IDENTIFICATION OF THE SEX OF HUMAN PREIMPLANTATION EMBRYOS IN 2 HOURSUSING AN IMPROVED SPREADING METHOD AND FLUORESCENT IN-SITU HYBRIDIZATION (FISH) USING DIRECTLY LABELED PROBES

Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos fo r preimplantation diagnosis of X-linked disease. With a modified sprea ding method and directly labelled fluorescent DNA probes, we have exam ined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggre gated 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 disagg regation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor a nd multiple signals were observed so that reliable diagnosis of sex wa s not possible. The data reported here confirm that by using an improv ed spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sex ing embryos for preimplantation diagnosis of X-linked disease.