This report summarizes our effort to test an mtDNA replacement in unfertilized human oocytes, initially developed and optimized in a monkey model. The results demonstrate that the ST procedure can be performed with high efficiency in human oocytes. Manipulated oocytes also supported high fertilization rates similar to that of controls. However, approximately half of the human ST zygotes exhibited abnormal fertilization primarily, as a result of excessive pronuclear numbers. This was an unexpected outcome that was not observed with monkey oocytes. Our follow up studies indicated that this is caused by the failure to complete meiosis and segregate chromosomes into the 2nd PB, likely due to premature activation. A set of haploid genetic material is normally discarded during asymmetrical cell division into the 2nd PB, while the other half forms the female pronucleus. By genetic analysis of ESCs derived from abnormally fertilized zygote, we confirmed the triploid nature and presence of two sets of female chromosomes.
The spindle-chromosomal apparatus in MII oocytes is an extremely sensitive structure that can be easily perturbed by physical or chemical manipulations. Our initial attempts to isolate and transplant monkey MII spindles were unsuccessful due to similar problems with spontaneous resumption of meiosis6
. Procedures were optimized to avoid this negative outcome and current ST protocols allow maintenance of an intact MII spindle and normal fertilization. It appears that human MII oocytes are more sensitive to spindle manipulations and further improvements and optimizations will be required for future clinical applications. Maintenance of meiotic spindles in MII oocytes is dependent on the activity of M-phase-specific kinases including maturation-promoting factor (MPF) and MAP kinase16
. Under normal conditions, sperm entry triggers degradation of kinase activities and chromosome segregation mediated by oscillations of intracellular Ca2+
. However, an influx of calcium induced by mechanical or chemical manipulations can induce parthenogenetic activation of oocytes and resumption of meiosis18
. Thus, ST manipulations in a medium without Ca2+
or supplementations with MG132 could potentially avoid problems with spontaneous activation19,20
Morphological evaluation of fertilization and early detection of abnormal pronuclear and/or polar body formation appears to be critical to separate normal and abnormal ST embryos. Blastocyst development and ESC isolation in normally fertilized ST zygotes was similar to controls. We also confirmed that all ESC lines derived from these ST embryos are karyotypically normal.
Two of the 9 ESC lines (22%) derived from non-manipulated oocytes also showed chromosomal abnormalities. Since aberrations were confined to the sex chromosomes (47XYY and 45XO), it is possible that this was induced by sperm carrying either two Y or without Y chromosome.
Despite the risk of abnormal pronuclear formation and aneuploidy in a portion of ST zygotes, embryo development and ESC isolation rates in normal ST zygotes are comparable to intact controls. Based on our estimates of retrieving on average 12 MII oocytes, 35% normal (2PN/2PB) fertilization rates, and 60% blastocyst development, at least 2 ST blastocysts suitable for transfers can be generated during a single cycle for each patient.
The safety of the ST procedure is also dependent on the amount of mutated mtDNA co-transferred with spindles. Importantly, mtDNA carryover in ST embryos and ESC lines is technically undetectable or below 1%. In most patients with mtDNA diseases, a threshold of 60% or higher of mutated mtDNA must be reached for clinical features to appear. Thus, it is unlikely that low mtDNA carryover during ST would cause disease in children. Segregation of mutated mtDNA to specific tissues during development and aging may hypothetically result in a significant accumulation of the mutant load. However, analysis of mtDNA carryover in monkey ST offspring discovered no detectable mtDNA segregation into different tissues9
. In addition, there were no changes in heteroplasmy levels during postnatal development of monkeys. Thus, carryover, segregation and tissue-specific accumulation of mutant mtDNA molecules in ST children seems unlikely to be a major concern.
Birth of a healthy monkey infant after oocyte freezing marks an important milestone in applying the ST technology to patients. Transplantation of vitrified spindles into fresh cytoplasms yields the best results, comparable to controls. However, fertilization of vitrified cytoplasts even with fresh spindles was compromised. These remarkable findings suggest that the damage after cryopreservation is confined mainly to the eggs’ cytoplasm, not to the chromosomes and spindles as commonly believed21
. Our observations also reveal another unexpected potential clinical application of the ST technique, suggesting that spindles in sub-optimally cryopreserved oocytes can be rescued by transplanting into fresh cytoplasts.
Follow up postnatal studies in four monkeys produced by ST provide convincing evidence that oocyte manipulation and mtDNA replacement procedures are compatible with normal development. These monkeys were derived by combining of nuclear and mtDNA from the two genetically distant subpopulations of rhesus macaques. Mitochondrial and nuclear genetic differences between these monkeys are considered to be as distant as those between some different primate species22
, thus imitating haplotype differences between humans. Concerns have been raised that nuclear and mtDNA incompatibilities between mtDNA patients and cytoplast donors may cause a “mismatch” and mitochondrial dysfunctions in ST children even in the absence of mutations15
. Based on our long-term observations, it is reasonable to speculate that nuclear-mtDNA interactions are conserved within species.
Pioneering work in nonhuman primates is critical for development and safety and efficacy evaluations of new treatments23,24
. It is important that scientists and clinicians further optimize ST protocols for human oocytes and assure these procedures are safe. It is also crucial that the FDA initiates careful review of these new developments. Such oversight will be important to establish safety and efficacy requirements and guide clinical trials. Current NIH funding restrictions surrounding these innovative reproductive technologies will also require amendments to support federally-funded clinical trials.