title: Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes.
abstract: Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos for preimplantation diagnosis of X-linked disease. With a modified spreading method and directly labelled fluorescent DNA probes, we have examined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggregated and 98 intact blastomeres obtained. The spreading efficiency was 96% and FISH signals were obtained from 97% of nuclei. In all cases, sibling blastomeres from the same embryo were the same sex. Mosaicism was observed in some embryos. Five cells which lysed during the disaggregation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor and multiple signals were observed so that reliable diagnosis of sex was not possible. The data reported here confirm that by using an improved spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sexing embryos for preimplantation diagnosis of X-linked disease.
authors.affiliation: Department of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
authors.forename: J C
authors.initials: JC
authors.lastname: Harper
authors.forename: E
authors.initials: E
authors.lastname: Coonen
authors.forename: F C
authors.initials: FC
authors.lastname: Ramaekers
authors.forename: J D
authors.initials: JD
authors.lastname: Delhanty
authors.forename: A H
authors.initials: AH
authors.lastname: Handyside
authors.forename: R M
authors.initials: RM
authors.lastname: Winston
authors.forename: A H
authors.initials: AH
authors.lastname: Hopman
mesh_terms: D001757:Blastomeres / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure; D015342:DNA Probes; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics; D008040:Genetic Linkage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D011247:Pregnancy; D011296:Prenatal Diagnosis; D012732:Sex Determination Analysis / Q000379:methods*; D013997:Time Factors; D014960:X Chromosome
chemical_list: D015342:DNA Probes