Mitochondrial Replacement Therapy Research Articles

Current and Emerging Clinical Treatment in Mitochondrial Disease.

Primary mitochondrial disease (PMD) is a group of complex genetic disorders that arise due to pathogenic variants in nuclear or mitochondrial genomes. Although PMD is one of the most prevalent inborn errors of metabolism, it often exhibits marked phenotypic variation and can therefore be difficult to recognise. Current treatment for PMD revolves around supportive and preventive approaches, with few disease-specific therapies available. However, over the last decade there has been considerable progress in our understanding of both the genetics and pathophysiology of PMD. This has resulted in the development of a plethora of new pharmacological and non-pharmacological therapies at varying stages of development. Many of these therapies are currently undergoing clinical trials. This review summarises the latest emerging therapies that may become mainstream treatment in the coming years. It is distinct from other recent reviews in the field by comprehensively addressing both pharmacological non-pharmacological therapy from both a bench and a bedside perspective. We highlight the current and developing therapeutic landscape in novel pharmacological treatment, dietary supplementation, exercise training, device use, mitochondrial donation, tissue replacement gene therapy, hypoxic therapy and mitochondrial base editing.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-020-00510-6.

Mitochondrial transfer from induced pluripotent stem cells rescues developmental potential of in vitro fertilized embryos from aging females†.

Conventional heterologous mitochondrial replacement therapy is clinically complicated by "tri-parental" ethical concerns and limited source of healthy donor oocytes or zygotes. Autologous mitochondrial transfer is a promising alternative in rescuing poor oocyte quality and impaired embryo developmental potential associated with mitochondrial disorders, including aging. However, the efficacy and safety of mitochondrial transfer from somatic cells remains largely controversial, and unsatisfying outcomes may be due to distinct mitochondrial state in somatic cells from that in oocytes. Here, we propose a potential strategy for improving in vitro fertilization (IVF) outcomes of aging female patients via mitochondrial transfer from induced pluripotent stem (iPS) cells. Using naturally aging mice and well-established cell lines as models, we found iPS cells and oocytes share similar mitochondrial morphology and functions, whereas the mitochondrial state in differentiated somatic cells is substantially different. By microinjection of isolated mitochondria into fertilized oocytes following IVF, our results indicate that mitochondrial transfer from iPS, but not MEF cells, can rescue the impaired developmental potential of embryos from aging female mice and obtain an enhanced implantation rate following embryo transfer. The beneficial effect may be explained by the fact that mitochondrial transfer from iPS cells not only compensates for aging-associated loss of mtDNA, but also rescues mitochondrial metabolism of subsequent preimplantation embryos. Using mitochondria from iPS cells as the donor, our study not only proposes a promising strategy for improving IVF outcomes of aging females, but also highlights the importance of synchronous mitochondrial state in supporting embryo developmental potential.

Mitochondrial replacement therapy: Genetic counselors' experiences, knowledge, and opinions

Mitochondrial disorders affect at least 1 in 5,000 individuals worldwide and are often incurable and fatal. Mitochondrial replacement therapy (MRT) is an in vitro fertilization technique used to prevent the transmission of mitochondrial disorders. Currently, MRT is the only approach that provides mothers who carry a pathogenic variant in their mitochondrial DNA (mtDNA), the opportunity to have a biological child without a mitochondrial disease. MRT involves the combination of nuclear DNA from the egg of the carrier mother and the cytoplasm from an oocyte donor, which contains healthy mitochondria. While MRT was approved for use in the UK in 2015, the ban on congressional funding for research on 'heritable genetic modification' has made MRT unavailable within the US borders. This survey-based study aimed to describe genetic counselors' experience, knowledge, and opinions about MRT. Additionally, we also assessed whether genetic counselors' comfort discussing MRT with patients, and feelings about clinical use of MRT in the United States changed after providing information about MRT compared with baseline. Responses were received from 139 genetic counselors in North America. Findings indicate low awareness and knowledge about MRT among participants. However, more participants expressed comfort with discussing MRT with patients and more participants were able to form opinions about statements about MRT after they were provided with information about MRT. This study is the first to assess genetic counselors' opinions toward MRT and suggests the need for more education about novel technologies such as MRT among genetic counselors.

Clinical translation of mitochondrial replacement therapy in Canada: a qualitative study of stakeholders’ attitudes

Mitochondrial replacement therapy (MRT) in Canada is considered a criminal offense according to article 5(1)(f) of the Assisted Human Reproduction Act (AHRA) (2004). The Act prohibits any practice that modifies the genome of “a human being or in vitro embryo such that the alteration is capable of being transmitted to descendants.” We carried out 32 semi-structured interviews with clinicians, researchers, patient groups, egg donors, and members of the public to explore their attitudes toward the clinical implementation of MRT in Canada. Our interview guide was informed by the socio-ethical, legal, and scientific literature of MRT. We used a thematic analysis to identify and analyze emerging themes and sub-themes. Our findings were divided into five broad themes: ( i) an outdated criminal ban, ( ii) motives for using MRT, ( iii) terminology, ( iv) practical and theoretical risks and benefits, and ( v) the feasibility of clinical translation in Canada. Although the public and stakeholders’ views on the feasibility of foreseeable translation of MRT in Canadian clinics varied, there was consensus on conducting an overdue review of the current AHRA ban on MRT.

Mitochondrial Replacement Therapy: Let the Science Decide

Mitochondrial replacement therapy (MRT) is an in vitro fertilization technique designed to prevent women who are carriers of mitochondrial diseases from passing on these heritable genetic diseases to their children. It is an innovative assisted reproductive technology that is only legal in a small number of countries. The United States has essentially stagnated all opportunities for research and clinical trials on MRT through a rider in H.R.2029 – Consolidated Appropriations Act, 2016. The rider bans clinical trials on all therapies in which a human embryo is intentionally altered to include a heritable genetic modification. This note argues that the rider should be amended to permit therapies such as MRT, which do not create artificial DNA sequences, while continuing to prohibit clinical trials on germline therapies that modify the sequence of a gene. MRT is distinct from the types of therapies that Congress intended to ban through the rider. Amending the rider would not automatically approve MRT trials, but rather allow the FDA to evaluate investigational new drug applications and determine whether individual trials may proceed. Without proper FDA oversight, carriers of mitochondrial diseases are denied access to a therapy that provides them with benefits they cannot enjoy by any other means, and researchers may look abroad to conduct the therapy illegally or dangerously. Further, the United States can look to other countries such as the United Kingdom as a model for how to proceed with research and trials on MRT in an ethical manner.

Mitochondrial enrichment in infertile patients: a review of different mitochondrial replacement therapies.

Poor ovarian responders exhibit a quantitative reduction in their follicular pool, and most cases are also associated with poor oocyte quality due to patient’s age, which leads to impaired in vitro fertilisation outcomes. In particular, poor oocyte quality has been related to mitochondrial dysfunction and/or low mitochondrial count as these organelles are crucial in many essential oocyte processes. Therefore, mitochondrial enrichment has been proposed as a potential therapy option in infertile patients to improve oocyte quality and subsequent in vitro fertilisation outcomes. Nowadays, different options are available for mitochondrial enrichment treatments that are encompassed in two main approaches: heterologous and autologous. In the heterologous approach, mitochondria come from an external source, which is an oocyte donor. These techniques include transferring either a portion of the donor’s oocyte cytoplasm to the recipient oocyte or nuclear material from the patient to the donor’s oocyte. In any case, this approach entails many ethical and safety concerns that mainly arise from the uncertain degree of mitochondrial heteroplasmy deriving from it. Thus the autologous approach is considered a suitable potential tool to improve oocyte quality by overcoming the heteroplasmy issue. Autologous mitochondrial transfer, however, has not yielded as many beneficial outcomes as initially expected. Proposed mitochondrial autologous sources include immature oocytes, granulosa cells, germline stem cells, and adipose-derived stem cells. Presently, it would seem that these autologous techniques do not improve clinical outcomes in human infertile patients. However, further trials still need to be performed to confirm these results. Besides these two main categories, new strategies have arisen for oocyte rejuvenation by improving patient’s own mitochondrial function and avoiding the unknown consequences of third-party genetic material. This is the case of antioxidants, which may enhance mitochondrial activity by counteracting and/or preventing oxidative stress damage. Among others, coenzyme-Q10 and melatonin have shown promising results in low-prognosis infertile patients, although further randomised clinical trials are still necessary.

Nasal administration of mitochondria reverses chemotherapy-induced cognitive deficits.

Up to seventy-five percent of patients treated for cancer suffer from cognitive deficits which can persist for months to decades, severely impairing quality of life. Although the number of cancer survivors is increasing tremendously, no efficacious interventions exist. Cisplatin, most commonly employed for solid tumors, leads to cognitive impairment including deficits in memory and executive functioning. We recently proposed deficient neuronal mitochondrial function as its underlying mechanism. We hypothesized nasal administration of mitochondria isolated from human mesenchymal stem cells to mice, can reverse cisplatin-induced cognitive deficits.Methods: Puzzle box, novel object place recognition and Y-maze tests were used to assess the cognitive function of mice. Immunofluorescence and high-resolution confocal microscopy were employed to trace the nasally delivered mitochondria and evaluate their effect on synaptic loss. Black Gold II immunostaining was used to determine myelin integrity. Transmission electron microscopy helped determine mitochondrial and membrane integrity of brain synaptosomes. RNA-sequencing was performed to analyse the hippocampal transcriptome.Results: Two nasal administrations of mitochondria isolated from human mesenchymal stem cells to mice, restored executive functioning, working and spatial memory. Confocal imaging revealed nasally delivered mitochondria rapidly arrived in the meninges where they were readily internalized by macrophages. The administered mitochondria also accessed the rostral migratory stream and various other brain regions including the hippocampus where they colocalized with GFAP+ cells. The restoration of cognitive function was associated with structural repair of myelin in the cingulate cortex and synaptic loss in the hippocampus. Nasal mitochondrial donation also reversed the underlying synaptosomal mitochondrial defects. Moreover, transcriptome analysis by RNA-sequencing showed reversal of cisplatin-induced changes in the expression of about seven hundred genes in the hippocampus. Pathway analysis identified Nrf2-mediated response as the top canonical pathway.Conclusion: Our results provide key evidence on the therapeutic potential of isolated mitochondria - restoring both brain structure and function, their capability to enter brain meninges and parenchyma upon nasal delivery and undergo rapid cellular internalization and alter the hippocampal transcriptome. Our data identify nasal administration of mitochondria as an effective strategy for reversing chemotherapy-induced cognitive deficits and restoring brain health, providing promise for the growing population of both adult and pediatric cancer survivors.

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

Mitochondrial DNA (mtDNA), as its name implies, is an embodiment of the mitochondrial genetic information that constitutes about 1% of the mammalian genome. It fills a vital niche in tracing matrilineality; the mtDNA is inherited solely from the mother and plays a crucial role in genealogical research. Even a single mutation in the mtDNA can have debilitating and life-altering consequences. Mothers carrying mtDNA mutations will inevitably pass it on to the future generation. Three-parent In vitro fertilization (IVF), a breakthrough technique, shows promising potential to prevent mothers with mtDNA defects from passing it on to their future generation, while also maintaining the genetic link to their posterity. In this review, I delve into the intricacies of this technique, compare and analyze the difference between maternal spindle transfer and pronuclear transfer, discuss the prospective therapeutic effects, and highlight the ethical concerns surrounding this procedure. Considering the various challenges and ethics of this contentious technique, the paper seeks to answer the rousing question – Is three-parent IVF the answer to preventing mitochondrial defects?

Germline transmission of donor, maternal and paternal mtDNA in primates.

What are the long-term developmental, reproductive and genetic consequences of mitochondrial replacement therapy (MRT) in primates? Longitudinal investigation of MRT rhesus macaques (Macaca mulatta) generated with donor mtDNA that is exceedingly distant from the original maternal counterpart suggest that their growth, general health and fertility is unremarkable and similar to controls. Mitochondrial gene mutations contribute to a diverse range of incurable human disorders. MRT via spindle transfer in oocytes was developed and proposed to prevent transmission of pathogenic mtDNA mutations from mothers to children. The study provides longitudinal studies on general health, fertility as well as transmission and segregation of parental mtDNA haplotypes to various tissues and organs in five adult MRT rhesus macaques and their offspring. MRT was achieved by spindle transfer between metaphase II oocytes from genetically divergent rhesus macaque populations. After fertilization of oocytes with sperm, heteroplasmic zygotes contained an unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mitochondrial (mt)DNA. MRT monkeys were grown to adulthood and their development and general health was regularly monitored. Reproductive fitness of male and female MRT macaques was evaluated by time-mated breeding and production of live offspring. The relative contribution of donor, maternal, and paternal mtDNA was measured by whole mitochondrial genome sequencing in all organs and tissues of MRT animals and their offspring. Both male and female MRT rhesus macaques containing unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mtDNA reached healthy adulthood, were fertile and most animals stably maintained the initial ratio of parental mtDNA heteroplasmy and donor mtDNA was transmitted from females to offspring. However, in one monkey out of four analyzed, initially negligible maternal mtDNA heteroplasmy levels increased substantially up to 17% in selected internal tissues and organs. In addition, two monkeys showed paternal mtDNA contribution up to 33% in selected internal tissues and organs. N/A. Conclusions in this study were made on a relatively low number of MRT monkeys, and on only one F1 (first generation) female. In addition, monkey MRT involved two wildtype mtDNA haplotypes, but not disease-relevant variants. Clinical trials on children born after MRT will be required to fully determine safety and efficacy of MRT for humans. Our data show that MRT is compatible with normal postnatal development including overall health and reproductive fitness in nonhuman primates without any detected adverse effects. 'Mismatched' donor mtDNA in MRT animals even from the genetically distant mtDNA haplotypes did not cause secondary mitochondrial dysfunction. However, carry-over maternal or paternal mtDNA contributions increased substantially in selected internal tissues / organs of some MRT animals implying the possibility of mtDNA mutation recurrence. This work has been funded by the grants from the Burroughs Wellcome Fund, the National Institutes of Health (RO1AG062459 and P51 OD011092), National Research Foundation of Korea (2018R1D1A1B07043216) and Oregon Health & Science University institutional funds. The authors declare no competing interests.

Jewish Ethical Reflections on the Promises and Challenges of Mitochondria Replacement Therapy

Abstract Orthodox Judaism is an ever-evolving religion, responding to changes in science, technology, medicine, etc. The evolution of thought within this religious tradition can be clearly examined in halakhic responses to assisted reproductive technology and the adoption of these technologies and procedures by the Orthodox Jewish community. A relatively new reproductive technology of great interest to the Orthodox community is mitochondrial replacement therapies (MRT). This article examines past and current decisions regarding reproductive technologies that are used when examining a new assisted reproductive technology. Additionally, close examination of these sources will elucidate whether MRT results in the conception and delivery of children who are “Jewish by birth” or how a child born via MRT can be considered Jewish, post-parturition.

Fetal mitochondrial DNA in maternal plasma in surrogate pregnancies: Detection and topology.

ObjectivesDue to the maternally‐inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and maternal mtDNA molecules in surrogate pregnancies.MethodsGenotypic differences between fetal and surrogate maternal mtDNA were used to identify the fetal and maternal mtDNA molecules in plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage‐end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in maternal plasma could be differentiated.ResultsFetal‐derived mtDNA molecules were mainly linear (median: 88%; range: 80%–96%), whereas approximately half of the maternal‐derived mtDNA molecules were circular (median: 51%; range: 42%–60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%–27%) than that of nuclear DNA (median: 20%; range: 9.7%–35%). The fetal‐derived linear mtDNA molecules were shorter than the maternal‐derived ones.ConclusionFetal mtDNA is present in maternal plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.

Mitochondrial replacement by genome transfer in human oocytes: Efficacy, concerns, and legality.

BackgroundPathogenic mitochondrial (mt)DNA mutations, which often cause life‐threatening disorders, are maternally inherited via the cytoplasm of oocytes. Mitochondrial replacement therapy (MRT) is expected to prevent second‐generation transmission of mtDNA mutations. However, MRT may affect the function of respiratory chain complexes comprised of both nuclear and mitochondrial proteins.MethodsBased on the literature and current regulatory guidelines (especially in Japan), we analyzed and reviewed the recent developments in human models of MRT.Main findingsMRT does not compromise pre‐implantation development or stem cell isolation. Mitochondrial function in stem cells after MRT is also normal. Although mtDNA carryover is usually less than 0.5%, even low levels of heteroplasmy can affect the stability of the mtDNA genotype, and directional or stochastic mtDNA drift occurs in a subset of stem cell lines (mtDNA genetic drift). MRT could prevent serious genetic disorders from being passed on to the offspring. However, it should be noted that this technique currently poses significant risks for use in embryos designed for implantation.ConclusionThe maternal genome is fundamentally compatible with different mitochondrial genotypes, and vertical inheritance is not required for normal mitochondrial function. Unresolved questions regarding mtDNA genetic drift can be addressed by basic research using MRT.

Powering life through MitoTechnologies: exploring the bio-objectification of mitochondria in reproduction

Mitochondria, the organelles providing the cell with energy, have recently gained greater public visibility in the UK and beyond, through the introduction of two reproductive technologies which involve their manipulation, specifically ‘mitochondrial donation’ to prevent the maternal transmission of inherited disorders, and ‘Augment’ to improve egg quality and fertility. Focusing on these two ‘MitoTechnologies’ and mobilising the conceptual framework of “bio-objectification”, we examine three key processes whereby mitochondria are made to appear to have a life of their own: their transferability, their optimisation of life processes and their capitalisation. We then explore the implications of their bio-objectification in the bioeconomy of reproduction. Drawing on publicly available material collected in two research projects, we argue that mitochondria become a biopolitical agent by contributing to the redefinition of life as something that can be boosted at the cellular level and in reproduction. Mitochondria are now presented as playing a key role for a successful and healthy conception through the development and promotion of MitoTechnologies. We also show how their “revitalising power” is invested with great promissory capital, mainly deriving from their ethical and scientific biovalue in the case of mitochondrial donation, and from the logics of assetisation, in the case of Augment.

The Conundrum of Poor Ovarian Response: From Diagnosis to Treatment.

Despite recent striking advances in assisted reproductive technology (ART), poor ovarian response (POR) diagnosis and treatment is still considered challenging. Poor responders constitute a heterogeneous cohort with the common denominator of under-responding to controlled ovarian stimulation. Inevitably, respective success rates are significantly compromised. As POR pathophysiology entails the elusive factor of compromised ovarian function, both diagnosis and management fuel an ongoing heated debate depicted in the literature. From the criteria employed for diagnosis to the plethora of strategies and adjuvant therapies proposed, the conundrum of POR still puzzles the practitioner. What is more, novel treatment approaches from stem cell therapy and platelet-rich plasma intra-ovarian infusion to mitochondrial replacement therapy have emerged, albeit not claiming clinical routine status yet. The complex and time sensitive nature of this subgroup of infertile patients indicates the demand for a consensus on a horizontally accepted definition, diagnosis and subsequent effective treating strategy. This critical review analyzes the standing criteria employed in order to diagnose and aptly categorize POR patients, while it proceeds to critically evaluate current and novel strategies regarding their management. Discrepancies in diagnosis and respective implications are discussed, while the existing diversity in management options highlights the need for individualized management.

SURVEY ON PERCEPTION OF GERMLINE GENE THERAPY AMONG JAPANESE MEDICAL STUDENT

Germline gene therapy (GGT) has potential to be radical treatment approach for monogenic genetic disorder. Because recent CRISPR technology enables us to edit human genome with high efficacy and lowest off-targeting, technical issues for GGT have been almost solved. Mitochondrial replacement therapy (MRT) is also only radical germline gene therapy for heritable mitochondrial disease. Japanese government has issued a statement prohibiting GGT, including MRT, and emphasizing the need for discussions in a wide range of perspectives.

COMPARISON OF THE STRUCTURE OF CHROMOSOMAL ABNORMALITIES IN THE EMBRYOS OBTAINED AFTER REPRODUCTIVE NUCLEAR TRANSFER VERSUS IVF-ICSI-PGT-A

The safety of reproductive nuclear transfer (or mitochondrial replacement therapy) in cases of IVF in patients with severe forms of infertility or a family history of mitochondrial diseases causes lively debate worldwide. Meanwhile, births of 13 children following this technique have already been announced. However, while the safety of the artificially created mitochondrial heteroplasmy is being discussed, the issue of possible induction of chromosomal abnormalities in the embryos caused by the manipulations with the nuclei at the stage of reconstruction is not addressed.

FIRST REGISTERED PILOT TRIAL TO VALIDATE THE SAFETY AND EFFECTIVENESS OF MATERNAL SPINDLE TRANSFER TO OVERCOME INFERTILITY ASSOCIATED WITH POOR OOCYTE QUALITY

Poor quality oocytes frequently fail to fertilise or produce embryos that arrest during their first days of culture in vitro. Oocyte cytoplasmic dysfunctions (including, but not limited to mitochondria) have been indicated as a major cause of this problem. Since maternal spindle transfer (MST) allows replacement of the entire cytoplasm of an affected oocyte, it holds promise for the enhancement of embryonic development. Recent studies in the mouse and in human oocytes donated for research have confirmed the technical feasibility of MST and provided reassurance concerning safety. Here we present results from the first registered pilot trial aiming to reveal whether MST has potential to overcome infertility caused by poor oocyte quality. This pilot trial (ISRCTN 11455145) was restricted to 25 patients, selected based on their multiple previous failed IVF attempts, in each case associated with massive embryo development arrest. Female age over 40y/o and severe male factor were exclusion criteria. Procedures were authorized by the National Authority of Assisted Reproduction (437/23.9.2016) and approved by the Hospital’s IRB. Informed consent was obtained from patients and donors to conduct all procedures and follow-up the children born. The meiotic spindle from patient’s oocytes was isolated in a minimal cytoplasmic volume and transferred to a previously enucleated donor oocyte. MST oocytes were inseminated by ICSI and cultured up to 6 days in a time-lapse incubator. Good morphology blastocysts underwent biopsy, aneuploidy testing and analysis of mitochondrial DNA (mtDNA) carryover levels. SNPs analysis and DNA fingerprints were used to confirm the origin of the nuclear genome and mtDNA in biopsied samples, amniotic fluid and somatic tissues of resulting children. A total of 25 MST cycles were performed in patients with an average age of 37.1 y/o and a mean number of previous failed IVF attempts of 5.7 (min 3 and max 11). The mean number of MII oocytes used for MST per patient was 4.4. MST was applied successfully in 113 of 123 oocytes (91.9%) used. Normal fertilization was confirmed in 76.1% (86/113) of injected oocytes and 52 of these developed into good quality blastocysts (60.5%). Genetic screening revealed 50% (26/52) of embryo biopsy specimens to be euploid and mtDNA carryover levels <1%. In 16/25 cases, at least one euploid blastocyst of good morphology was obtained. Thus far, single blastocyst transfers were performed in 9 patients, resulting in 6 clinical pregnancies (66.7%). Two patients have delivered a healthy child and 3 more pregnancies are ongoing. Genetic analyses of the biopsied cells, amniotic fluid and samples collected after birth (blood, urine, saliva, cord blood, placenta) confirmed the parentage of the children and the origin of the donated mtDNA. Follow-up studies are being performed on the children born. Given the difficult reproductive history of the patients, results are encouraging. However, more carefully controlled pilot trials and follow-up studies are needed to provide more insights into the efficacy of MST to overcome infertility.

Mitochondrial transplantation-a possible therapeutic for mitochondrial dysfunction?: Mitochondrial transfer is a potential cure for many diseases but proof of efficacy and safety is still lacking.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

Germline nuclear transfer in mice may rescue poor embryo development associated with advanced maternal age and early embryo arrest.

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.

Improvement of early developmental competence of postovulatory-aged oocytes using metaphase II spindle injection in mice.

PurposeAssisted reproductive technology (ART) is a widely applied fertility treatment. However, the developmental competence of aged oocytes from women of a late reproductive age is seriously reduced and the aged oocytes often fail in fertilization even when ART is used. To resolve this problem, we examined usefulness of a new method “the metaphase II spindle transfer (MESI)” as ART using mouse oocytes.MethodsThis work was composed of two experiments. First, 24 hours after collection, embryos from oocytes (1‐day‐old oocytes, called postovulatory‐aged oocytes), were observed, after intracytoplasmic sperm injection (ICSI), and it was found that they were not able to reach the blastocyst stage. Next, the metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes were injected into cytoplasts from oocytes just collected, using piezo pulses to generate reconstructed oocytes. This procedure was named metaphase II spindle injection (MESI).ResultsAfter ICSI, embryos from the reconstructed oocytes (32/105), which contained the genes of 1‐day‐old oocytes, were able to develop into the blastocyst stage. The fragmentation rate after ICSI was 28.6%. Thus, the developmental competence of 1‐day‐old oocytes was improved by MESI.ConclusionsThe MESI method has the potential to improve the success rate of infertility treatments for women of a late reproductive age.