E-GEOD-11206 - Transcription profiling of mouse blastocyst after embryo biopsy
Submitted on 17 April 2008, released on 11 June 2008, last updated on 10 June 2011
Preimplantation Genetic Testing (PGT), which encompasses both Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic Screening (PGS), is a form of prenatal screening done on embryos conceived through assisted reproduction techniques (ART) prior to the initiation of pregnancy to ensure that only select embryos are used for transfer. PGT is typically performed on 8-cell embryos derived from either in vitro fertilization or intracytoplasmic sperm injection (ICSI) followed by extended culture. PGT requires a highly invasive embryo biopsy procedure that involves 1) incubating embryos in divalent-cation-deficient medium to disrupt cell adhesion, 2) breaching the protective zona pellucida with acid Tyrode’s, laser drilling, or mechanical force and 3) aspirating one or two blastomeres. In this study we developed a mouse model of the embryo biopsy procedure inherent to PGT to determine the effect of various aspects of the procedure (incubation in Ca2+/Mg2+-free medium (CMF), acid Tyrode’s treatment, blastomere aspiration), performed individually or in combination, on global patterns of gene expression in the resulting blastocysts. Experiment Overall Design: The comprehensive embryo biopsy procedure was done as outlined below and was modified according to the specific experimental treatment being performed. Experiment Overall Design: Eight-cell embryos with rounded blastomeres and no fragmentation were initially incubated in CMF-Chatot-Ziomek-Brinster medium for 5 min at 37oC in a humidified atmosphere of 5% CO2 in air to loosen cell-cell contacts and then transferred to CMF-Dulbecco’s Phosphate-Buffered Saline. The biopsies were done on the stage of a Nikon Eclipse TE2000 microscope equipped with Hoffman optics and motorized Eppendorf micromanipulators. No more than three embryos were manipulated outside of the incubator at any time. Embryos were held in place by gentle suction on an Eppendorf Sterile VacuTip holding pipette (15 μm diameter), and a dual-holder system was used to control the pipette containing acid Tyrode’s (6 μm diameter) and the biopsy pipette (15 μm diameter). The zona pellucida was breached by gentle expulsion of a steady stream of acid Tyrode’s solution (pH 1.6), and a single blastomere with a clear nucleus was removed by aspiration. Following each respective treatment, embryos were cultured individually in 10 μl drops of Potassium Simplex Optimization Medium supplemented with amino acids at 37oC in a humidified atmosphere of 5% CO2 in air until 96 h post-hCG injection. Four replicates of these five experimental groups, each containing 20 embryos, were generated for a total of 20 samples and 400 embryos. Experiment Overall Design: To determine the effect of the various aspects of the embryo biopsy procedure on global patterns of gene expression, total RNA was extracted from each set of 20 embryos in each treatment group at 96 h post-hCG injection. This total RNA was then used to prepare target cRNA by linear two-round amplification as described previously. The final yield of biotinylated cRNA/20 embryos ranged from 34 μg -78 μg, of which 20 μg was fragmented and submitted to the University of Pennsylvania Microarray Facility where the samples were serially hybridized to the MOE430v2 GeneChip. Quality control parameters for all samples were within the following ranges: scale factor: 0.27-0.47, percent of genes detected: 44-51%, actin 3’/5’ signal ratio: 1.5-4.2, and GAPDH 3’/5’ signal ratio: 3.1-4.3. The GC-RMA algorithm was used to quantify and normalize microarray signals using Stratagene Array Assist Lite, Version 3.4. The resulting .chp files were imported to GeneSpring (v7.31, Agilent Technologies) where probe sets were filtered to retain those that were flagged as P (present) in at least 3 of the 20 samples. The data was log2-transformed and subjected to analysis using Partek Genomics Suite (v6.3, Partek, Inc).
transcription profiling by array, unknown experiment type