and time-dependent increases in mitochondrial incorporation into myoblasts. This resulted in elongated mitochondrial networks and an enhancement of bioenergetic profile of the host cells. Mitochondrial donation also rejuvenated the functional capacities of the myoblasts when respiration efficiency and lysosomal function were inhibited by complex I inhibitor rotenone and bafilomycin A, respectively. Mitochondrial transfer was accomplished via tunneling nanotubes, extracellular vesicles, gap junctions, and by macropinocytosis internalization. Murine muscle mitochondria were also effectively transferred to human fibroblast cells having mitochondrial DNA mutations, resulting in augmented mitochondrial dynamics and metabolic functions. This improved cell function by diminishing reactive oxygen species (ROS) emission in the diseased cells. Our findings suggest that mitochondria from donor skeletal muscle can be integrated in both healthy and functionally compromised host cells leading to mitochondrial structural refinement and respiratory boost. This mitochondrial trafficking and bioenergetic reprogramming to maintain and revitalize tissue homeostasis could be a useful therapeutic strategy in treating diseases.NEW & NOTEWORTHY In our study, we have shown the potential of mouse skeletal muscle intermyofibrillar mitochondria to be transplanted in myoblasts and human fibroblast cells having mitochondrial DNA mutations. This resulted in an augmentation of mitochondrial dynamics and enhancement of bioenergetic profile in the host cells. Our findings suggest that mitochondria from donor skeletal muscle can be integrated into both healthy and functionally compromised host cells leading to mitochondrial structural refinement and respiratory boost.

Current and Future Landscape in Genetic Therapies for Leber Hereditary Optic Neuropathy.

Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial genetic disease that causes blindness in young adults. Over 50 inherited mitochondrial DNA (mtDNA) variations are associated with LHON; however, more than 95% of cases are caused by one of three missense variations (m.11778 G > A, m.3460 G > A, and m.14484 T > C) encoding for subunits ND4, ND1, and ND6 of the respiration complex I, respectively. These variants remain silent until further and currently poorly understood genetic and environmental factors precipitate the visual loss. The clinical course that ensues is variable, and a convincing treatment for LHON has yet to emerge. In 2015, an antioxidant idebenone (Raxone) received European marketing authorisation to treat visual impairment in patients with LHON, and since then it was introduced into clinical practice in several European countries. Alternative therapeutic strategies, including gene therapy and gene editing, antioxidant and neurotrophic agents, mitochondrial biogenesis, mitochondrial replacement, and stem cell therapies are being investigated in how effective they might be in altering the course of the disease. Allotopic gene therapies are in the most advanced stage of development (phase III clinical trials) whilst most other agents are in phase I or II trials or at pre-clinical stages. This manuscript discusses the phenotype and genotype of the LHON disease with complexities and peculiarities such as incomplete penetrance and gender bias, which have challenged the therapies in development emphasising the most recent use of gene therapy. Furthermore, we review the latest results of the three clinical trials based on adeno-associated viral (AAV) vector-mediated delivery of NADH dehydrogenase subunit 4 (ND4) with mitochondrial targeting sequence, highlighting the differences in the vector design and the rationale behind their use in the allotopic transfer.

The Parliamentary Inquiry into Mitochondrial Donation Law Reform (Maeve’s Law) Bill 2021 in Australia: A Qualitative Analysis

Recently, Australia became the second jurisdiction worldwide to legalize the use of mitochondrial donation technology. The Mitochondrial Donation Law Reform (Maeve’s Law) Bill 2021 allows individuals with a family history of mitochondrial disease to access assisted reproductive techniques that prevent the inheritance of mitochondrial disease. Using inductive content analysis, we assessed submissions sent to the Senate Committee as part of a programme of scientific inquiry and public consultation that informed drafting of the Bill. These submissions discussed a range of bioethical and legal considerations of central importance to the political debate. Significantly, submissions from those with a first-hand experience of mitochondrial disease, including clinicians and those with a family history of mitochondrial disease, were in strong support of this legislation. Those in support of the Bill commended the two-staged approach and rigorous licencing requirements as part of the Bill’s implementation strategy. Submissions which outlined arguments against the legislation either opposed the use of these techniques in general or opposed aspects of the implementation strategy in Australia. These findings offer a window into the ethical arguments and perspectives that matter most to those Australians who took part in the Senate inquiry into mitochondrial donation. The insights garnered from these submissions may be used to help refine policy and guidelines as the field progresses.

Eight Strategies to Engineer Acceptance of Human Germline Modifications.

Until recently, scientific consensus held firm that genetically manipulated embryos created through methods including Mitochondrial Replacement Therapy or human germline genome editing should not be used to initiate a pregnancy. In countries that have relevant laws pertaining to heritable human germline modifications, the vast majority prohibit or restrict this practice. In the last several years, scholars have observed a transformation of scientific and policy restrictions with insistent calls for creating a regulatory pathway. Multiple stakeholders highlight the role of social consensus and public engagement for governance of heritable human germline modifications. However, in the drive to gain public acceptance and lift restrictions, some proponents provide distorted or misleading narratives designed to influence public perception and incrementally shift the consensus. This article describes eight discrete strategies that proponents employ to influence framing, sway public opinion, and revise policymaking of human germline modifications in a manner that undermines honest engagement.

Exploring the therapeutic potential of the mitochondrial transfer-associated enzymatic machinery in brain degeneration.

Mitochondrial dysfunction is a central event in the pathogenesis of several degenerative brain disorders. It entails fission and fusion dynamics disruption, progressive decline in mitochondrial clearance, and uncontrolled oxidative stress. Many therapeutic strategies have been formulated to reverse these alterations, including replacing damaged mitochondria with healthy ones. Spontaneous mitochondrial transfer is a naturally occurring process with different biological functions. It comprises mitochondrial donation from one cell to another, carried out through different pathways, such as the formation and stabilization of tunneling nanotubules and Gap junctions and the release of extracellular vesicles with mitochondrial cargoes. Even though many aspects of regulating these mechanisms still need to be discovered, some key enzymatic regulators have been identified. This review summarizes the current knowledge on mitochondrial dysfunction in different neurodegenerative disorders. Besides, we analyzed the usage of mitochondrial transfer as an endogenous revitalization tool, emphasizing the enzyme regulators that govern this mechanism. Going deeper into this matter would be helpful to take advantage of the therapeutic potential of mitochondrial transfer.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs)domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

P-730 Detection of mitochondrial reversal following meiotic spindle transfer: a finding of importance for mitochondrial replacement therapies used for the purpose of avoiding mtDNA disease transmission

Abstract Study question Following transfer of the meiotic spindle from a patient’s oocyte into an enucleated donor oocyte, do relative levels of patient and donor mtDNA remain stable? Summary answer mtDNA transferred along with the patient’s spindle typically remain at a low level. However, disproportionate expansion of the patient’s mtDNA can occur in some cases. What is known already Mitochondrial DNA disorders are caused by mutations in the mitochondrial genome, disrupting ATP production. They have few treatment options and no cure. All mitochondria are derived from the oocyte, therefore mtDNA disorders are maternally inherited. It has been proposed that disease transmission could be avoided if female mtDNA mutation carriers underwent meiotic spindle transfer (MST), removing the chromosomes (on the spindle) from an affected oocyte and transferring them into the healthy cytoplasm of a donor oocyte. However, research using embryonic stem cells has suggested that the small population of mitochondria transferred along with the spindle can sometimes undergo dramatic expansion. Study design, size, duration 25 infertile couples were enrolled in a prospective pilot study evaluating MST as a treatment for infertility. The female participants had a mean age of 37.2, and an average of 6.4 previous unsuccessful IVF cycles (range 3-11) characterised by extremely poor embryo development and without any previous pregnancy. Importantly, none of the patients were carriers of a mtDNA disorder. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. Participants/materials, setting, methods Metaphase-II-spindles from patient oocytes were transferred into enucleated donor oocytes. Reconstructed oocytes underwent ICSI and the resulting embryos were transferred at the blastocyst stage. Mitochondrial genomes were sequenced to identify polymorphisms differing between the patient and oocyte donor. These variations were quantified in the embryos (blastocyst biopsies), during pregnancy (amniocentesis), in newborns (cord blood, cord tissue, urine), at 3-6 months and at one year (saliva, urine, blood), revealing the relative amounts of each mitochondrial type. Main results and the role of chance This MST pilot study resulted in the birth of six children, indicating that the procedure is compatible with the production of viable embryos, capable of producing healthy live births. The patient’s mtDNA was shown to represent &amp;lt;1% of the total in all blastocysts produced, confirming that MST is highly reproducible and that relatively few mitochondria are transferred along with the spindle. For five of the six children, the proportion of the total mtDNA attributable to the patient appeared to be stable, remaining at very low levels in all of the samples from later developmental stages. However, in one child the small quantity of mtDNA transferred along with the spindle increased disproportionately with respect to the mtDNA of the oocyte donor, ultimately representing 30-60% of the total at birth, depending on the tissue tested. The precise timing of the expansion of one type of mtDNA at the expense of the other is not known but occurred sometime between the blastocyst stage and birth. By the time of birth, the levels of donor and patient mtDNA appeared to have stabilised (no further increases were seen at 6 months and one year). All of the children born remain developmentally normal and healthy. Limitations, reasons for caution After using MST, several pregnancies were achieved for patients with a long history of unsuccessful IVF attempts, associated with poor oocyte quality and a failure to produce blastocysts. However, this small pilot study lacked the controls necessary for a definitive evaluation of MST as a tool for infertility treatment. Wider implications of the findings All children born following MST were healthy. However, our results clearly demonstrate that a substantial degree of mtDNA ‘reversal’ is possible. Consequently, mitochondrial replacement therapies used for avoidance of mtDNA disorders might not always be successful, even when initial levels of mutant mtDNA in reconstructed oocytes are very low. Trial registration number ISRCTN11455145

O-208 Maternal spindle transfer restores the developmental competence of in vitro aged oocytes with diminished metabolic activity identified by hyperspectral imaging

Abstract Study question Can the developmental competence of poor-quality oocytes identified by hyperspectral imaging be restored by maternal spindle transfer? Summary answer Maternal spindle transfer can be efficiently assisted by hyperspectral imaging to identify and subsequently repair the developmental competence of poor-quality oocytes. What is known already Oocyte cytoplasmic dysfunctions (including, but not limited to the mitochondria) are a major cause for poor embryonic development. Given that maternal spindle transfer (MST) allows the replacement of the entire cytoplasm of an oocyte, it is a promising method for the enhancement of oocyte developmental competence. However, accurate diagnostic tools for oocyte quality assessment are lacking. Recently, a novel hyperspectral imaging approach has been shown to classify oocytes non-invasively based on their metabolic profile, which could assist in the evaluation of oocyte developmental potential prior to MST. Study design, size, duration Oocytes were obtained from young female mice and analyzed blindly by hyperspectral imaging either immediately after collection (fresh group, n = 122), after overnight culture (in-vitro aged group, n = 58) or on a group of MST oocytes (n = 36), where the meiotic spindle from in-vitro aged oocytes was transferred into enucleated fresh oocytes. Oocytes were inseminated by ICSI and cultured in-vitro individually. Correlations between the oocyte’s metabolic profiles and rate of development to the blastocyst stage were performed. Participants/materials, setting, methods Oocytes were collected from 6-10 weeks-old superovulated B6CBAF1 females. MST was performed in manipulation medium supplemented with cytochalasin B. After enucleation, karyoplasts were exposed to an inactivated Sendai virus solution to promote membrane fusion. ICSI was performed using a Piezo-Drill actuator. Hyperspectral imaging was done in the near-infra-red regime to avoid photoxicity. Image processing was done using dimensionality reduction, which was then feed to an Akaike Information Criterion algorithm to classify the different metabolic subpopulations. Main results and the role of chance Fresh oocytes showed metabolic profiles that significantly differed from the profiles of in-vitro aged oocytes, which were characterized by a pronounced diminished metabolic activity. While in the fresh group, 67% of the oocytes developed into expanded blastocysts, none of the in-vitro aged oocytes with low metabolism produced a blastocyst (p &amp;lt; 1x10-05). Interestingly, MST oocytes that were imaged 1h after nuclear reconstruction, showed a restored metabolic spectrum that resembled the profile from fresh non-manipulated controls. This restoration of the metabolic activity was concomitantly associated with an enhancement of developmental competence, with expanded blastocyst rates (63%) comparable to those of fresh non-manipulated controls (p = 0.668). Blastocyst developmental rates of fresh oocytes after hyperspectral imaging were comparable to oocytes from the same cohort that were not imaged and used as controls. Limitations, reasons for caution The metabolic profiles obtained from these experimental groups need to be correlated with implantation rates in mouse. The impaired embryo developmental pattern characteristic of poor-quality oocytes may not be exclusively attributed to metabolic deficiencies, so caution must be exerted when generalizing these results to oocytes with other cytoplasmic dysfunctions. Wider implications of the findings MST experiments confirm that the developmental decline of these oocytes is primarily attributed to abnormal function of cytoplasmic factors involved in metabolism, while the nuclear genome remains developmentally competent. MST can benefit from hyperspectral imaging to identify oocytes with impaired developmental potential within a cohort. Trial registration number not applicable

P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes

Abstract Study question Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice? Summary answer Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes. What is known already In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts. Study design, size, duration The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer. Participants/materials, setting, methods Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls. Main results and the role of chance In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p &amp;lt; 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p &amp;lt; 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p &amp;lt; 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p &amp;lt; 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p &amp;lt; 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p &amp;lt; 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p &amp;lt; 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing. Limitations, reasons for caution Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique. Wider implications of the findings Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human. Trial registration number Not applicable