title: Obtaining metaphase spreads from single blastomeres for PGD of chromosomal rearrangements.
abstract: It has previously been shown that it is possible to obtain metaphase chromosomes from single blastomeres converted into metaphase in the cytoplasm of a mouse zygote. This method is highly labour intensive and cannot be performed outside the preimplantation genetic diagnosis (PGD) laboratory, so to overcome these limitations, a method was developed for obtaining metaphase spreads from single biopsied blastomeres using different chemicals. The substances tested were calyculin A, caffeine, paclitaxel and colcemid in a total of 496 disaggregated and 234 biopsied blastomeres from day 3 embryos. It was demonstrated that the optimal method involved a combined use of 'selective biopsy' (selection of the biopsied blastomere according to morphological criteria) and exposure to caffeine. This resulted in shortening the mean incubation time of biopsied blastomeres, with a metaphase formation rate of 80%. The method is simple for obtaining metaphases from single blastomeres, and may be implemented in clinical practice of PGD for structural rearrangements.
authors.affiliation: Reproductive Genetics Institute, 2825 North Halsted Street, Chicago, IL 60657, USA.
authors.forename: Artem
authors.initials: A
authors.lastname: Shkumatov
authors.forename: Valeriy
authors.initials: V
authors.lastname: Kuznyetsov
authors.forename: Jeanine
authors.initials: J
authors.lastname: Cieslak
authors.forename: Yuri
authors.initials: Y
authors.lastname: Ilkevitch
authors.forename: Yury
authors.initials: Y
authors.lastname: Verlinsky
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002869:Chromosome Aberrations; D003470:Culture Media / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D003703:Demecolcine / Q000494:pharmacology; D046149:Embryo Culture Techniques; D006801:Humans; D008387:Marine Toxins; D008677:Metaphase; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D019836:Preimplantation Diagnosis; D015053:Zygote / Q000378:metabolism
chemical_list: D003470:Culture Media; D008387:Marine Toxins; D010080:Oxazoles; D002110:Caffeine; C059041:calyculin A; D017239:Paclitaxel; D003703:Demecolcine