Rebekah S Zimmerman1, Chaim Jalas2, Xin Tao3, Anastasia M Fedick4, Julia G Kim4, Russell J Pepe4, Lesley E Northrop4, Richard T Scott5, Nathan R Treff5. 1. The Foundation for Embryonic Competence, Basking Ridge, New Jersey. Electronic address: rzimmerman@feclabs.org. 2. The Foundation for Embryonic Competence, Basking Ridge, New Jersey; Bonei Olam, New York, New York. 3. The Foundation for Embryonic Competence, Basking Ridge, New Jersey. 4. Reproductive Medicine Associates of New Jersey, Basking Ridge, New Jersey. 5. 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.
Abstract
OBJECTIVE: To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). DESIGN: Prospective and blinded. SETTING: Not applicable. PATIENT(S): Couples indicated for preimplantation genetic diagnosis (PGD). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. RESULT(S): 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). CONCLUSION(S): 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.
OBJECTIVE: To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). DESIGN: Prospective and blinded. SETTING: Not applicable. PATIENT(S): Couples indicated for preimplantation genetic diagnosis (PGD). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. RESULT(S): 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). CONCLUSION(S): 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: Antonio Capalbo; Valeria Romanelli; Danilo Cimadomo; Laura Girardi; Marta Stoppa; Lisa Dovere; Domenico Dell'Edera; Filippo Maria Ubaldi; Laura Rienzi Journal: J Assist Reprod Genet Date: 2016-07-16 Impact factor: 3.412
Authors: Karen Sermon; Antonio Capalbo; Jacques Cohen; Edith Coonen; Martine De Rycke; Anick De Vos; Joy Delhanty; Francesco Fiorentino; Norbert Gleicher; Georg Griesinger; Jamie Grifo; Alan Handyside; Joyce Harper; Georgia Kokkali; Sebastiaan Mastenbroek; David Meldrum; Marcos Meseguer; Markus Montag; Santiago Munné; Laura Rienzi; Carmen Rubio; Katherine Scott; Richard Scott; Carlos Simon; Jason Swain; Nathan Treff; Filippo Ubaldi; Rita Vassena; Joris Robert Vermeesch; Willem Verpoest; Dagan Wells; Joep Geraedts Journal: Mol Hum Reprod Date: 2016-06-02 Impact factor: 4.025