Carmen Rubio1, Luis Navarro-Sánchez2, Carmen M García-Pascual2, Olcay Ocali3, Danilo Cimadomo4, William Venier5, Gerardo Barroso6, Laura Kopcow7, Mustafa Bahçeci8, Marcos Iuri Roos Kulmann9, Lourdes López10, Emilio De la Fuente11, Roser Navarro12, Diana Valbuena13, Denny Sakkas3, Laura Rienzi4, Carlos Simón14. 1. Valencia, Spain. R&D Department, Igenomix. Electronic address: carmen.rubio@igenomix.com. 2. Valencia, Spain. R&D Department, Igenomix. 3. Boston, USA. IVF laboratory, Boston IVF. 4. Rome, Italy. Clinica Valle Giulia, Genera center for reproductive medicine. 5. San Diego, USA. IVF laboratory. San Diego Fertility Center. 6. Mexico, IVF Clinical department, Escuela Superior de Medicina Instituto Politécnico Nacional y Centro de Reproducción Arcos S.C. NASCERE. 7. Buenos Aires, Argentina, Reproductive Genetics Department, Pregna Medicina Reproductiva. 8. Istanbul, Turkey, Clinical department. Bahçeci Fertility. 9. Porto Alegre, Brazil. Embryology Department, Nilo Frantz Reproductive Medicine. 10. Madrid, Spain, IVF Laboratory. PROCREATEC by IVF Spain. 11. Valencia, Spain, Clinical Studies Department, Igenomix. 12. Valencia, Spain, Bioinformatics Department, Igenomix. 13. Valencia, Spain, Medical Department, Igenomix. 14. Valencia, Spain. Igenomix. Department of Pediatrics, Obstetrics and Gynecology, Universidad de Valencia. INCLIVA, Valencia, Spain. Department of Obstetrics and Gynecology, BIDMC, Harvard University, MA, USA.
Abstract
BACKGROUND: The recent identification of embryo cell-free DNA in the spent blastocyst media opened a new era of possibilities for non-invasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing limited numbers of embryos reported variable concordance between embryo cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE(S): 1) To evaluate the concordance and reproducibility of testing embryo cell-free DNA versus trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts. 2) To assess the contribution of the inner cell mass and/or trophectoderm to the embryo cell-free DNA released to the culture media. STUDY DESIGN: This is the interim analysis of a prospective, observational study among eight in vitro fertilization centers, on four continents, to assess consistency between non-invasive embryo aneuploidy testing of embryo cell-free DNA and conventional trophectoderm biopsy. Analysis included 1,301 day-6/7 blastocysts obtained in 406 IVF cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional PGT-A analysis was performed, comprising of trophectoderm biopsy and blastocyst vitrification. Embryo cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryo cell-free DNA analyses were 78.2% (866/1,108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Indeed, concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3%, and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1,108), and false-positive rate was 12.4% (137/1,108). Interestingly, the two fertilization techniques provided similar sensitivity (80.9% vs. 87.9%) and specificity (78.6% vs. 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryo cell-free DNA with trophectoderm and inner cell mass suggest that the embryo cell-free DNA originates from both compartments of the embryo. CONCLUSION(S): Non-invasive analysis of embryo cell-free DNA analyzed in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A non-invasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.
BACKGROUND: The recent identification of embryo cell-free DNA in the spent blastocyst media opened a new era of possibilities for non-invasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing limited numbers of embryos reported variable concordance between embryo cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE(S): 1) To evaluate the concordance and reproducibility of testing embryo cell-free DNA versus trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts. 2) To assess the contribution of the inner cell mass and/or trophectoderm to the embryo cell-free DNA released to the culture media. STUDY DESIGN: This is the interim analysis of a prospective, observational study among eight in vitro fertilization centers, on four continents, to assess consistency between non-invasive embryo aneuploidy testing of embryo cell-free DNA and conventional trophectoderm biopsy. Analysis included 1,301 day-6/7 blastocysts obtained in 406 IVF cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional PGT-A analysis was performed, comprising of trophectoderm biopsy and blastocyst vitrification. Embryo cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryo cell-free DNA analyses were 78.2% (866/1,108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Indeed, concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3%, and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1,108), and false-positive rate was 12.4% (137/1,108). Interestingly, the two fertilization techniques provided similar sensitivity (80.9% vs. 87.9%) and specificity (78.6% vs. 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryo cell-free DNA with trophectoderm and inner cell mass suggest that the embryo cell-free DNA originates from both compartments of the embryo. CONCLUSION(S): Non-invasive analysis of embryo cell-free DNA analyzed in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A non-invasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.
Authors: Katalin Gombos; Bence Gálik; Krisztina Ildikó Kalács; Krisztina Gödöny; Ákos Várnagy; Donát Alpár; József Bódis; Attila Gyenesei; Gábor L Kovács Journal: Int J Mol Sci Date: 2021-02-28 Impact factor: 5.923