SEXING SIBLING MOUSE BLASTOMERES BY POLYMERASE CHAIN-REACTION AND FLUORESCENT IN-SITU HYBRIDIZATION

The diagnosis of human genetic diseases at preimplantation stages allows the transmission of genetic anomalies in high risk couples to be prevented. Embryo sexing can be carried out using the polymerase chain reaction (PCR) or by fluorescent in-situ hybridization (FISH). However, it is still questionable whether the results obtained from a single analysed cell are fully predictive of the genetic characteristics of the whole embryo. We have isolated all the blastomeres from 4-cell stage mouse embryos and analysed them by PCR and FISH to assess the consistency of the results obtained. One half of each embryo (2/4-embryo) was spread and fixed to be processed by FISH, while the blastomeres of the other half embryo were isolated (1/4 + 1/4-embryo) and individually processed for PCR. We have determined the efficiency of both techniques when applied to different cells of the same embryo. We were able to amplify 92.5% of the embryos. Of these, 91.8% were classified as male or female, while the remaining 8.2% gave contradictory results (one male and its sibling blastomere female). Of the 40 embryos analysed by FISH, 97.5% could be classified as male or female with a sex distribution close to the expected 1:1. When comparing both techniques, 24 out of 36 embryos gave the same result. In nine cases the results did not coincide, and in three embryos comparison was not possible because of PCR contradictory results.