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


      To validate a next-generation sequencing (NGS)–based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS).


      Retrospective blinded study.


      Reference laboratory.


      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.



      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.


      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%).


      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.

      Key Words

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        • Lathi R.B.
        • Westphal M.D.
        • Milki A.A.
        Aneuploidy in the miscarriages of infertile women and the potential benefit of preimplantation genetic diagnosis.
        Fertil Steril. 2008; 89: 353-357
        • Ebner T.
        • Moser M.
        • Sommergruber M.
        • Tews G.
        Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review.
        Hum Reprod Update. 2003; 9: 251-262
        • Munne S.
        • Wells D.
        • Cohen J.
        Technology requirements for preimplantation genetic diagnosis to improve assisted reproduction outcomes.
        Fertil Steril. 2010; 94: 408-430
        • Wilton L.
        Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review.
        Prenat Diagn. 2002; 22: 512-518
        • Mastenbroek S.
        • Twisk M.
        • van der Veen F.
        • Repping S.
        Preimplantation genetic screening: a systematic review and meta-analysis of RCTs.
        Hum Reprod Update. 2011; 17: 454-466
        • Rubio C.
        • Bellver J.
        • Rodrigo L.
        • Bosch E.
        • Mercader A.
        • Vidal C.
        • et al.
        Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials.
        Fertil Steril. 2013; 99: 1400-1407
        • Harper J.
        • Coonen E.
        • De Rycke M.
        • Fiorentino F.
        • Geraedts J.
        • Goossens V.
        • et al.
        What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee.
        Hum Reprod. 2010; 25: 821-823
        • Harper J.C.
        • Harton G.
        The use of arrays in PGD/PGS.
        Fertil Steril. 2010; 94: 1173-1177
        • Gutierrez-Mateo C.
        • Colls P.
        • Sanchez-Garcia J.
        • Escudero T.
        • Prates R.
        • Ketterson K.
        • et al.
        Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos.
        Fertil Steril. 2011; 95: 953-958
        • Fiorentino F.
        • Spizzichino L.
        • Bono S.
        • Biricik A.
        • Kokkali G.
        • Rienzi L.
        • et al.
        PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization.
        Hum Reprod. 2011; 26: 1925-1935
        • Fragouli E.
        • Wells D.
        • Whalley K.M.
        • Mills J.A.
        • Faed M.J.
        • Delhanty J.D.
        Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies.
        Cytogenet Genome Res. 2006; 114: 30-38
        • Wilton L.
        • Voullaire L.
        • Sargeant P.
        • Williamson R.
        • McBain J.
        Preimplantation aneuploidy screening using comparative genomic hybridization or fluorescence in situ hybridization of embryos from patients with recurrent implantation failure.
        Fertil Steril. 2003; 80: 860-868
        • Schoolcraft W.B.
        • Fragouli E.
        • Stevens J.
        • Munne S.
        • Katz-Jaffe M.G.
        • Wells D.
        Clinical application of comprehensive chromosomal screening at the blastocyst stage.
        Fertil Steril. 2010; 94: 1700-1706
        • Treff N.R.
        • Su J.
        • Tao X.
        • Levy B.
        • Scott Jr., R.T.
        Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays.
        Fertil Steril. 2010; 94: 2017-2021
        • Johnson D.S.
        • Gemelos G.
        • Baner J.
        • Ryan A.
        • Cinnioglu C.
        • Banjevic M.
        • et al.
        Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24 h protocol.
        Hum Reprod. 2010; 25: 1066-1075
        • Treff N.R.
        • Tao X.
        • Ferry K.M.
        • Su J.
        • Taylor D.
        • Scott Jr., R.T.
        Development and validation of an accurate quantitative real-time polymerase chain reaction–based assay for human blastocyst comprehensive chromosomal aneuploidy screening.
        Fertil Steril. 2012; 97: 819-824
        • Wells D.
        • Alfarawati S.
        • Fragouli E.
        Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH.
        Mol Hum Reprod. 2008; 14: 703-710
        • Treff N.R.
        • Levy B.
        • Su J.
        • Northrop L.E.
        • Tao X.
        • Scott Jr., R.T.
        SNP microarray–based 24 chromosome aneuploidy screening is significantly more consistent than FISH.
        Mol Hum Reprod. 2010; 16: 583-589
        • Scott Jr., R.T.
        • Ferry K.
        • Su J.
        • Tao X.
        • Scott K.
        • Treff N.R.
        Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study.
        Fertil Steril. 2012; 97: 870-875
        • Northrop L.E.
        • Treff N.R.
        • Levy B.
        • Scott Jr., R.T.
        SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.
        Mol Hum Reprod. 2010; 16: 590-600
        • Forman E.J.
        • Tao X.
        • Ferry K.M.
        • Taylor D.
        • Treff N.R.
        • Scott Jr., R.T.
        Single embryo transfer with comprehensive chromosome screening results in improved ongoing pregnancy rates and decreased miscarriage rates.
        Hum Reprod. 2012; 27: 1217-1222
        • Fragouli E.
        • Katz-Jaffe M.
        • Alfarawati S.
        • Stevens J.
        • Colls P.
        • Goodall N.N.
        • et al.
        Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure.
        Fertil Steril. 2010; 94: 875-887
        • Yang Z.
        • Liu J.
        • Collins G.S.
        • Salem S.A.
        • Liu X.
        • Lyle S.S.
        • et al.
        Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.
        Mol Cytogenet. 2012; 5: 24
        • Scott Jr., R.T.
        • Upham K.M.
        • Forman E.J.
        • Hong K.H.
        • Scott K.L.
        • Taylor D.
        • et al.
        Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.
        Fertil Steril. 2013; 100: 697-703
        • Handyside A.H.
        • Wells D.
        Single nucleotide polymorphisms and next generation sequencing.
        in: Gardner D.K. Sakkas D. Seli E. Wells D. Human gametes and preimplantation embryos: assessment and diagnosis. Springer Science Business Media, New York2013: 135-146
        • Handyside A.H.
        24-chromosome copy number analysis: a comparison of available technologies.
        Fertil Steril. 2013; 100: 595-602
        • Yin X.
        • Tan K.
        • Vajta G.
        • Jiang H.
        • Tan Y.
        • Zhang C.
        • et al.
        Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts.
        Biol Reprod. 2013; 88: 1-6
        • Fiorentino F.
        • Kokkali G.
        • Biricik A.
        • Stavrou D.
        • Ismailoglu B.
        • De Palma R.
        • et al.
        Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations.
        Fertil Steril. 2010; 94: 2001-2011
        • Knapp M.
        • Stiller M.
        • Meyer M.
        Generating barcoded libraries for multiplex high-throughput sequencing.
        Methods Mol Biol. 2012; 840: 155-170
        • Raczy C.
        • Petrovski R.
        • Saunders C.T.
        • Chorny I.
        • Kruglyak S.
        • Margulies E.H.
        • et al.
        iSAAC: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms.
        Bioinformatics. 2013; 29: 2041-2043
        • Quinlan A.R.
        • Hall I.M.
        BEDtools: A flexible suite of utilities for comparing genomic features.
        Bioinformatics. 2010; 26: 841-842
        • Li H.
        • Handsaker B.
        • Wysoker A.
        • Fennell T.
        • Ruan J.
        • Homer N.
        • et al.
        The sequence alignment/map (SAM) format and SAMtools.
        Bioinformatics. 2009; 25: 2078-2079
        • Li H.
        • Durbin R.
        Fast and accurate short read alignment with Burrows-Wheeler transform.
        Bioinformatics. 2009; 25: 1754-1760
        • Treff N.R.
        • Fedick A.
        • Tao X.
        • Devkota B.
        • Taylor D.
        • Scott Jr., R.T.
        Evaluation of targeted next-generation sequencing–based preimplantation genetic diagnosis of monogenic disease.
        Fertil Steril. 2013; 99: 1377-1384

      Linked Article

      • Next-generation sequencing: the dawn of a new era for preimplantation genetic diagnostics
        Fertility and SterilityVol. 101Issue 5
        • Preview
          The term next-generation sequencing (NGS) does not describe a single method, but rather encompasses a range of techniques, underpinned by several technologies, some of which differ significantly from others. The feature that unifies the various NGS strategies is that all have the capacity to deliver vast quantities of DNA sequence information from the samples analyzed, with data generated rapidly and at relatively low cost. To put the power of NGS in perspective, consider that the sequencing of the first human genome took approximately 13 years to complete and cost in excess of $100,000,000.
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