Abstract
BackgroundCarriers of balanced translocations are at high risk for unbalanced gametes which can result in recurrent miscarriages or birth defects. Preimplantation genetic diagnosis (PGD) is often offered to select balanced embryos. This selection is currently mainly performed by array CGH on blastomeres. Current methodology does not take into account the phase of the cell cycle, despite the variable copy number status of different genomic regions in S phase.ResultsCell lines derived from 3 patients with different chromosomal imbalances were used to evaluate the accuracy of single cell array CGH. The different cell cycle phases were sorted by flow cytometry and 10 single cells were picked per cell line per cell cycle phase, whole genome amplified and analyzed by BAC arrays, the most commonly used platform for PGD purposes. In contrast to G phase, where the imbalances were efficiently identified, less than half of the probes in the regions of interest indicated the presence of the aberration in 17 S-phase cells, resulting in reduced accuracy.ConclusionsThe results demonstrate that the accuracy to detect segmental chromosomal imbalances is reduced in S-phase cells, which could be a source of misdiagnosis in PGD. Hence, the cell cycle phase of the analyzed cell is of great importance and should be taken into account during the analysis. This knowledge may guide future technological improvements.
Highlights
Carriers of balanced translocations are at high risk for unbalanced gametes which can result in recurrent miscarriages or birth defects
After sorting of the different cell cycle phases by flow cytometry, the genomic content of populations of cells, as an internal control for the sorting procedure, as well as individual S- and G0/G1-phase cells were analyzed by array based comparative genomic hybridization (aCGH) using 24Sure + Bacterial artificial chromosome (BAC) arrays, the most commonly used arrays for Preimplantation genetic diagnosis (PGD) purposes
Populations of S-phase cells result in more scattered aCGH profiles compared to G0/G1-phase cell populations To compare the aCGH profiles of S- and G0/G1-phase cells and as an internal control for the successful separation of the corresponding subpopulations by Fluorescenceactivated cell sorting (FACS), we hybridized genomic DNA extracted from many cells of S- and G0/G1-phase populations against control DNA of the opposite sex using BAC arrays
Summary
Carriers of balanced translocations are at high risk for unbalanced gametes which can result in recurrent miscarriages or birth defects. Preimplantation genetic diagnosis (PGD) is often offered to select balanced embryos. This selection is currently mainly performed by array CGH on blastomeres. Up to 15% of the couples confront fertility problems and 1% of the couples attempting to conceive a child experience recurrent miscarriage (RM), defined as the loss of at least three consecutive pregnancies [1]. In order to avoid miscarriages or the possibility of an affected child, preimplantation genetic diagnosis (PGD) can be offered to select those embryos which are chromosomally balanced. Preimplantation genetic screening (PGS) to Overall, during the 10 years of data collection by the European Society of Human Reproduction and Embryology (ESHRE), there have been 27,630 cycles to oocyte retrieval (OR) reported, that resulted in 202,357 fertilized oocytes and the transfer of 35,944 embryo. For 16% of these cycles the indication was chromosomal abnormalities [9]
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