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Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts

      Background

      The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach.

      Objective

      This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media.

      Study Design

      This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization 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 preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic 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

      Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. 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% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 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 embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo.

      Conclusion

      Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive 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.

      Key words

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      References

        • Lo Y.M.
        • Corbetta N.
        • Chamberlain P.F.
        • et al.
        Presence of fetal DNA in maternal plasma and serum.
        Lancet. 1997; 350: 485-487
        • Lefkowitz R.B.
        • Tynan J.A.
        • Liu T.
        • et al.
        Clinical validation of a noninvasive prenatal test for genomewide detection of fetal copy number variants.
        Am J Obstet Gynecol. 2016; 215: 227.e1-227.e16
        • Qiao L.
        • Yu B.
        • Liang Y.
        • et al.
        Sequencing shorter cfDNA fragments improves the fetal DNA fraction in noninvasive prenatal testing.
        Am J Obstet Gynecol. 2019; 221: 345.e1-345.e11
        • Bianchi D.W.
        • Parker R.L.
        • Wentworth J.
        • et al.
        DNA sequencing versus standard prenatal aneuploidy screening.
        N Engl J Med. 2014; 370: 799-808
        • Williams 3rd, J.
        • Rad S.
        • Beauchamp S.
        • et al.
        Utilization of noninvasive prenatal testing: impact on referrals for diagnostic testing.
        Am J Obstet Gynecol. 2015; 213: 102.e1-102.e6
        • Petersen A.K.
        • Cheung S.W.
        • Smith J.L.
        • et al.
        Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory.
        Am J Obstet Gynecol. 2017; 217: 691.e1-691.e6
        • Di Renzo G.C.
        • Bartha J.L.
        • Bilardo C.M.
        Expanding the indications for cell-free DNA in the maternal circulation: clinical considerations and implications.
        Am J Obstet Gynecol. 2019; 220: 537-542
        • Rubio C.
        • Rodrigo L.
        • Garcia-Pascual C.
        • et al.
        Clinical application of embryo aneuploidy testing by next-generation sequencing.
        Biol Reprod. 2019; 101: 1083-1090
        • Gleicher N.
        • Vidali A.
        • Braverman J.
        • et al.
        Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos.
        Reprod Biol Endocrinol. 2016; 14: 54
        • Shamonki M.I.
        • Jin H.
        • Haimowitz Z.
        • Liu L.
        Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.
        Fertil Steril. 2016; 106: 1312-1318
        • Feichtinger M.
        • Vaccari E.
        • Carli L.
        • et al.
        Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study.
        Reprod Biomed Online. 2017; 34: 583-589
        • Xu J.
        • Fang R.
        • Chen L.
        • et al.
        Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.
        Proc Natl Acad Sci USA. 2016; 113: 11907-11912
        • Vera-Rodriguez M.
        • Diez-Juan A.
        • Jimenez-Almazan J.
        • et al.
        Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.
        Hum Reprod. 2018; 33: 745-756
        • Ho J.R.
        • Arrach N.
        • Rhodes-Long K.
        • et al.
        Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos.
        Fertil Steril. 2018; 110: 467-475.e2
        • Yeung Q.S.Y.
        • Zhang Y.X.
        • Chung J.P.W.
        • et al.
        A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM).
        J Assist Reprod Genet. 2019; 36: 1609-1621
        • Huang L.
        • Bogale B.
        • Tang Y.
        • Lu S.
        • Xie X.S.
        • Racowsky C.
        Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.
        Proc Natl Acad Sci USA. 2019; 116: 14105-14112
        • Rubio C.
        • Rienzi L.
        • Navarro-Sánchez L.
        • et al.
        Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.
        Fertil Steril. 2019; 112: 510-519
        • Rubio C.
        • Bellver J.
        • Rodrigo L.
        • et al.
        In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.
        Fertil Steril. 2017; 107: 1122-1129
        • Li P.
        • Song Z.
        • Yao Y.
        • et al.
        Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization.
        Sci Rep. 2018; 8: 9275
        • Kuznyetsov V.
        • Madjunkova S.
        • Antes R.
        • et al.
        Evaluation of a novel non-invasive preimplantation genetic screening approach.
        PLoS One. 2018; 13e0197262
        • Jiao J.
        • Shi B.
        • Sagnelli M.
        • et al.
        Minimally invasive preimplantation genetic testing using blastocyst culture medium.
        Hum Reprod. 2019; 34: 1369-1379
        • Hammond E.R.
        • McGillivray B.C.
        • Wicker S.M.
        • et al.
        Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.
        Fertil Steril. 2017; 107: 220-228.e5
        • Belandres D.
        • Shamonki M.
        • Arrach N.
        Current status of spent embryo media research for preimplantation genetic testing.
        J Assist Reprod Genet. 2019; 36: 819-826
        • Minasi M.G.
        • Fabozzi G.
        • Casciani V.
        • et al.
        Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on 1128 sibling human zygotes.
        J Assist Reprod Genet. 2015; 32: 215-220
        • Tannus S.
        • Cohen Y.
        • Henderson S.
        • et al.
        Fresh transfer of day 5 slow-growing embryos versus deferred transfer of vitrified, fully expanded day 6 blastocysts: which is the optimal approach?.
        Hum Reprod. 2019; 34: 44-51
        • Bourdon M.
        • Pocate-Cheriet K.
        • Finet de Bantel A.
        • et al.
        Day 5 versus day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes.
        Hum Reprod. 2019; 34: 1948-1964
        • Tiegs A.W.
        • Sun L.
        • Patounakis G.
        • Scott R.T.
        Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as day 5 and day 6 blastocysts.
        Hum Reprod. 2019; 34: 1632-1639

      Supplemental References

        • Gardner D.K.
        • Stevens J.
        • Sheehan C.B.
        • Schoolcraft W.B.
        Analysis of blastocyst morphology.
        in: Elder K. Cohen J. Human Preimplantation Embryo Selection. Informa Healthcare, London2007: 79-87
        • Rubio C.
        • Rienzi L.
        • Navarro-Sánchez L.
        • et al.
        Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.
        Fertil Steril. 2019; 112: 510-519
        • Capalbo A.
        • Rienzi L.
        Mosaicism between trophectoderm and inner cell mass.
        Fertil Steril. 2017; 107: 1098-1106