Abstract

Mitochondrial DNA replacement in human oocytes

Seven volunteers (age 21-32 years) underwent ovarian stimulation and a total of 106 mature MII oocytes were retrieved (range of 7–28, or a mean of 15 oocytes per donor cycle). Participants were synchronized in 3 separate experiments, so that at least two fresh oocyte cohorts were available on the same day for reciprocal ST. We selected a total of 65 MII oocytes for the ST procedure and 33 served as non-manipulated controls (Fig. 1a). We successfully transferred the spindle surrounded by a membrane and small amount of cytoplasm (karyoplast), between 64 oocytes (98%; Fig. 1a, b). Sixty oocytes survived fertilization by intracytoplasmic sperm injection (ICSI; 94%) and 44 formed visible pronuclei (73%). These outcomes were similar to controls, where 32 oocytes survived ICSI (97%) and 24 (75%) formed pronuclei (Fig. 1b). Microscopic evaluations determined that almost half of ST zygotes (21/44, 48%) contained normal 2 pronuclei and 2 polar bodies (2PN/2PB) (Fig. 2a). However, the remaining ST zygotes displayed an irregular number of pronuclei and/or polar bodies (Fig. 2a). Abnormal fertilization was also observed in the intact control group, albeit at a lower incidence (3/24, 13%).

Blastocyst formation rate in the normally fertilized ST group (13/21, 62%) was statistically similar to controls (16/21, 76%). However, the majority of abnormal ST zygotes arrested with only 26% (6/23) reaching blastocysts (Suppl. Table 1). Interestingly, blastocyst development of 3PN/1PB zygotes was noticeably higher (4/11, 36%) compared to other abnormally fertilized ST groups (Suppl. Table 1).

Derivation and genetic analysis of human ESCs

To provide additional insights into the developmental competence of ST-produced human embryos and to obtain sufficient material for molecular and cytogenetic analyses, we derived ESCs from blastocysts. Nine ESC lines (hESO lines) were established from 16 control blastocysts (56%; Fig. 1b). This ESC derivation rate is significantly higher than currently reported for embryos donated by IVF patients⁷. Similarly, 13 ST blastocysts developed from normally fertilized zygotes produced 5 ESC lines (38%; hESO-ST lines). We also plated 4 ST blastocysts that originated from 3PN/1PB zygotes and derived one ESC line (25%; Suppl. Table 1).

The ESCs derived from the ST embryos displayed normal morphology and were indistinguishable from controls (Suppl. Fig. 1a, b). All ESCs expressed standard pluripotency markers, including OCT-4, SOX2, SSEA-4, TRA-1-81 and TRA-1-60 (Suppl. Fig. 1a, b). Following injection into immunodeficient mice, experimental ESCs formed teratoma tumors consisting of cells and tissues representing all three germ layers (Suppl. Fig. 1c). Detailed analysis of nuclear DNA employing microsatellite markers confirmed that all hESO-ST lines inherited their chromosomes from the spindle donor oocytes (Fig. 3a, Table 1, Suppl. Fig. 2a, d). Analysis of mtDNA confirmed that all hESO-ST cell lines derived their mtDNA from the cytoplast donors (Fig. 3b, Table 1, Suppl. Fig. 2b,e, and 3)^6,8.

As reported previously⁶, small amounts of mtDNA is usually co-transferred with the karyoplast during the ST causing a low carryover heteroplasmy. In clinical situations this may result in the transmission of mutant mtDNA to ST embryos and children. Therefore we conducted both qualitative and quantitative mtDNA assays to determine the degree of mtDNA carryover in ST embryos and ESC lines. We identified mtDNA sequence differences between oocyte donors and unique restriction enzyme recognition sites for RFLP assay ⁶. For example, mtDNA of the egg donor 1 (spindle contributor for hESO-ST2, -ST3 and -ST4) possessed a unique EcoRV digestion sequence GATATC (Suppl. Fig. 4a). In contrast, egg donor 2 (mtDNA contributor) carried a single nucleotide polymorphism GATACC precluding enzyme recognition. The results confirmed that mtDNA in ST cell lines was exclusively derived from the cytoplast donors with no detectable mtDNA carryover (Table 1, Suppl. Fig. 2c and 4a). We also utilized more sensitive ARMS-qPCR that allows measuring heteroplasmy below 1%^9,10. The mean mtDNA carryover in ST oocytes and embryos was 0.5% (SD: ± 0.4; range 0-0.9%) and in hESO-ST cell lines was 0.6% (SD: ±0.9; range 0-1.7%) (Suppl. Fig. 4b, Table 2). These results are consistent with our previous data from a nonhuman primate⁶ and suggest negligible mtDNA carryover in ST offspring.

Cytogenetic analyses, using G-banding, indicated that all 5 hESO-ST lines derived from normal ST embryos contained diploid male or female karyotypes, with no evidence of detectable numerical or structural chromosomal abnormalities (Table 1 and Suppl. Fig. 5). However, 2 of 9 lines derived from control embryos displayed numerical aberrations. Notably, hESO-6 carried a 47XYY karyotype, while hESO-9 was 45XO (Suppl. Table 3 and Suppl. Fig. 6). G-banding revealed that hESO-ST6, the cell line derived from abnormally fertilized ST zygote, contained abnormal triploid female chromosome complement (Suppl. Fig. 5). In addition, detailed microsatellite analysis of nuclear DNA in this cell line confirmed the presence of three alleles for most STR loci (Suppl. Table 4). Based on the allele inheritance, we concluded that the triploid karyotype was caused by retention of the genetic material of the 2nd polar body. This was consistent with observation of the extra pronucleus but lack of the second polar body in the zygote.

Origin of abnormal fertilization in human zygotes produced by ST

Abnormal fertilization observed in some human ST oocytes was unexpected since this was not observed in monkey studies ⁶. Therefore, additional experiments were conducted to investigate possible underlying mechanisms. Genetic analysis of the hESO-ST6 cell line hinted that some ST oocytes and embryos retain extra chromosomes that are normally extruded into the 2^nd polar body. This was likely caused by suboptimal conditions during the ST procedure that disturbed spindle integrity. Initially, we focused on the effect of cytochalasin B (CB), a microfilament inhibitor known to inhibit cytokinesis and to block the 2^nd PB extrusion in oocytes^11,12. CB is used acutely during the ST procedure and oocytes are thoroughly rinsed before fertilization, but residual CB could interrupt the 2^nd PB extrusion. Therefore, we extended incubation time between ST and ICSI, or decreased CB concentration. However, abnormal fertilization persisted even in the absence of CB (Suppl. Tables 5, 6) suggesting that abnormal meiotic segregation is not likely caused by CB exposure.

We next addressed if oocyte polarity during displacement of spindles leads to abnormal meiosis. Typically, spindles in MII oocytes are adjacent to the 1st polar bodies. However, during the ST procedure, karyoplasts are reintroduced on the opposite side (referred to as 180 degree)^5,6. We reintroduced spindles next to or at the 90 degree from the 1^st PB (Suppl. Fig. 7). However, ST zygotes exhibited similar pronuclear abnormalities (Suppl. Fig. 7).

Lastly, we reasoned that human meiotic spindles may undergo premature activation during the ST manipulations leading to incomplete resumption of meiosis after fertilization. Spindle morphology and meiotic stage was analysed in intact and ST human oocytes following immunolabeling with α- and β-tubulins. Analysis demonstrated that in some ST oocytes spindles already progressed to the late anaphase II, whereas all control oocytes maintained uniform metaphase II (Fig. 2b).

Oocyte cryopreservation prior to ST

Current ST protocols use fresh oocytes and require that both patient and healthy mtDNA egg donors undergo synchronous retrievals. However, it is difficult to manage the same-day egg retrievals due to differences in the ovarian cycle and responses to gonadotropins. In addition, an equal number of patient and donor eggs retrieved would be ideal to avoid oocyte wastage. Therefore, oocyte freezing, storage and thawing will be critical for clinical applications of the ST. Recent advances in oocyte vitrification procedures suggest that cryopreserved human MII oocytes can be used in clinical IVF practice with the same efficiency as fresh eggs^13,14. To evaluate the feasibility of using cryopreserved oocytes for ST, we turned to the nonhuman primate model. We tested a commercially available vitrification kit (CRYOTOP) and determined that survival and recovery of rhesus macaque MII oocytes post-thaw is high. After ISCI, 72% formed pronuclei, but only 6% developed to blastocysts (P<0.05) (Table 2). Thus, this cryopreservation method compromises blastocyst development, since blastocyst formation of fresh oocytes from the same cohort was 52% (Table 2)^6,9.

We next conducted reciprocal ST between fresh and frozen-thawed monkey oocytes and examined fertilization and embryo development (Suppl. Fig. 8). When, fresh spindles were transplanted into vitrified cytoplasts, fertilization after ICSI was impaired (50%) compared to controls (91%) (Table 2). Moreover, all embryos in this ST group arrested before reaching blastocysts, while 57% controls progressed to blastocysts. These ST results were similar to that seen with frozen-thawed intact controls (Table 2). However, when spindles from vitrified oocytes were transferred into fresh cytoplasts, fertilization (88%) and blastocyst formation (68%) rates were similar to fresh controls (Table 2). These results suggest that vitrification causes damage primarily within the cytoplasm rather than to the spindle apparatus.

To further evaluate developmental potential, we plated 6 ST blastocysts derived from vitrified spindles onto feeder cells and established two ESC lines (33%). We transplanted 4 ST blastocysts from vitrified spindles into a recipient that resulted in the timely birth of a healthy infant (Suppl. Fig. 8).

Postnatal development of monkey ST offspring

While the technical feasibility of mtDNA replacement is documented for human embryos and ESCs, questions remain whether a “mismatch” between mtDNA and nuclear DNA haplotypes may cause mitochondrial dysfunctions in ST children¹⁵. To address these concerns, we conducted a 3-year follow up studies on monkey ST offspring born in 2009⁶.

The growth and development of four healthy infants following ST procedure was evaluated during the postnatal period (Suppl. Fig. 9a). Their overall health, including routine blood and body weight measurements monitored from birth to 3 years were comparable to age-matched controls. The values for haemoglobin, red blood and white blood cell counts, mean corpuscular volume, and haemoglobin concentrations were all within normal ranges (Suppl. Table 7). We also measured blood chemistry and arterial blood gas parameters and demonstrated that metabolic status of ST offspring is comparable to controls (Suppl. Table 6). In addition, the body weight gains for the ST juvenile monkeys were similar to that of age-matched controls (Suppl. Fig. 9b). We also confirmed that ATP levels and mitochondrial membrane potential (ΔΨm) in skin fibroblasts were similar to controls (Suppl. Fig. 10). Finally, there were no significant changes in mtDNA carryover and heteroplasmy in blood and skin samples with age (Suppl. Fig. 9c).

Discussion

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 2^nd PB, likely due to premature activation. A set of haploid genetic material is normally discarded during asymmetrical cell division into the 2^nd 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⁺ concentrations¹⁷. 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 activation^19,20.

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 treatments^23,24. 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.

Methods Summary

The study protocols were approved by both the OHSU Embryonic Stem Cell Research Oversight Committee and the Institutional Review Board. Mature oocytes were donated by volunteers and ST procedures were carried out as described^5,6. Oocytes were fertilized, cultured to blastocysts and used for ESC isolation. Detailed methods are described in Supplementary information at www.nature.com/nature.

Supplementary Material

Acknowledgements

Footnotes

References