Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease

Abstract

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

Figure 1. Pronuclear transfer using abnormally fertilised human zygotes

a-g, Transfer of two pronuclei between human zygotes. a, Schematic diagram showing recipient zygote (one pronucleus which is removed) and donor zygote (three pronuclei, two of which are removed and fused with the recipient zygote). b, Recipient zygote containing a single pronucleus (marked with arrow) which is removed by a biopsy pipette to leave an enucleated zygote d. c, Donor zygote with three pronuclei (marked with arrows) and two of these pronuclei removed as karyoplasts e. f, Enucleated recipient zygote with two pronuclear karyoplasts from the donor zygote (arrows) prior to fusion. g, Recipient zygote 20 minutes after transfer already showing fusion of the karyoplast membranes (arrow). h, Development of unmanipulated abnormally fertilised zygotes (n=76; black bars), one pronuclear (n=44; grey bars) and two pronuclear (n=36; white bars) transfer embryos. i, Day 7 hatched blastocyst containing two donor pronuclei. Scale bars are 50μm.

Figure 2. MtDNA analysis of pronuclear transfer embryos

a, Schematic diagram showing the potential transfer of donor zygote mtDNA to the recipient zygote. b, Sequence electropherograms of mtDNA non-coding control region in donor and recipient zygotes with the sequence variant used for last hot cycle PCR-RFLP assay highlighted. c, Scheme of RFLP designed using the sequence variant. d, Last hot cycle-PCR RFLP analysis of donor mtDNA carry-over detected in two pronuclear transfer embryos with products separated by 12% nondenaturing polyacrylamide gel electrophoresis. U: undigested, C1 and C2: controls (C1: donor embryo for E3, recipient embryo for E1 and E2; C2: donor embryo for E1 and E2, recipient embryo for E3). e, MtDNA copy number in human mature oocytes.

Figure 3. MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryos

a, Last hot cycle PCR RFLP of individual blastomeres from a pronuclear transfer embryo showing variable levels of mtDNA donor genotype in individual blastomeres. The arrow indicates the band representing carry-over mtDNA. b, Analysis of levels of donor mtDNA carry-over in individual blastomeres from 8 embryos prior to modifications to minimise levels of donor mtDNA in pronuclear karyoplasts. In some embryos not all blastomeres could be collected. Figures represent the percentage mtDNA carry-over in individual blastomeres following pronuclear transfer. nd: non-detectable. c, Image of pronuclear karyoplasts after additional manipulation showing minimal amount of donor cytoplasm when compared with Figure 1e. Scale bar 25μm. d, Last hot cycle PCR RFLP of individual blastomeres from a pronuclear transfer embryo showing no detectable levels of mtDNA donor genotype in individual blastomeres. The arrow indicates the band representing carry-over mtDNA. e, Analysis of levels of donor mtDNA carry-over in individual blastomeres from 9 embryos following improvements to pronuclear karyoplast removal. In some embryos, not all blastomeres could be collected. Figures represent the percentage of mtDNA carry-over in individual blastomeres following pronuclear transfer. nd: non-detectable.