A microsurgical technique for enucleation of multipronuclear human zygotes

Abstract

Tripronuclear human zygotes were used to determine the feasibility of an enucleation procedure without the use of cytoskeleton inhibitors to obtain intact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zygotes manipulated, 100% of the zygotes survived after removal of one pronucleus and 90.1% after complete enucleation, proving the reliability of the proposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 87.5% of cultured cytoplasts showed morphological changes, which included cleavage into two 'blastomeres', three 'blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison with the diploid human zygotes from the same harvests, which had undergone the first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes.

Self-correction in tripronucleated human embryos

“…of development, suggests that the selective microsurgical epronucleation of the surplus pronucleus is necessary in order to restore the heteroparental diploid condition in IVF-TPN embryos (4)(5)(6)(7)(8)(9)(10).…”

“…Restoration of diploidy and heteroparentality in IVF-TPN embryos can be achieved by selective microsurgical removal of one of the surplus male pronuclei (i.e., epronucleation of the pronucleus located far from the 2PB [4][5][6][7][8][9][10]). However, the appropriateness of extending this epronucleation procedure to human ICSI-TPN eggs is controversial, because there are no morphologic indicators of the parental origin of every pronucleus.…”

Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos

“…In humans, the removal of an extra male pronucleus has been performed with (5)(6)(7)11) or without (7,8,11) intervention on the cytoskeletal organization.…”

Fertilization abnormalities following human in vitro fertilization and intracytoplasmic sperm injection

“…The microsurgical removal of a single pronucleus from 3PN zygotes has been applied successfully in human IVF (Ivakhnenko et al, 2000). Enucleation of tripronucleate zygotes is most useful in patients with an especially high rate of hyperploidy, and in low responders with very few oocytes.…”