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

Craven L, Tuppen HA, Greggains GD, Harbottle SJ, Murphy JL, Cree LM, Murdoch AP, Chinnery PF, Taylor RW, Lightowlers RN, Herbert M, Turnbull DM - Nature (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Mitochondrial Research Group, Institute for Ageing and Health, Newcastle upon Tyne NE2 4HH, UK.

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.

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MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryosa, 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.
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Figure 3: MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryosa, 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.

Mentions: 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 development121314, we found marked variation in the mtDNA copy number (Figure 2e) 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 (Figure 3a, b). 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 (Figure 3b). 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 (Figure 3c). 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 (Figure 3d, e). 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 (Fig 3e). These levels of mtDNA are equivalent to those seen in unaffected individuals in epidemiological studies1.


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

Craven L, Tuppen HA, Greggains GD, Harbottle SJ, Murphy JL, Cree LM, Murdoch AP, Chinnery PF, Taylor RW, Lightowlers RN, Herbert M, Turnbull DM - Nature (2010)

MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryosa, 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.
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Related In: Results  -  Collection

Show All Figures
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Figure 3: MtDNA analysis of individual blastomeres disaggregated from pronuclear transfer embryosa, 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.
Mentions: 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 development121314, we found marked variation in the mtDNA copy number (Figure 2e) 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 (Figure 3a, b). 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 (Figure 3b). 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 (Figure 3c). 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 (Figure 3d, e). 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 (Fig 3e). These levels of mtDNA are equivalent to those seen in unaffected individuals in epidemiological studies1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Mitochondrial Research Group, Institute for Ageing and Health, Newcastle upon Tyne NE2 4HH, UK.

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.

Show MeSH
Related in: MedlinePlus