Chromosomal analysis of blastocysts from balanced chromosomal rearrangement carriers

Abstract

Balanced chromosomal rearrangements (CRs) are among the most common genetic abnormalities in humans. In the present study, we have investigated the degree of consistency between the chromosomal composition of the blastocyst inner cell mass (ICM) and trophectoderm (TE) in carriers with balanced CR, which has not been previously addressed. As a secondary aim, we have also evaluated the validity of cleavage-stage preimplantation genetic diagnosis (PGD) based on fluorescence in situ hybridization (FISH) of blastocysts from CR carriers. Blastocyst ICM and TE were screened for chromosomal aneuploidy and imbalance of CR-associated chromosomes based on whole-genome copy number variation analysis by low-coverage next-generation sequencing (NGS) following single-cell wholegenome amplification by multiple annealing and looping-based amplification cycling. The NGS results were analyzed without knowledge of cleavage-stage FISH results. NGS results for blastocyst ICM and TE from CR carriers were 86.49% (32/37) consistent. Of the 1702 (37!46) chromosomes examined, 99.47% (1693/1702) showed consistency. However, only 40.0% (18/45) of all embryos had consistent results for chromosomes involved in CR, as determined by blastocyst NGS and cleavage-stage FISH. Of the 85 CR-affected chromosomes analyzed by FISH, 37.65% (32/85) were incongruous with NGS results, with 87.5% (28/32) showing imbalanced composition by FISH but balanced composition by NGS. These results indicate that chromosomal composition of blastocyst ICM and TE in balanced CR carriers is highly consistent, and that PGD based on cleavage-stage FISH is inaccurate; therefore, using blastocyst TE biopsies for NGS-based PGD is recommended for identifying chromosomal imbalance in embryos from balanced CR carriers.

Identification of balanced chromosomal rearrangements previously unknown among participants in the 1000 Genomes Project: implications for interpretation of structural variation in genomes and the future of clinical cytogenetics

“…Due to similarity in the G-banding pattern and size of the exchanged segments, chromosome analysis did not readily identify the translocation ( Figure 2A ). Therefore, metaphase FISH 17 was performed using BAC probes (SpectrumOrange: RP11-7D23 at 16q24.3, SpectrumGreen: RP11-526M7 at 17q25.1 and SpectrumRed: RP11-135N5 at 17p13.3) in more than 100 cells confirming the t(16;17) ( Figure 2C ).…”

“…G-banded chromosome analysis was performed using standard protocols for more than 100 cells in each EBV-B cell line 16 . Fluorescence in situ hybridization (FISH) was performed for NA18612 using standard procedures with BAC clones labeled by nick translation with SpectrumOrange or SpectrumRed, SpectrumGreen dUTP (Abbott Molecular, Des Plaines, IL) 16 , 17 . BAC clones were selected from the UCSC Genome Browser.…”

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

“…Blastocyst trophectoderm biopsy has been increasingly used and widely accepted in the PGS field due to its relatively low invasiveness, compared with performing blastomere biopsy at the cleavage stage. Chromosome screening on all 24 chromosomes has been mostly used on patients with advanced maternal age, recurrent pregnancy loss, repeated implantation failure, as well as on patients with abnormal karyotype such as balanced translocation and Robertsonian translocation (32)(33)(34)(35)(36)(37). Extensive use of PGS on all IVF/ICSI cycles has been hotly debated in the past few years (38)(39)(40)(41) due to the invasiveness of the biopsy procedure itself, particularly regarding the potential harm on the trophectoderm and possible compromise of implantation potential, as well as potential concerns on long-term effects on the offspring, which are very difficult to assess.…”

“…1). The MALBAC-NGS protocol has been previously validated in performing PGS with cleavage-stage and blastocyst-stage biopsies (29)(30)(31), and is increasingly used for single-gene PGD combined with chromosomal PGS (30,32). Similarly, we performed MALBAC-NGS on all 24 chromosomes from the corresponding D5 whole embryos, which we used as the gold standard to evaluate the chromosome screening results from the culture media.…”

Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human preimplantation embryos

“…However, while current NGS-based CCS methods are valuable, they are still limited to preimplantation genetic testing for aneuploidy. For example, CCS is not able to resolve whether an embryo with balanced ploidy has normal karyotype or is a carrier of a chromosomal translocation (18,19,21,24,25). Transferring a translocation-carrying embryo of balanced ploidy causes propagation of the translocation karyotype into the next generation, in addition to all the associated pregnancy risks (26,27).…”