Mitochondrial diseases, caused by pathogenic variants in the mitochondrial genome, affect about one in 5000 live births. It can result in severe multisystem disorders. These mutations may be heteroplasmic or homoplasmic. Preimplantation genetic testing (PGT) enables the selection of embryos with lower levels of heteroplasmy. But it is not effective for females with high levels of mutation. An alternative approach is mitochondrial donation using pronuclear transfer (PNT), which involves transferring the nuclear genetic material from the fertilized egg of an affected female into an enucleated donor zygote containing healthy mitochondria. Â
This study aimed to evaluate the clinical application of PNT and PGT in reducing the risk of mitochondrial DNA (mtDNA) disease transmission. It also assesses parameters as pregnancy rates, embryo development, live births, neonatal mtDNA levels, and fertilization outcomes. Â
A total of 32 females with a known pathogenic mtDNA variant received case-specific approval from the UK Human Fertilisation and Embryology Authority (HFEA) to undergo PNT, and 25 underwent oocyte retrieval. In parallel, 39 females underwent PGT. Patients with heteroplasmic variants underwent PGT, while those with high-heteroplasmy or homoplasmic variants were offered PNT. Â
Embryos were generated using intracytoplasmic sperm injection (ICSI) and biopsied on the 3rd day to assess heteroplasmy levels in the PGT group. Embryos with heteroplasmy below 30% were prioritized for cryopreservation or transfer. The pronuclei of the patient zygotes were inserted into enucleated donor zygotes about 8 to 13 hours after fertilization in PNT. Embryos were cultured, assessed, and selected for transfer based on inferred heteroplasmy risk and morphology. Heteroplasmy levels in the newborns were evaluated postnatally using next-generation sequencing and blood spot analysis. This study employed statistical methods, including chi-square tests, Fisher’s exact test, unpaired t-tests, and Tukey’s multiple comparison test.Â
The results showed that clinical pregnancies were achieved in 8 of 22 patients (36%) using PNT and 16 of 39 (41%) using PGT. PNT resulted in 8 live births and one with an ongoing pregnancy. PGT was produced in 18 live births. Fertilization rates were lower in the PNT groups (48.5%) compared to donor eggs (63.8%) and PGT patient eggs (66.5%), with p-values <0.001. The reduction in fertilization efficiency was attributed to the effects of vitrification and the biochemical impact of mtDNA mutations. Embryo development to the blastocyst stage was comparable between groups; however, PNT embryos had a higher proportion of top and good-quality blastocysts on day 6. In terms of neonatal results, heteroplasmy levels in PNT infants ranged from undetectable to16%. In all 6 cases, the reduction in maternal pathogenic mtDNA was above 95%. In 2 cases, it was from 77 to 88%. Data were accessible for 10 to 18 infants for PGT, and all had heteroplasmy levels below 7% with most showing undetectable levels. No congenital anomalies were identified, although one cardiac anomaly was found in each group, both in newborns with heteroplasmy below 5%.Â
This study demonstrates that both PGT and PNT are effective strategies for reducing the transmission of pathogenic mitochondrial DNA (mtDNA) variants. While PGT is more efficient in establishing pregnancies, PNT is an important alternative for females with homoplasmic or high-mtDNA variants. This result supports the use of these methods in a regulated and ethically guided manner. PGT is a well-established method that yields high success rates and low neonatal heteroplasmy. However, long-term follow-up of children born through PGT and PNT remains essential to confirm their safety and optimize protocols that decrease mitochondrial carryover and improve reproductive outcomes. Â
Reference: Hyslop LA, Blakely EL, Aushev M, et al. Mitochondrial Donation and Preimplantation Genetic Testing for mtDNA Disease. N Engl J Med. 2025. doi:10.1056/NEJMoa2415539.Â
