Mitochondrial Donation and Preimplantation Genetic Testing for mtDNA Disease

Abstract

Background: Children born to women who carry pathogenic variants in mitochondrial DNA (mtDNA) are at risk for a range of clinical syndromes collectively known as mtDNA disease. Mitochondrial donation by pronuclear transfer involves transplantation of nuclear genome from a fertilized egg from the affected woman to an enucleated fertilized egg donated by an unaffected woman. Thus, pronuclear transfer offers affected women the potential to have a genetically related child with a reduced risk of mtDNA disease.

Methods: We offered mitochondrial donation (by pronuclear transfer) or preimplantation genetic testing (PGT) to a series of women with pathogenic mtDNA variants who sought to reduce the transmission of these variants to their children. Patients with heteroplasmy (variants present in a proportion of copies of mtDNA) were offered PGT, and patients with homoplasmy (variants present in all copies of mtDNA) or elevated heteroplasmy were offered pronuclear transfer.

Results: Clinical pregnancies were confirmed in 8 of 22 patients (36%) and 16 of 39 patients (41%) who underwent an intracytoplasmic sperm injection procedure for pronuclear transfer or for PGT, respectively. Pronuclear transfer resulted in 8 live births and 1 ongoing pregnancy. PGT resulted in 18 live births. Heteroplasmy levels in the blood of the 8 infants whose mothers underwent pronuclear transfer ranged from undetectable to 16%. Levels of the maternal pathogenic mtDNA variant were 95 to 100% lower in 6 newborns and 77 to 88% lower in 2 newborns than in the corresponding enucleated zygotes. Heteroplasmy levels were known for 10 of the 18 infants whose mothers underwent PGT and ranged from undetectable to 7%.

Conclusions: We found that mitochondrial donation through pronuclear transfer was compatible with human embryo viability. An integrated program involving pronuclear transfer and PGT was effective in reducing the transmission of homoplasmic and heteroplasmic pathogenic mtDNA variants. (Funded by NHS England and others.).

Figure 1. pronuclear transfer and PGT treatment pathways.

(A) Schematic of the pronuclear-transfer pathway and progression of the 32 patients approved through the stages of egg storage, pronuclear transfer treatment and embryo transfer. (B) Schematic of the PGT pathway and progression of the 39 patients through the stages of ICSI, embryo biopsy and embryo transfer.

Figure 2. Oocyte retrieval and fertilization outcomes of pronuclear transfer patient, PGT patient and donor eggs.

(A) Number of oocytes per egg retrieval for pronuclear-transfer and PGT patients. (D) Number of oocytes per egg retrieval for egg donors. (B) (C) Comparison of proportions of normally fertilized patient (pronuclear transfer and PGT) and donor eggs (P<0.001, Chi-Square).

Figure 3. pronuclear transfer procedure and comparison of pronuclear transfer and PGT embryo development.

(A) Schematic illustrating the pronuclear-transfer procedure. (B) Images showing enucleation and fusion of patient karyoplast and donor egg cytoplast; scale bar = 20µm. (C) Comparison of proportions of pronuclear-transfer and PGT embryos developing to the blastocyst stage. (D) Images showing examples of top and good quality pronuclear-transfer blastocysts; scale bar = 20µm. (E) Comparison of the proportions of top and good quality blastocysts after pronuclear transfer and PGT showing an increased proportion of top and good quality pronuclear-transfer blastocysts on day 5 (P=0.04; Chi-square) and day 6 (P=0.006; Chi-square).

Figure 4. Clinical outcomes after pronuclear transfer and PGT.

(A) Proportion of patients who had embryos available for intrauterine transfer after ICSI for pronuclear transfer or PGT. (B) Graph showing the overall proportion of patients who has a clinical pregnancy after pronuclear transfer and PGT. (C) Graph showing the proportions embryo transfers (fresh and frozen) that resulted in a positive pregnancy test and clinical pregnancy after pronuclear transfer and PGT. Pronuclear-transfer patients had a reduced incidence of clinical pregnancy (P=0.05; Chi-Square). (D) Graph showing an increased proportion of biochemical pregnancies after pronuclear transfer (P=0.04; Fisher’s exact).