title: Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles.
abstract: OBJECTIVE
  Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer?
  
  
  CONCLUSIONS
  Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies.
  
  
  BACKGROUND
  The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge.
  
  
  METHODS
  The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes.
  
  
  METHODS
  Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software.
  
  
  RESULTS
  A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies.
  
  
  CONCLUSIONS
  Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable.
  
  
  CONCLUSIONS
  This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available.
  
  
  BACKGROUND
  No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare.
  
  
  BACKGROUND
  Not applicable.
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy fiorentino@laboratoriogenoma.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Fiorentino
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Bono
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy.
authors.forename: Anil
authors.initials: A
authors.lastname: Biricik
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Nuccitelli
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Cotroneo
authors.affiliation: Molecular Genetics Laboratory, 'GENOMA', Via di Castel Giubileo, 11, 00138 Rome, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Cottone
authors.affiliation: Illumina, Inc., Cambridge, UK.
authors.forename: Felix
authors.initials: F
authors.lastname: Kokocinski
authors.affiliation: Illumina, Inc., Cambridge, UK.
authors.forename: Claude-Edouard
authors.initials: CE
authors.lastname: Michel
authors.affiliation: Reproductive Medicine, European Hospital, Via Portuense, 700, 00149 Rome, Italy.
authors.forename: Maria Giulia
authors.initials: MG
authors.lastname: Minasi
authors.affiliation: Reproductive Medicine, European Hospital, Via Portuense, 700, 00149 Rome, Italy.
authors.forename: Ermanno
authors.initials: E
authors.lastname: Greco
mesh_terms: D000328:Adult; D000782:Aneuploidy; D004311:Double-Blind Method; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D005260:Female; D015894:Genome, Human; D006801:Humans; D008875:Middle Aged; D019836:Preimplantation Diagnosis / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: array-comparative genomic hybridization; clinical outcomes; comprehensive chromosome screening; next-generation sequencing; preimplantation genetic screening