Validation of Copy Number Variation Sequencing for Detecting Chromosome Imbalances in Human Preimplantation Embryos1

Abstract

Chromosome aneuploidies commonly arise in embryos produced by assisted reproductive technologies and represent a major cause of implantation failure and miscarriage. Currently, preimplantation genetic diagnosis (PGD) is performed by array-based methods to identify euploid embryos for transfer to the patient. We speculated that a combination of next-generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing. To meet this challenge, we developed a high-resolution copy number variation (CNV) sequencing pipeline suitable for single-cell analysis. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy, and segmental imbalances in 24 whole genome amplification samples from PGD embryos that were originally diagnosed by gold standard array comparative genomic hybridization. In addition, CNV-Seq was capable of detecting, mapping, and accurately quantifying terminal chromosome imbalances down to 1 Mb in size originating from abnormal segregation of translocation chromosomes. These validation studies indicate that CNV-Seq displays the hallmarks of an accurate and reliable embryo test with the potential to further improve the overall efficacy of PGD.

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations

“…In vitro fertilization (IVF) and embryo biopsy procedures were performed for 21 patients at the Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing, using previously published procedures [19]. In brief, fertilization of MII oocytes was achieved by intracytoplasmic sperm injection of a single sperm.…”

“…In their first IVF and PGD cycle, all couples produced a cohort of embryos for PGD (Supplementary Table 1). Biopsy samples were amplified by WGA and embryonic DNA products subjected to NGS using CNV-Seq technology [19].…”

“…Further, in those embryos that are diagnosed as balanced, there is the possibility of additional incidental aneuploidies, including whole or partial chromosomal gains and losses, mosaicism, and segmental imbalances in other chromosomes that are known to commonly arise by either non-disjunction errors [15] or breakage-fusion-bridge cycles [16]. So far, the technology developed for PGD of translocations has primarily focused on the detection of unbalanced chromosome derivatives and incidental whole or partial chromosomal aneuploidies using 24-chromosome testing either by chromosome microarray analysis [11,12] or next-generation sequencing (NGS) [13,[17][18][19][20]. We speculated that a higherresolution technology with the capacity to additionally detect more subtle chromosomal abnormalities might further benefit patients undertaking PGD cycles for translocations.…”

“…Recently, we have developed a novel quantitative NGS method termed copy number variation sequencing (CNVSeq) for fine analysis of embryonic chromosomes [19]. In a series of validation studies using cell lines with terminal deletions associated with chromosome disease syndromes [21], as well as whole genome amplification (WGA) products with known segmental translocation imbalances defined by array comparative genomic hybridization (CGH) [19,22], we demonstrated that all the small CNVs tested in the range of 1-5 Mb could be reliably, accurately, and reproducibly detected.…”

“…In a series of validation studies using cell lines with terminal deletions associated with chromosome disease syndromes [21], as well as whole genome amplification (WGA) products with known segmental translocation imbalances defined by array comparative genomic hybridization (CGH) [19,22], we demonstrated that all the small CNVs tested in the range of 1-5 Mb could be reliably, accurately, and reproducibly detected. In addition, we have further validated CNV-Seq for the measurement of mosaicism in trophectoderm biopsy samples [23].…”