MtDNA mutations are maternally transmitted7
. MtDNA is present in all cells in multiple copies and in patients with mtDNA disease either all mtDNA copies are mutated (termed homoplasmy) or there is a mixture of wild-type and mutated mtDNA (termed heteroplasmy)8
. Studies of human pedigrees with transmitted mtDNA mutations have shown that clinical disease is only seen in those patients with high loads of mutated mtDNA in affected tissues (usually greater than 60% mutated mtDNA)9,10
. There has been very limited success in developing effective treatment for mtDNA disease and genetic counselling combined with prenatal or pre-implantation genetic diagnosis is increasingly being offered to women who carry pathogenic mtDNA mutations11
. However, these techniques will only be of value to women who have low levels of mtDNA mutations in oocytes.
Following the granting of a research licence by the Human Fertilisation and Embryology Authority (UK), and informed consent by the donors, we used abnormally fertilised (unipronuclear or tripronuclear) human zygotes (one cell embryos) generated from a human IVF programme to study the feasibility of pronuclear transfer to prevent mtDNA disease transmission from mother to child. Unipronuclear and tripronuclear zygotes are not normally used in fertility treatment. Our studies involved the transfer of one or two pronuclei between abnormally fertilised zygotes (, Supplementary Figure 1
). Following treatment with cytoskeletal inhibitors (nocodazole and cytochalasin B), pronuclei were removed from a donor zygote within a karyoplast containing a small volume of cytoplasm. Karyoplasts were placed under the zona pellucida
of a recipient zygote and were fused using inactivated viral envelope proteins of the Hemagglutinating Virus of Japan (HVJ-E). Reconstituted zygotes were cultured for 6-8 days to monitor development in vitro
Pronuclear transfer using abnormally fertilised human zygotes
We first confirmed that pronuclear transfer between human zygotes was associated with a change in the nuclear genotype of the embryo by analysing microsatellite markers. In all embryos studied, informative markers confirmed that the reconstituted pronuclear transfer embryo contained donor embryo nuclear genotype (see Supplementary Table 1
). We then determined if pronuclear transfer was compatible with onward development in vitro
. This was complicated by the fact that abnormally fertilised zygotes have limited potential for development to the blastocyst stage in vitro
(17%) compared with normally fertilised embryos (32%). Nonetheless, following pronuclear transfer, zygotes showed onward development with 10 out of 44 (22.7%) of one pronuclear transfer zygotes and 8 out of 36 (22.2%) of two pronuclear transfer zygotes developing to >8 cell stage. We found no difference in embryo development at any stage whether we transferred one or two pronuclei. Following two pronuclear transfer, 8.3% of abnormally fertilised embryos developed to the blastocyst stage (). This is approximately 50% of the blastocyst rate for unmanipulated abnormally fertilised embryos; as there is no reliable morphological indicator to distinguish between the male and female pronucleus in the human zygote, it is likely that the decline in blastocyst formation is partly due to absence of either a maternal or paternal genome.
Having established that pronuclear transfer is compatible with onward development of human embryos, we next determined the carry-over of donor mtDNA genotype in the reconstituted pronuclear transfer embryos (). We sequenced the non-coding mtDNA control region from both the pronuclear donor and pronuclear recipient embryos () and identified polymorphic mtDNA variants which were unique to donor or recipient embryo, thereby allowing the determination of mtDNA carry-over in the pronuclear transfer embryo. Hot last cycle-PCR RFLP assays were developed specifically for these mtDNA variants () and used to analyse mtDNA extracted from whole embryos. We found that there was variation in the amount of mtDNA genotype from the donor zygote which is transferred to the two pronuclear transfer embryo (8.1% ±7.6; mean ± SD n=8) ().
MtDNA analysis of pronuclear transfer embryos
There are many factors which could affect the carry-over of mtDNA following pronuclear transfer. We therefore studied the mtDNA copy number present in human oocytes. Similar to the results in mice and previous studies of human oocytes at various stages of development12
, we found marked variation in the mtDNA copy number () and this may contribute to variation in level of mtDNA carry-over. Previous studies have investigated heteroplasmy levels in blastomeres obtained from donated heteroplasmic embryos and have reported variation of 0-19% between individual blastomeres from the same embryo11,15
. We therefore determined whether the proportion of donor mtDNA genotype also varied between blastomeres in the reconstituted embryos following transfer of two pronuclei (). In 1/8 embryos there was no detectable donor mtDNA in any blastomere. In the other seven embryos which contained donor zygote mtDNA, there was variation in level of donor mtDNA genotype between blastomeres (). Although this variation is similar to previous reports on heteroplasmic human embryos11,15
, we wished to minimise the carry-over of donor zygote mtDNA and therefore explored techniques to reduce the amount of cytoplasm contained within the pronuclear karyoplast. We focused on careful manipulation of the pronuclear karyoplast and we were able to remove the pronuclei with a minimal amount of cytoplasm (). Using hot last cycle-PCR RFLP assays we demonstrated that the mtDNA carry-over was significantly lower (P<0.005), with 4/9 embryos containing undetectable levels of mtDNA carry-over (). The average mtDNA carry-over in all remaining embryos was <2% (mean 1.68 ± 1.81% mean ± SD n=9). These embryos also revealed much less variation in mtDNA carry-over between individual blastomeres (). These levels of mtDNA are equivalent to those seen in unaffected individuals in epidemiological studies1
MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryos
Very recently a related technique, metaphase II spindle transfer between unfertilised metaphase II oocytes, has been reported using non-human primate oocytes16
. This resulted in the birth of live offspring in which the authors were unable to detect donor mtDNA using a less sensitive assay than we have used (lower limit of detection was 3% compared with <0.5%). Whilst our optimised techniques of pronuclear extraction resulted in <3% carry-over, we were nonetheless interested to determine whether the technique of metaphase II spindle transfer might offer the possibility of further reducing the level of mtDNA carry-over. We therefore measured the mtDNA copy number in karyoplasts containing the metaphase II spindle from freshly harvested human oocytes donated to research. We found no significant difference in the mtDNA copy number between metaphase II spindle karyoplasts (13222 ± 5733 mean ± SEM, n=21) compared with double pronuclear karyoplasts (18316 ±4336 mean ± SEM n=12). The wide variation within both groups of karyoplasts is likely due to the vastly different copy numbers observed in human oocytes (). We conclude from this that both approaches would be effective in greatly reducing the risk of mtDNA disease.
Our studies show that in human zygotes, pronuclear transfer has the potential to “treat” human mtDNA disease at a genetic level. The recent development of metaphase II spindle transfer has confirmed in non-human primates that this closely related method also holds great promise. The comparative value of both techniques has not been established in the same animal model or human oocytes, but both have potential advantages. The metaphase II spindle is smaller and technically easier to remove. However, it is not surrounded by a membrane and without the use of a DNA stain, it would be difficult to eliminate the possibility that some chromosomes may not be aligned on the metaphase plate or associated with the spindle as has been previously reported in human oocytes from older women 17
and in response to exposure to ambient conditions18
. Studies in mice have shown that pronuclear transfer limits mtDNA transfer to subsequent generations19
. In addition, the pronuclei are easier to visualise than the metaphase II spindle but they are also larger and their manipulation may induce more cellular trauma. Our studies in human zygotes have been particularly challenging since working with abnormally fertilised zygotes is technically more difficult than using normally fertilised (two pronuclear) zygotes and is less likely to yield normal embryos due to abnormal chromosomal constitution20
. Despite these problems we observed development of the manipulated embryos at approximately 50% of the abnormal embryos which have not been manipulated and shown either no detectable or very low levels of mtDNA carry-over.
In view of the lack of available treatment for these patients and their families21
, preventing the transmission of mtDNA disease is a priority. Whilst mtDNA mutations are common, pronuclear or metaphase II spindle transfer is unlikely to be of value for asymptomatic individuals or those with mild mtDNA disease in the family. However, in some families, mtDNA disease can affect multiple family members with catastrophic consequences22
– for these families pronuclear transfer may be an option that mothers who carry mtDNA mutations would consider. MtDNA mutations which are maternally inherited are either homoplasmic or heteroplasmic and high loads of mutated mtDNA are necessary before there is clinical disease (usually >60% of total mtDNA)8
. We have shown that we can generate human embryos with donor mtDNA carry-over at levels well below the disease threshold and indeed unlikely to be detected except with very sensitive genetic techniques. With inherited mtDNA mutations there is little evidence of increasing levels of mutated mtDNA in tissues with time, in fact the opposite occurs with loss of mutation in some tissues23
, and thus the very low levels of mtDNA carry-over detected in some embryos will not cause mtDNA disease.
We believe the data presented in this paper on human zygotes and their development show that pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. Manipulation of human oocytes and zygotes has the potential to cause chromosomal or epigenetic abnormalities24
in the developing embryo which require further study to ensure the safety of different techniques. We believe our study in human zygotes and embryos represents a major advance towards preventing transmission of disease in patients with mtDNA mutations.