Amplification of representative c-DNA pools from single human oocytes and pronucleate embryos

In the human embryo, geneexpression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study in- dividual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between indivi- dual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in tran- script abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including b-actin (expressed in 24/28 oocytes,6/6 pronuclear embryos), the integrins b1 (17/28 oocytes, 6/6 pronuclear embryos) and b5 (8/28 oo-cytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear em- bryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regu- lator BAX increased in human oocytes following pro- longed periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.