Noninvasive preimplantation genetic testing for aneuploidy in spent culture medium as a substitute for trophectoderm biopsy
2021; Elsevier BV; Volume: 115; Issue: 4 Linguagem: Inglês
10.1016/j.fertnstert.2021.02.045
ISSN1556-5653
AutoresCarmen Rubio, Catherine Racowsky, David H. Barad, Richard T. Scott, Carlos Simón,
Tópico(s)Genetic Syndromes and Imprinting
ResumoSince the first study that identified embryonic cfDNA in spent blastocyst media (1Shamonki 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: 1312-1318Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), its potential as a new noninvasive preimplantation genetic test for aneuploidy (niPGT-A) to assess embryo chromosome copy number has become evident. Since then, several studies have explored the concordance between the copy number of this cfDNA and amplified DNA obtained from polar bodies (2Feichtinger M. Vaccari E. Carli L. Wallner E. Mädel U. Figl K. 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: 583-589Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), trophectoderm biopsies (teBxs) (3Ho J.R. Arrach N. Rhodes-Long K. Ahmady A. Ingles S. Chung K. et al.Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos.Fertil Steril. 2018; 110: 467-475Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 4Vera-Rodriguez M. Diez-Juan A. Jimenez-Almazan J. Martinez S. Navarro R. Peinado V. et al.Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.Hum Reprod. 2018; 33: 745-756Crossref PubMed Scopus (69) Google Scholar, 5Yeung Q.S.Y. Zhang Y.X. Chung J.P.W. Lui W.T. Kwok Y.K.Y. Gui B. 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: 1609-1621Crossref PubMed Scopus (28) Google Scholar, 6Rubio C. Rienzi L. Navarro-Sánchez L. Cimadomo D. García-Pascual C.M. Albricci L. et al.Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.Fertil Steril. 2019; 112: 510-519Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar, 8Lledó B, Morales R, Ortiz JA, Rodriguez-Arnedo A, Ten J, Castillo JC, et al. Consistent results of non-invasive pre-implantation genetic testing for aneuploidy (niPGT-A) of human embryos using two different techniques for chromosomal analysis. Reprod Biomed Online. In press.Google Scholar, 9Rubio C. Navarro-Sánchez L. García-Pascual C.M. Ocali O. Cimadomo D. Venier W. et al.Multicenter prospective study of concordance between embryo cell-free DNA and trophectoderm biopsies from 1,301 human blastocysts.Am J Obstet Gynecol. 2020; 223: 751.e1-751.e13Abstract Full Text Full Text PDF Scopus (22) Google Scholar), and whole blastocysts (3Ho J.R. Arrach N. Rhodes-Long K. Ahmady A. Ingles S. Chung K. et al.Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos.Fertil Steril. 2018; 110: 467-475Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar, 10Xu J. Fang R. Chen L. Chen D. Xiao J.P. Yang W. 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: 11907-11912Crossref PubMed Scopus (100) Google Scholar, 11Yin B, Zhang H, Xie J, Yao Y, Wei Y, Zhang C, et al. Validation of preimplantation genetic tests for aneuploidy (PGT-A) with DNA from spent culture media (SCM): concordance assessment and implication. Reprod Biol Endocrinol. In press.Google Scholar). The reported informativity rates of cfDNA in terms of successful amplification and interpretable results using next-generation sequencing (NGS) ranged between 80% and 100%. The concordance rates of cfDNA with DNA from teBxs varied widely, up to 89.1% for thawed, biopsied blastocysts (7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar). Interestingly, the cfDNA concordance rate with the DNA of whole blastocysts reached 93.8% (7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar). This result strongly suggested that embryo cfDNA is robust and can be more representative of embryo chromosomal status than teBxs (7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar). Further, our recent study (9Rubio C. Navarro-Sánchez L. García-Pascual C.M. Ocali O. Cimadomo D. Venier W. et al.Multicenter prospective study of concordance between embryo cell-free DNA and trophectoderm biopsies from 1,301 human blastocysts.Am J Obstet Gynecol. 2020; 223: 751.e1-751.e13Abstract Full Text Full Text PDF Scopus (22) Google Scholar) obtained concordance rates of cfDNA similar to those of teBx and inner cell mass (ICM) biopsy (87.5% and 84.5%, respectively). Apoptosis during preimplantation development has been proposed as a potential mechanism for embryo DNA shedding because of the embryo’s rapid, dynamic transformation during late preimplantation development (12Hardy K. Spanos S. Becker D. Iannelli P. Winston R.M. Stark J. From cell death to embryo arrest: mathematical models of human preimplantation embryo development.Proc Natl Acad Sci USA. 2001; 98: 1655-1660Crossref PubMed Scopus (156) Google Scholar). Apoptotic DNA is compromised because of the degradation processes and, therefore, does not provide robust diagnostic information. However, cfDNA exhibits high concordance with teBxs and whole blastocysts. Further, in cases of low-quality blastocysts (e.g., aneuploids), high degrees of apoptosis are expected to lead to shedding of high quantities of DNA. However, this is not the case; the informativity and concordance rates in moderate- or low-quality blastocysts are similar to those of good-quality blastocysts (13Kuznyetsov V. Madjunkova S. Abramov R. Antes R. Ibarrientos Z. Motamedi G. et al.Minimally invasive cell-free human embryo aneuploidy testing (miPGT-A) utilizing combined spent embryo culture medium and blastocoel fluid—towards development of a clinical assay.Sci Rep. 2020; 10: 1-2Crossref PubMed Scopus (11) Google Scholar). In a recent study, we obtained similar concordance rates of cfDNA with teBx and ICM biopsy (87.5% and 84.5%, respectively) (9Rubio C. Navarro-Sánchez L. García-Pascual C.M. Ocali O. Cimadomo D. Venier W. et al.Multicenter prospective study of concordance between embryo cell-free DNA and trophectoderm biopsies from 1,301 human blastocysts.Am J Obstet Gynecol. 2020; 223: 751.e1-751.e13Abstract Full Text Full Text PDF Scopus (22) Google Scholar), in addition to similar DNA quantity in media from euploid and aneuploid blastocysts, as observed previously (4Vera-Rodriguez M. Diez-Juan A. Jimenez-Almazan J. Martinez S. Navarro R. Peinado V. et al.Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.Hum Reprod. 2018; 33: 745-756Crossref PubMed Scopus (69) Google Scholar). Taken together, these findings instead supported the hypothesis that cfDNA secretion is a physiologic phenomenon for each embryo, independent of their quality and ploidy status and not related to self-correction by apoptotic events. Noninvasive tests are the ultimate goal in medicine, and indeed, noninvasive prenatal testing has changed the practice of prenatal medicine. In in vitro fertilization (IVF) programs, incorporating preimplantation genetic test for aneuploidy (PGT-A) improves ongoing pregnancy rates per transfer and shortens the time to pregnancy while decreasing miscarriage rates in couples experiencing infertility (14Neal S.A. Morin S.J. Franasiak J.M. Goodman L.R. Juneau C.R. Forman E.J. et al.Preimplantation genetic testing for aneuploidy is cost-effective, shortens treatment time, and reduces the risk of failed embryo transfer and clinical miscarriage.Fertil Steril. 2018; 110: 896-904Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Somigliana E. Busnelli A. Paffoni A. Vigano P. Riccaboni A. Rubio C. Capalbo A. Cost-effectiveness of preimplantation genetic testing for aneuploidies.Fertil Steril. 2019; 111: 1169-1176Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, the current practice for PGT-A is invasive, relying on embryo manipulation (teBX) that requires dedicated equipment and highly trained personnel and, therefore, encompasses substantial commitment in terms of time and cost. Moreover, recent studies have associated teBx with a significant increase in pre-eclampsia (16Zhang W.Y. von Versen-Höynck F. Kapphahn K.I. Fleischmann R.R. Zhao Q. Baker V.L. Maternal and neonatal outcomes associated with trophectoderm biopsy.Fertil Steril. 2019; 112: 283-290Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and hypertensive disorders (17Makhijani R. Bartels C.B. Godiwala P. Bartolucci A. DiLuigi A. Nulsen J. et al.Impact of trophectoderm biopsy on obstetric and perinatal outcomes following frozen–thawed embryo transfer cycles.Hum Reprod. 2021; 36: 340-348Crossref PubMed Scopus (11) Google Scholar). An efficient and reliable noninvasive approach could offer the benefits of invasive PGT-A without the accompanying technical and financial burdens. Further, the noninvasive approach could be offered as a prioritization model, in which the testing of cfDNA in culture media, in combination with morphology, could enable “ranking” of viable blastocysts, most likely resulting in a healthy baby. This model can promote blastocyst prioritization so that providers and couples can select the best blastocyst for the first embryo transfer and reserve the others (rather than discard) for subsequent transfers. With advances in scientific and technical knowledge, research on biomedical microelectromechanical systems is also developing toward lab-on-a-chip devices. The niPGT-A approach might lead to a part of IVF development moving toward automation because the additional steps required to remove cumulus cell contamination and collect a spent culture medium can be easily automated. Several publications have provided evidence for the fact that embryonic cfDNA of blastocysts cultured under different conditions yields blastocyst concordance rates similar to those of teBxs (9Rubio C. Navarro-Sánchez L. García-Pascual C.M. Ocali O. Cimadomo D. Venier W. et al.Multicenter prospective study of concordance between embryo cell-free DNA and trophectoderm biopsies from 1,301 human blastocysts.Am J Obstet Gynecol. 2020; 223: 751.e1-751.e13Abstract Full Text Full Text PDF Scopus (22) Google Scholar). However, a strong objection is that an expanded culture till day 6 is required to obtain sufficient cfDNA quantity for robust results (6Rubio C. Rienzi L. Navarro-Sánchez L. Cimadomo D. García-Pascual C.M. Albricci L. et al.Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.Fertil Steril. 2019; 112: 510-519Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Meyers J. Laliberte J. Choudhary K. Yasuyama N. Sifuentes C.J. Xianget W. et al.Assessment of cell-free DNA from embryo spent media and its effects on non-invasive PGT-A concordance to conventional PGT-A and calculated copy number noise.Fertil Steril. 2020; 114: e422Abstract Full Text Full Text PDF Google Scholar). Accumulated evidence has indicated using aneuploidy testing that vitrified day-6 blastocysts have ongoing pregnancy rates similar to those of day-5 blastocysts (19Kaye L. Anspach E. Bartolucci A. Nulsen J. Benadiva C. Engmann L. Pregnancy rates for single embryo transfer (SET) of day 5 and day 6 blastocysts after cryopreservation by vitrification and slow freeze.J Assist Reprod Genet. 2017; 34: 913-919Crossref PubMed Scopus (22) Google Scholar). Tiegs et al. (20Tiegs A.W. Sun L. Patounakis G. Scott Jr., 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: 1632-1639Crossref PubMed Scopus (24) Google Scholar) showed no differences in sustained implantation rates between day-5 and day-6 single embryo transfers in PGT-A cases, with deferred embryo transfer. This was confirmed by a meta-analysis that concluded using PGT-A that implantation, clinical pregnancy, live birth, and ongoing pregnancy rates were similar for euploid day-5 and day-6 blastocyst transfers (21Bourdon M. Pocate-Cheriet K. de Bantel A.F. Grzegorczyk-Martin V. Hoffet A.A. Arboet E. et al.Day 5 versus day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes.Hum Reprod. 2019; 34: 1948-1964Crossref PubMed Scopus (26) Google Scholar). These results align with those of previous reports that used PGT-A, with similar outcomes for day-5 or day-6 euploid transfers (22Cimadomo D. Soscia D. Vaiarelli A. Maggiulli R. Capalbo A. Ubaldi F.P. et al.Looking past the appearance: a comprehensive description of the clinical contribution of poor-quality blastocysts to increase live birth rates during cycles with aneuploidy testing.Hum Reprod. 2019; 34: 1206-1214Crossref PubMed Scopus (22) Google Scholar, 23Sacchi L. Albani E. Cesana A. Smeraldi A. Parini V. Fabiani M. et al.Preimplantation genetic testing for aneuploidy improves clinical, gestational, and neonatal outcomes in advanced maternal age patients without compromising cumulative live-birth rate.J Assist Reprod Genet. 2019; 36: 2493-2504Crossref PubMed Scopus (23) Google Scholar, 24Rubino P. Tapia L. Ruiz de Assin R. Mazmanian K. Guan L. Dearden L. et al.Trophectoderm biopsy protocols can affect clinical outcomes: time to focus on the blastocyst biopsy technique.Fertil Steril. 2020; 113: 981-989Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 25Friedenthal J. Maxwell S.M. Tiegs A.W. Besser A.G. McCaffrey C. Munné S. et al.Clinical error rates of next generation sequencing and array comparative genomic hybridization with single thawed euploid embryo transfer.Eur J Med Genet. 2020; 63: 103852Crossref PubMed Scopus (8) Google Scholar). A recent study evaluating the effect of the day of cryopreservation showed that in frozen transfers of euploid embryos assessed by PGT-A, the day of cryopreservation did not significantly affect live birth rates (26Shear M.A. Vaugham D.A. Modest A.M. Seidler E.A. Leung A.Q. Hacker M.R. et al.Blast from the past: is morphology useful in PGT-A tested and untested frozen embryo transfers?.Reprod Biomed Online. 2020; 41: 981-989Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The remaining question is the potential for this technology to improve its reproductive outcome compared with that of standard IVF. Class I evidence should be obtained from randomized trials comparing the efficacy of niPGT-A with that of current IVF practice without PGT-A for different patient age ranges. In addition, nonselection studies can help reveal the reproductive potential of different patterns of results obtained after cfDNA sequencing. Such data can help inform a scoring system for each pattern according to its capability of resulting in a healthy live birth so as to facilitate its use in clinics. Following the first report of successful live births after the transfer of embryos selected based on an analysis of cfDNA in a culture medium (10Xu J. Fang R. Chen L. Chen D. Xiao J.P. Yang W. 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: 11907-11912Crossref PubMed Scopus (100) Google Scholar), numerous studies have shown the high reliability of detection and amplification of cfDNA for niPGT-A (27Brouillet S. Martinez G. Coutton C. Hamamah S. Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review.Reprod Biomed Online. 2020; 40: 779-796Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). However, the assessment of copy number variation may be hindered in some cfDNA samples because of noisy profiles (6Rubio C. Rienzi L. Navarro-Sánchez L. Cimadomo D. García-Pascual C.M. Albricci L. et al.Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.Fertil Steril. 2019; 112: 510-519Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar, 13Kuznyetsov V. Madjunkova S. Abramov R. Antes R. Ibarrientos Z. Motamedi G. et al.Minimally invasive cell-free human embryo aneuploidy testing (miPGT-A) utilizing combined spent embryo culture medium and blastocoel fluid—towards development of a clinical assay.Sci Rep. 2020; 10: 1-2Crossref PubMed Scopus (11) Google Scholar) and/or contamination due to either the culture medium’s constituents (4Vera-Rodriguez M. Diez-Juan A. Jimenez-Almazan J. Martinez S. Navarro R. Peinado V. et al.Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.Hum Reprod. 2018; 33: 745-756Crossref PubMed Scopus (69) Google Scholar, 28Hammond E.R. McGillvary B.C. Wicker S.M. Peek J.C. Shelling S.N. Stone P. et al.Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.Fertil Steril. 2017; 107: 220-228Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 29Li P. Song Z. Yao Y. Huang T. Mao R. Huang J. et al.Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization.Sci Rep. 2018; 8: 9275Crossref PubMed Scopus (25) Google Scholar) or cumulus cells (2Feichtinger M. Vaccari E. Carli L. Wallner E. Mädel U. Figl K. 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: 583-589Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Vera-Rodriguez M. Diez-Juan A. Jimenez-Almazan J. Martinez S. Navarro R. Peinado V. et al.Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.Hum Reprod. 2018; 33: 745-756Crossref PubMed Scopus (69) Google Scholar, 30Capalbo A. Ubaldi F.M. Rienzi L. Scott R. Treff N. Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities.Hum Reprod. 2017; 32: 492-498PubMed Google Scholar). Although typically, >95% of teBxs provide informative results (31Leaver M. Wells D. Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?.Hum Reprod Update. 2020; 26: 16-42Crossref PubMed Scopus (37) Google Scholar), NGS studies of cells obtained from disaggregated blastocysts indicated that approximately 50% of embryos may exhibit some level of mosaicism either among trophectoderm (TE) cells and/or between the ICM and TE lineage (32Popovic M. Dhaenens L. Boel A. Menten B. Heindryckx B. Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma.Hum Reprod Update. 2020; 26: 313-334Crossref PubMed Scopus (46) Google Scholar). Because only a few TE cells were analyzed, mosaicism inherently confounded the diagnostic accuracy of invasive PGT-A with teBx (iPGT-A) (33Paulson R.J. Preimplantation genetic screening: What is the clinical efficiency?.Fertil Steril. 2017; 108: 228-230Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), as highlighted by numerous reports on healthy births resulting from the transfer of embryos that were classified as “abnormal” based on iPGT-A (34Greco E. Minasi M.G. Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.N Engl J Med. 2015; 373: 2089-2090Crossref PubMed Scopus (284) Google Scholar, 35Patrizio P. Shoham G. Shoham Z. Leong M. Barad D.H. Gleicher N. Worldwide live births following the transfer of chromosomally “Abnormal” embryos after PGT/A: results of a worldwide web-based survey.J Assist Reprod Genet. 2019; 36: 1599-1607Crossref PubMed Scopus (36) Google Scholar). Because of the potential for genetic discordance between ICM and TE, the evaluation of the relative reliability of niPGT-A compared with that of iPGT-A for determining the chromosomal constitution of ICM should use the corresponding embryo or ICM as a control gold standard. When data from 4 such available studies assessing cfDNA in a spent culture medium with or without blastocyst fluid (BF) were combined (7Huang 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: 14105-14112Crossref PubMed Scopus (58) Google Scholar, 29Li P. Song Z. Yao Y. Huang T. Mao R. Huang J. et al.Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization.Sci Rep. 2018; 8: 9275Crossref PubMed Scopus (25) Google Scholar, 36Kuznyetsov V. Madjunkova S. Antes R. Abramov R. Motamedi G. et al.Evaluation of a novel non-invasive preimplantation genetic screening approach.PLoS One. 2018; 13e0197262Crossref PubMed Scopus (46) Google Scholar, 37Chen J, Jia L, Li T, Guo Y, He S, Zhang Z, et al. Diagnostic efficiency of blastocyst culture medium in noninvasive preimplantation genetic testing (niPGT). F S Rep. In press.Google Scholar; Supplemental Table 1 [available online]), the false-positive rate (FPR) was significantly improved for niPGT-A versus that for iPGT-A (P = .042) (Table 1). When only 3 studies in which the medium was collected after blastocyst culture (day 5 or 6 or day 6 or 7) (29Li P. Song Z. Yao Y. Huang T. Mao R. Huang J. et al.Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization.Sci Rep. 2018; 8: 9275Crossref PubMed Scopus (25) Google Scholar, 36Kuznyetsov V. Madjunkova S. Antes R. Abramov R. Motamedi G. et al.Evaluation of a novel non-invasive preimplantation genetic screening approach.PLoS One. 2018; 13e0197262Crossref PubMed Scopus (46) Google Scholar, 37Chen J, Jia L, Li T, Guo Y, He S, Zhang Z, et al. Diagnostic efficiency of blastocyst culture medium in noninvasive preimplantation genetic testing (niPGT). F S Rep. In press.Google Scholar) were analyzed, the performance of niPGT-A was superior to that of iPGT-A in terms of FPR (P = .008), positive predictive value (P = .034), and concordance for both embryo ploidy (P = .039) and chromosome copy number variation (P = .024) (Table 2). These data indicated that niPGT-A was less prone to errors associated with embryo mosaicism and was more reliable than iPGT-A, a conclusion consistent with the 3-fold increase in ongoing pregnancy rate (>20-week gestation) for transferred embryos classified as euploid based on both niPGT-A and iPGT-A versus those classified as euploid based on iPGT-A but classified as aneuploid based on niPGT-A (6Rubio C. Rienzi L. Navarro-Sánchez L. Cimadomo D. García-Pascual C.M. Albricci L. et al.Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.Fertil Steril. 2019; 112: 510-519Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Table 1Comparison of the performance of niPGT-A and iPGT-A for PGT-A, regardless of the days of culture before sample collection.Performance characteristicniPGT-A (n = 139)iPGT-A (n = 138)P valueFPR24.4% (10/41)46.5% (20/43).042FNR3.1% (3/98)2.1% (2/95)1.000PPV90.5% (95/105)82.3% (93/113).115NPV91.9% (34/37)94.1% (32/34)1.000Sensitivity96.9% (95/98)97.9% (93/95)1.000Specificity75.6% (31/41)60.5% (26/43).165Concordance for embryo ploidy90.6% (126/139)84.1% (116/138).107Concordance for whole chromosome CN71.9% (100/139)67.4% (93/138).233Note: The comparison includes all 4 studies assessing the performance characteristics of niPGT-A and iPGT-A on whole embryo from samples (spent medium with or without BF) after culture from day 3 to 5, day 5 to 6, or day 6 to 7. P values were derived using the chi-square and Fisher’s exact tests, with P <.05 considered statistically significant. BF = blastocyst fluid; CN = copy number; FNR = false-negative rate; FPR = false-positive rate; iPGT-A = invasive preimplantation genetic test for aneuploidy; niPGT-A = noninvasive preimplantation genetic test for aneuploidy; NPV = negative predictive value; PPV = positive predictive value; PGT-A = preimplantation genetic testing for aneuploidy. Open table in a new tab Table 2Comparison of the performance of niPGT-A and iPGT-A for PGT-A using samples collected following blastocyst culture.Performance characteristicniPGT-A (n = 102)iPGT-A (n = 100)P valueFPR21.7% (5/23)62.5% (15/24).008FNR1.3% (1/79)0.0% (0/76)1.000PPV94.0% (78/83)83.5% (76/91).034NPV95.5% (21/22)100.0% (18/18)1.000Sensitivity98.7% (78/79)100.0% (76/76)1.000Specificity78.3% (18/23)50.0% (12/24).069Concordance for embryo ploidy94.1% (96/102)85.0% (85/100).039Concordance for whole chromosome CN81.4% (83/102)67.0% (67/100).024Note: The comparison includes 3 studies assessing the performance characteristics of niPGT-A and iPGT-A on whole embryo after the collection of samples (spent medium with or without BF) at the blastocyst stage (culture day 5 to 6 or day 6 to 7). P values were derived using the chi-square and Fisher’s exact tests, with P <.05 considered statistically significant. BF = blastocyst fluid; CN = copy number; FNR = false-negative rate; FPR = false-positive rate; iPGT-A = invasive preimplantation genetic test for aneuploidy; niPGT-A = noninvasive preimplantation genetic test for aneuploidy; NPV = negative predictive value; PPV = positive predictive value; PGT-A = preimplantation genetic testing for aneuploidy. Open table in a new tab Note: The comparison includes all 4 studies assessing the performance characteristics of niPGT-A and iPGT-A on whole embryo from samples (spent medium with or without BF) after culture from day 3 to 5, day 5 to 6, or day 6 to 7. P values were derived using the chi-square and Fisher’s exact tests, with P <.05 considered statistically significant. BF = blastocyst fluid; CN = copy number; FNR = false-negative rate; FPR = false-positive rate; iPGT-A = invasive preimplantation genetic test for aneuploidy; niPGT-A = noninvasive preimplantation genetic test for aneuploidy; NPV = negative predictive value; PPV = positive predictive value; PGT-A = preimplantation genetic testing for aneuploidy. Note: The comparison includes 3 studies assessing the performance characteristics of niPGT-A and iPGT-A on whole embryo after the collection of samples (spent medium with or without BF) at the blastocyst stage (culture day 5 to 6 or day 6 to 7). P values were derived using the chi-square and Fisher’s exact tests, with P <.05 considered statistically significant. BF = blastocyst fluid; CN = copy number; FNR = false-negative rate; FPR = false-positive rate; iPGT-A = invasive preimplantation genetic test for aneuploidy; niPGT-A = noninvasive preimplantation genetic test for aneuploidy; NPV = negative predictive value; PPV = positive predictive value; PGT-A = preimplantation genetic testing for aneuploidy. Cell-free DNA secretion is related to ploidy self-correction; this is supported by the fact that clinical and ongoing pregnancy rates were higher following the transfer of embryos classified as euploid based on iPGT-A but without cfDNA detected in their BF compared with the transfer of those with BF cfDNA detected (38Magli M.C. Albanese C. Crippa A. Tabanelli C. Ferraretti A.P. Gianaroli L. Deoxyribonucleic acid detection in blastocoelic fluid: a new predictor of embryo ploidy and viable pregnancy.Fertil Steril. 2019; 111: 77-85Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and that in a murine model (39Bolton H. Graham S.J.L. Van der Aa N. Kumar P. Theunis K. Gallardo E.F. et al.Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.Nat Commun. 2016; 7: 11165Crossref PubMed Scopus (190) Google Scholar), the frequency of apoptosis in ICM of aneuploid cell clones was increased compared with that of ICM of euploid clones (41.4% vs. 19.5%, respectively, P<.0001); both the frequencies were much higher than those in their TE counterparts (3.3% vs. 0.6% for euploid and aneuploid clones, respectively, P<.001). The apoptotic origin of cfDNA was supported by the finding that BF cfDNA comprises 2 peaks, a dominant peak of 160–220 bas
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