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SEMINAL CONTRIBUTION Development and validation of an accurate quantitative real-time polymerase chain reaction–based assay for human blastocyst comprehensive chromosomal aneuploidy screening Nathan R. Treff, Ph.D.,a,b Xin Tao, M.Sc.,a Kathleen M. Ferry, B.Sc.,a Jing Su, M.Sc.,a Deanne Taylor, Ph.D.,a and Richard T. Scott Jr., M.D., H.C.L.D.a,b a Reproductive Medicine Associates of New Jersey, Morristown; and b Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology, and Reproductive Science, University of Medicine and Dentistry of New Jersey– Robert Wood Johnson Medical School, New Brunswick, New Jersey Objective: To develop and validate a quantitative real-time polymerase chain reaction (qPCR)–based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy. Design: Prospective, randomized, and blinded. Setting: Academic center for reproductive medicine. Patient(s): Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening. Intervention(s): None. Main Outcome Measure(s): Consistency of qPCR diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarray- based diagnoses of human blastocysts. Result(s): Samples from nine cell lines with well characterized karyotypes were diagnosed by qPCR with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blas- tocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qPCR in 98.6% of cases (70/71). Overall euploidy (n ¼ 37) and aneuploidy (n ¼ 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours. Conclusion(s): These
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