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Introduction: Fluorescent in situ hybridization (FISH) is currently the main method used for aneuploidy preimplantation genetic screening (PGS). The main limitation of FISH analysis is that it usually includes only 5-10 chromosome- specific probes, compared to the 23 chromosome pairs of the human cell. This fact along with the high incidence of mosaicism in human embryos could be the explanation for the low success rates of PGS. The development of microarray technology combined with whole genome amplification (WGA) has recently emerged as a promising solution for comprehensive aneuploidy screening of the whole genome of a single cell, since array-CGH allows for the analysis of the full chromosome content and the diagnosis of small genomic imbalances. This study aims to evaluate the use of single-cell array-CGH for the molecular karyotyping of pre-implantation embryos in a pre-clinical setting, to investigate if by analyzing one blastomere we can identify a karyotypically fully normal embryo and to determine the levels of mosaicism in human embryos. Materials and Methods: The study design is blinded, including two operators. Nine good quality supernumerary IVF/ICSI frozen embryos donated for research were thawed at day 3 of embryo development, were disaggregated and all the blastomeres (n ¼ 42) numbered randomly by the first operator. The blastomeres were then subjected to multiple displacement amplification (MDA) to amplify the whole genome of each single cell. The second operator carried out the array-CGH using BAC/PAC arrays of 1 MB resolution (VIB, Leuven). As a reference, genomic DNA was used that was extracted from peripheral blood of male and female donors. Both test and reference DNA samples were labeled by random primer labeling using Cy5- and Cy3-labeled dCTPs. The labeled DNAs were co-precipitated and the slides hybridized in a humid chamber at 378C for 24 to 72 hours. The slides were then washed and scanned and the log2 of the Cy5 to Cy3 fluorescent intensity ratios were calculated and plotted. Results: Array CGH was carried out for the 35 blastomeres that were successfully amplified by MDA and array data were analysed. Before decoding, an attempt was made to assign the single blastomeres to the embryo they were assumed to come from. After decoding of the samples, the following results were obtained: all the blastomeres of two embryos were of normal karyotype. One embryo of which five blastomeres were analysed showed four cells with a normal karyotype 46,XX and one blastomere with 47,XX,þ13. The remaining 24 blastomeres came from six embryos and they were totally chaotic, since that all blastomeres in each embryo showed an abnormal and different karyotype precluding classification per originating embryo prior to decoding. Conclusions: These preliminary results show that array-CGH can be reliably used at the single cell level and diagnose whole chromosome aneuploidies, and moreover identifies chromosomal aberrations such as partial duplication or deletion of chromosome arms. Our results confirm the highprevalence of mosaicism in well developing human embryos and that a variety of different abnormalities can co-exist in an embryo. Most importantly, we could blindly identify the ten normal blastomeres from three embryos. If these nine embryos had been analyzed for PGS, we would have correctly identified the two fully normal embryos, but not the mosaic 46,XX/47,XX,þ13. We are currently analyzing a larger cohort of embryos and we will confirm the diagnosis by multiplex PCR on all the trisomic chromosomes. |
