Limits...
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.

Show MeSH

Related in: MedlinePlus

Pronuclear transfer using abnormally fertilised human zygotesa-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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2875160&req=5

Figure 1: Pronuclear transfer using abnormally fertilised human zygotesa-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.

Mentions: 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 (Figure 1, 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 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)

Pronuclear transfer using abnormally fertilised human zygotesa-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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875160&req=5

Figure 1: Pronuclear transfer using abnormally fertilised human zygotesa-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.
Mentions: 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 (Figure 1, 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.

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