title: Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.
abstract: OBJECTIVE
  To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR).
  
  
  METHODS
  Prospective and blinded.
  
  
  METHODS
  Not applicable.
  
  
  METHODS
  Couples indicated for preimplantation genetic diagnosis (PGD).
  
  
  METHODS
  None.
  
  
  METHODS
  Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening.
  
  
  RESULTS
  The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33).
  
  
  CONCLUSIONS
  This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification.
authors.affiliation: The Foundation for Embryonic Competence, Basking Ridge, New Jersey. Electronic address: rzimmerman@feclabs.org.
authors.forename: Rebekah S
authors.initials: RS
authors.lastname: Zimmerman
authors.affiliation: The Foundation for Embryonic Competence, Basking Ridge, New Jersey; Bonei Olam, New York, New York.
authors.forename: Chaim
authors.initials: C
authors.lastname: Jalas
authors.affiliation: The Foundation for Embryonic Competence, Basking Ridge, New Jersey.
authors.forename: Xin
authors.initials: X
authors.lastname: Tao
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey.
authors.forename: Anastasia M
authors.initials: AM
authors.lastname: Fedick
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey.
authors.forename: Julia G
authors.initials: JG
authors.lastname: Kim
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Pepe
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey.
authors.forename: Lesley E
authors.initials: LE
authors.lastname: Northrop
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey; Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, Basking Ridge, New Jersey.
authors.forename: Richard T
authors.initials: RT
authors.lastname: Scott
authors.affiliation: Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey; Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, Basking Ridge, New Jersey.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Treff
mesh_terms: D000782:Aneuploidy; D001706:Biopsy; D002869:Chromosome Aberrations; D004624:Embryo Transfer; D005260:Female; D005298:Fertility; D005307:Fertilization in Vitro; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007246:Infertility / Q000175:diagnosis / Q000503:physiopathology / Q000628:therapy*; D008297:Male; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011247:Pregnancy; D018873:Pregnancy Rate; D019836:Preimplantation Diagnosis / Q000379:methods*; D011446:Prospective Studies; D015203:Reproducibility of Results; D016896:Treatment Outcome
keywords: Single gene disorder; monogenic disorder; preimplantation genetic diagnosis; trophectoderm