P–800 Generation of artificial oocytes by two distinct mechanisms

Study question Are haploid genome replication and somatic cell haploidization feasible mechanisms for generating parentally genotyped oocytes? Summary answer Artificial oocytes can be generated by haploid genome replication and somatic cell haploidization. The latter is more efficient and capable of generating live offspring. What is known already A low number of mature oocytes is one of the major limitations to treating infertile women who have impaired ovarian reserve. Although it has been proposed that competent oocytes can be created by a phenomenon known as somatic cell haploidization (SCH), its clinical value has yet to be examined due to its poorly understood mechanism. On the other hand, spindle transfer has been clinically applied for mitochondrial replacement therapy. Therefore, we propose to utilize G2-phase haploid pseudo-blastomere (HpB), generated by parthenogenesis, as a nuclear donor to create oocyte replica. Study design, size, duration In the past 7 months, individual G0 phase cumulus cells (CCs) were transferred into 1,066 ooplasts for SCH. HpBs obtained from the activation of 80 oocytes were transferred into 464 ooplasts. Both cohorts were ICSI-inseminated and placed in the time lapse for embryo development. Another 379 unmanipulated oocytes were ICSI-inseminated, serving as control. Pre-implantation development was monitored and compared for both neogametogenesis techniques. Fully expanded blastocysts were transferred to obtain live pups. Participants/materials, setting, methods CCs were isolated from the cumulus oophorus of B6D2F1 mice. HpBs were obtained via oocyte activation, cultured to the 8-cell stage, and subsequently treated by nocodazole to synchronize at the G2-phase. In two experimental groups, CCs or HpBs were individually transferred into the perivitelline space of the ooplasts with inactivated Sendai virus. Reconstructed oocytes presenting with a pseudo-meiotic spindle were fertilized by piezo-actuated ICSI. Blastocysts were transferred into a pseudo-pregnant CD–1 surrogate to obtain pups. Main results and the role of chance A total of 1,769 oocytes underwent enucleation to generate ooplasts, with a survival rate of 97%. Survived ooplasts were allocated to SCH (n = 1,034) and HpB-SCNT (n = 458). To generate HpBs, 80 unmanipulated oocytes were activated; 58 of them progressed to the 8-cell stage and generated 464 HpB for SCNT. For SCH, CCs were selected based on morphology with a diameter <10 micron. Nuclear transfer of CCs and HpB yielded survival rates of 98.6% and 93.2%, respectively. Following SCH and HpB-SCNT, spindle development for SCH and HpB-SCNT was comparable at 63.5% for SCH and 66.7% for HpB-SCNT. The ICSI survival rates for SCH and HpB-SCNT were 58.9% and 64.9%, respectively, but lower than the control at 73.9% (P < 0.001). Fertilization rates for SCH and HpB-SCNT were also comparable at 61.3% and 64.3%, respectively, but lower than the control at 89.6% (P < 0.00001). Full pre-implantation development was achieved for both experimental groups. While the SCH group yielded a development rate of 24.6% (n = 94), the HpB-SCNT group yielded a lower rate at 12.4% (n = 23) (P < 0.001), both lower than the control (71.7%, P < 0.00001); however, the morphokinetics of the embryo development was retained. To date, only 3 live pups were obtained from SCH group. Limitations, reasons for caution While these techniques to manufacture oocytes are very new and highly experimental, our findings show a lower blastulation rate for oocytes generated by HpB. Both techniques require refinement and improvement of reliability and consistency before they can be considered a feasible technique for human reproduction. Wider implications of the findings: The study confirms the potential to create artificial oocytes capable of supporting full pre-implantation development and, in some cases, live pups. If further streamlining of both procedures demonstrates their safety, they may both represent a viable option to generate de novo gametes Trial registration number N/A

mtDNA Heteroplasmy: Origin, Detection, Significance, and Evolutionary Consequences

Mitochondrial DNA (mtDNA) is predominately uniparentally transmitted. This results in organisms with a single type of mtDNA (homoplasmy), but two or more mtDNA haplotypes have been observed in low frequency in several species (heteroplasmy). In this review, we aim to highlight several aspects of heteroplasmy regarding its origin and its significance on mtDNA function and evolution, which has been progressively recognized in the last several years. Heteroplasmic organisms commonly occur through somatic mutations during an individual’s lifetime. They also occur due to leakage of paternal mtDNA, which rarely happens during fertilization. Alternatively, heteroplasmy can be potentially inherited maternally if an egg is already heteroplasmic. Recent advances in sequencing techniques have increased the ability to detect and quantify heteroplasmy and have revealed that mitochondrial DNA copies in the nucleus (NUMTs) can imitate true heteroplasmy. Heteroplasmy can have significant evolutionary consequences on the survival of mtDNA from the accumulation of deleterious mutations and for its coevolution with the nuclear genome. Particularly in humans, heteroplasmy plays an important role in the emergence of mitochondrial diseases and determines the success of the mitochondrial replacement therapy, a recent method that has been developed to cure mitochondrial diseases.

Mitochondrial transfer from iPS 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 IVF outcomes of aging female patients via mitochondrial transfer from 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: 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.

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.

Germline transmission of donor, maternal and paternal mtDNA in primates

STUDY QUESTION What are the long-term developmental, reproductive and genetic consequences of mitochondrial replacement therapy (MRT) in primates? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.

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.

Should mitochondrial replacement therapy be funded by the National Health Service?

A clinical trial on mitochondrial replacement therapy (MRT) is currently being conducted and if this technique proves effective, National Health Service (NHS) England will fund MRT through the highly specialised services (HSS) funding stream. This paper considers whether MRT should be publicly funded by the NHS. Given the current financial pressure the NHS is experiencing, a comprehensive discussion is essential. There is yet to be a thorough discussion on MRT funding, perhaps because this is a small-scale issue and presumed to be covered by the HSS budget. However, the source of funding has not been confirmed due to the trial’s incompletion. Upon its completion, reasoned decisions need to be made over the allocation of scarce NHS resources. It is therefore important to consider the following arguments in advance. Three arguments given against NHS funding of MRT will be evaluated. The first argument against NHS funding examines the HSS overspending its budget in an underfunded NHS, suggesting funding must be carefully reprioritised. Second, the ethical issue of allowing public access to a technique with insufficient evidence behind it will be explored. The final point considers the option of privately funding MRT and how this would affect the treatment’s development. After illustrating the weaknesses of such arguments, it will be concluded that MRT should be funded by the NHS.