Analysis of human preimplantation embryos and aging oocytes by cDNA microarrays

Abstract

Objective: To develop methods to analyze single oocytes and preimplantation embryos by cDNA microarrays 1) to define differences between oocytes from younger and older reproductive-aged women, and 2) to examine the cascade of gene expression that follows fertilization. Design: Descriptional study of human oocyte and embryo gene expression. Materials/Methods: These studies were approved by the UCSF Committee on Human Research. Consenting patients from the UCSF in vitro fertilization (IVF) program were candidates for these studies. Donated immature (germinal vesicle) oocytes from patients undergoing ICSI were stripped of granulosa cells and frozen individually in tubes. Embryos were obtained from patients who had completed their families and wished their remaining embryos be donated to research rather than destroyed. RNA was prepared using PicoPure RNA Isolation Kit (Arcturus Engineering), and amplified using a high sensitivity proprietary kit (collaborative effort, Arcturus Engineering). Cy3 (control) and Cy5 fluorescently-labeled probes were reverse-transcribed from amplified RNA. Two microarray chips (∼22,000 genes and ESTs each) were hybridized, washed, and scanned using the GenePix 4000B scanner. To test the amplification protocol, RNA from two oocytes was mixed then divided into two tubes for independent amplification. Analysis was performed using the Pearson Correlation. Oocytes from women 30 years and less were compared to oocytes from women 40 years and older, and to embryos at day 1 (pronuclear) and day 3 of development. Analysis was performed using GenePix 3.0, Significance Analysis of Microarrays (Stanford), and Cluster/TreeView (Lawrence Berkeley National Lab) software. Results: RNA amplification using these techniques was 1.8 million-fold, sufficient for multiple microarray experiments. The amplification control yielded a correlation coefficient of 98.3%, demonstrating reproducibility of the amplification methods. SAM analysis identified the most differentially expressed genes; more importantly, however, it revealed that biological and experimental variability can be overcome by relatively few experiments. Few genes are differentially expressed between young and old oocytes, suggesting that any differences are likely functional or at the protein level. Striking differences are evident by day 1 of embryo development, and this pattern contrasts greatly with those at day 3. Cluster analysis clearly demonstrates unique expression profiles among the different stages of development tested and has identified potential candidate genes for further study. Conclusions: cDNA microarray analysis of extremely small samples is now technologically feasible. These studies have shown few differences in the RNA between young and old eggs, but robust changes at the pronuclear stage of embryo development following fertilization. Future studies will more fully define the differences between the oocytes of younger and older women, and the different stages of preimplantation development. Supported by: Reproductive Scientist Development Program and Arcturus Engineering (amplification kits).