Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos
Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos
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DOI：
10.1016/j.fertnstert.2014.01.051
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发表时间：
2014-05-01
影响因子：
6.7
通讯作者：
Michel, Claude-Edouard
中科院分区：
文献类型：
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作者：
Fiorentino, Francesco;Biricik, Anil;Michel, Claude-Edouard
目的：验证基于下一代测序（NGS）的24-染色体非整倍体筛查方法，并研究其对植入前遗传筛查（PGS）的适用性。设计：回顾性盲法研究。设置：参考实验室。患者：核型确定的染色体异常单细胞和全基因组扩增（WGA）产物，先前通过阵列比较基因组杂交分析（阵列CGH），选自68个临床PGS周期，胚胎在卵裂期活检。干预：无。主要结果测量：基于NGS的非整倍体诊断与单细胞常规核型分析或单卵裂球阵列CGH诊断的一致性。结果：用基于NGS的方案对来自单卵裂球的18个单细胞和190个WGA产物进行盲法评价。总共评估了4，992条染色体，其中402条携带拷贝数不平衡。NGS对非整倍体判定的特异性（染色体拷贝数分配的一致性）为99.98%（95%置信区间[CI] 99.88%-100%），灵敏度为100%（95% CI 99.08%-100%）。NGS对非整倍体胚胎的特异性（24-染色体诊断一致性）为100%（95%CI 94.59%-100%），灵敏度为100%（95%CI 97.39%-100%）。鉴于与已建立的方法（如阵列CGH）的高度一致性，NGS已经证明了一种强大的高通量方法，可用于生殖医学的临床应用，具有降低成本和提高精度的潜在优势。(C)2014年，美国生殖医学会（American Society for Reproductive Medicine）
Objective: To validate a next-generation sequencing (NGS)-based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS).Design: Retrospective blinded study.Setting: Reference laboratory.Patient(s): Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage.Intervention(s): None.Main Outcome Measure(s): Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres.Result(s): Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%-100%) with a sensitivity of 100% (95% CI 99.08%-100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%-100%) with a sensitivity of 100% (95% CI 97.39%-100%).Conclusion(s): This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision. (C) 2014 by American Society for Reproductive Medicine.