Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos

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• 作者：Francesco Fiorentino ; Ph.D.^a ; ^{fiorentino@laboratoriogenoma.it" class="auth_mail} ; Anil Biricik ; M.Sc.^a ; Sara Bono ; B.Sc.^a ; Letizia Spizzichino ; B.Sc.^a ; Ettore Cotroneo ; B.Sc.^a ; Giuliano Cottone ; B.Sc.^a ; Felix Kokocinski ; Ph.D.^b ; Claude-Edouard Michel ; Ph.D.^b
• 关键词：Comprehensive chromosome screening ; preimplantation genetic screening ; array comparative genomic hybridization ; next-generation sequencing ; embryo aneuploidy
• 刊名：Fertility and Sterility
• 出版年：May 2014
• 年：2014
• 卷：101
• 期：5
• 页码：1375-1382.e2
• 全文大小：1628 K

文摘

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.