Fish reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts reveals high accuracy of diagnosis and no sign of mosaicism or preferential allocation

Abstract

To study the blastocyst genetic constitution (possible chromosomal mosaicism between ICM and TE and within TE), after isolating ICM by a novel method.and to evaluate array-CGH accuracy against FISH testing Cross-sectional study. 32 blastocysts diagnosed as genetically abnormal by array-CGH were donated to research. ICM was isolated from the trophectoderm (TE) using a novel mechanical separation which was confirmed by KRT18 immunostaining. 24 of the 32 embryo had single or double aneuploidy and 8 aneusomic for unbalanced product of translocation or for long single arm deletions/duplications but with normal ploidy (by aCGH). After biopsy, individual samples were fixed by Carnoy method and analyzed with conventional 9-chromosomes FISH and locus specific probes. We reanalyzed 32 ICM and 96 TE samples (3 separate groups of cells from each TE of the same embryo). Looking at the single chromosome level, 28 out of 28 aneuploidies diagnosed by a-CGH were confirmed by FISH reanalysis both in ICM and TE samples resulting in a analytical sensitivity of 100%(95%CI 90.2-100). For almost all embryos, the reported aneuploidies were constitutive. As regard to chromosomes diagnosed as disomic, only 2 out of 248 (specificity 99.1 95%CI 97.4-99.8) were misdiagnosed by a-CGH, a trisomy 13 in an unbalanced embryo from reciprocal translocation carrier observed in a constitutive state and a mosaic nullisomy/monosomy 16 in a double aneuploid embryo. Consequently, only one out of 32 embryos was misdiagnosed as false negative. Only in one case we observed a confined trisomy 17 in all nuclei of a TE sample from a trisomic embryo for chromosome 13. In all other embryos ICM and TE samples were concordant and confirmed the original a-CGH diagnosis. Our data show a very high analytical accuracy of a-CGH to diagnose embryo chromosomal constitution. Concordance between ICM and TE was reported for all embryos analyzed, showing no sign of mosaicism or preferential allocation at blastocyst stage.