The “mosaic” embryo: misconceptions and misinterpretations in preimplantation genetic testing for aneuploidy
2021; Elsevier BV; Volume: 116; Issue: 5 Linguagem: Inglês
10.1016/j.fertnstert.2021.06.027
ISSN1556-5653
Autores Tópico(s)Assisted Reproductive Technology and Twin Pregnancy
ResumoPreimplantation genetic testing for aneuploidy (PGT-A) remains one of the most controversial topics in reproductive medicine. With more than 40% of in vitro fertilization cycles in the United States reportedly involving PGT, both those in favor of and those opposed to PGT-A have significant interest in the efficacy of PGT-A. Ongoing issues include what patient population, if any, benefits from PGT-A, the true frequency of chromosomal mosaicism, whether embryonic aneuploidies self-correct, and how practitioners manage embryos designated as “mosaic.” This review addresses several misconceptions and misinterpretations of data surrounding the genetic analysis and prediction of mosaicism in the preimplantation embryo. Preimplantation genetic testing for aneuploidy (PGT-A) remains one of the most controversial topics in reproductive medicine. With more than 40% of in vitro fertilization cycles in the United States reportedly involving PGT, both those in favor of and those opposed to PGT-A have significant interest in the efficacy of PGT-A. Ongoing issues include what patient population, if any, benefits from PGT-A, the true frequency of chromosomal mosaicism, whether embryonic aneuploidies self-correct, and how practitioners manage embryos designated as “mosaic.” This review addresses several misconceptions and misinterpretations of data surrounding the genetic analysis and prediction of mosaicism in the preimplantation embryo. DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/33151 DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/33151 Preimplantation genetic testing (PGT) requires the application of significant and often error-prone DNA amplification methods to obtain a result. In addition, the process by which cells are obtained from the embryo and the quality of the embryo from which cells are obtained for PGT vary widely. These and other methodological challenges result in difficulty in distinguishing true signal from technical noise. The application of PGT to the diagnosis of monogenic disorders (PGT-M) serves as an example of the ability to develop methods that improve accuracy and overcome these and other technical challenges. Several important advances, including parallel analysis of mutation-linked markers and prevention and detection of DNA contamination, have helped avoid misdiagnoses and improve accuracy. In contrast, the uninformed introduction of “mosaic" diagnoses has led to a significant reduction in the accuracy of preimplantation genetic testing for aneuploidy (PGT-A). Clinical manifestation of this type of inaccuracy is exemplified by the STAR trial (1Munné S. Kaplan B. Frattarelli J.L. Child T. Nakhuda G. Shamma F.N. et al.Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial.Fertil Steril. 2019; 112: 1071-1079.e7Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), which reported an overall 49% (unusually high) aneuploidy rate and failed to demonstrate clinical utility in patients under the age of 35 years. Laboratories in the study that included “mosaic" diagnoses reported aneuploidy rates of 33% to 72%, whereas laboratories not including “mosaic" diagnoses reported aneuploidy rates of 0% to 43% in the same age group. This indicates that the inclusion of mosaic diagnoses results in overestimation of the presence of chromosomal abnormalities. False positive mosaicism undoubtedly plays a significant role in the failure to demonstrate improved outcomes with PGT-A, particularly in patients under the age of 35 years. Mosaicism in the preimplantation embryo is defined as a mixture of cells with different karyotypes. The most commonly used methods of predicting mosaicism involve the use of arbitrary chromosome copy number thresholds (i.e., from 20% to 80%) (2Cram D.S. Leigh D. Handyside A. Rechitsky L. Xu K. Harton G. et al.PGDIS position statement on the transfer of mosaic embryos 2019.Reprod Biomed Online. 2019; 39: e1-e4PubMed Scopus (61) Google Scholar) from next-generation sequencing data (Fig. 1). If a chromosome’s copy number falls outside the threshold for a normal disomic copy number of 2 (i.e., between 1.8 and 2.2) and outside the threshold for uniform monosomy or trisomy (i.e., below 1.2 or above 2.8), that chromosome is determined to fall in the “mosaic range” (1.2–1.8 or 2.2–2.8). In fact, several studies have published chromosome copy number data after analyzing artificial mixtures of cell lines with different karyotypes, thereby mimicking an aneuploid/euploid mosaicism scenario in a trophectoderm biopsy (3Goodrich D. Xing T. Tao X. Lonczak A. Zhan Y. Landis J. et al.Evaluation of comprehensive chromosome screening platforms for the detection of mosaic segmental aneuploidy.J Assist Reprod Genet. 2017; 34: 975-981Crossref PubMed Scopus (27) Google Scholar, 4Greco 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 (312) Google Scholar, 5Maxwell S.M. Colls P. Hodes-Wertz B. McCulloh D.H. McCaffrey C. Wells D. et al.Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing.Fertil Steril. 2016; 106: 1414-1419.e5Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Intermediate chromosome copy number profiles are indeed observed for mosaic aneuploidies in cell line mixtures, and researchers have considered these data as “preclinical validation” for the detection of mosaicism in a trophectoderm biopsy. Unfortunately, it is inaccurate to assume that an embryo is truly mosaic according to this “intermediate copy number” strategy (6Paulson R.J. Treff N.R. Isn't it time to stop calling preimplantation embryos mosaic?.F S Rep. 2020; 1: 164-165Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Although mosaicism can be detected with the use of copy number thresholds, analysis of cell lines is an inadequate representation of the technical variation introduced by a trophectoderm biopsy. A more rigorous system to investigate the presence of mosaicism in a blastocyst involves performing multiple biopsies on the same embryo (7Capalbo 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). This method is obviously incompatible with diagnosing mosaicism on a transferable embryo, but it provides superior evidence of bona fide mosaicism. A systematic review of studies that performed rebiopsies of embryos deemed mosaic on the basis of intermediate copy numbers revealed that most of those embryos (57%) were unlikely to have been mosaic (8Marin D. Xu J. Treff N.R. Preimplantation genetic testing for aneuploidy: a review of published blastocyst reanalysis concordance data.Prenat Diagn. 2021; 41: 545-553Crossref PubMed Scopus (10) Google Scholar). In addition, many embryos with chromosome copy numbers in the intermediate “mosaic range" have been found to have a meiotic origin of aneuploidy consistent with uniform aneuploidy, further confirming the inaccuracy of predicting mosaicism by these methods (9Handyside A.H. McCollin A. Summers M.C. Ottolini C.S. Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos.Prenat Diagn. 2021; 41: 525-535Crossref PubMed Scopus (4) Google Scholar). When aneuploidy originates from a meiotic error, all cells in the resulting embryo will possess the same aneuploidy (uniform). In addition, recent studies have demonstrated that embryos deemed “low-level mosaic” have equivalent reproductive potential to embryos deemed euploid (10Capalbo A. Poli M. Rienzi L. Girardi L. Cimadomo D. Benini F. et al.A prospective double-blinded non-selection trial of reproductive outcomes and chromosomal normalcy of newborns derived from putative low/moderate-degree mosaic IVF embryos.medRxiv. 2021; (2021.02.07.21251201)Google Scholar, 11Rubino P. Li X. Ruiz De Assin Alonso R. Mazmanian K. Guan L. Dearden L. et al.Embryos classified as low-grade mosaic (<50%) after preimplantation genetic screening (PGS) by means of high resolution next-generation screening (hr-NGS), can have the same competence of producing healthy newborns as euploid embryos.Fertil Steril. 2018; 109: e46-e47Abstract Full Text Full Text PDF Google Scholar). Although there are several examples of individual studies that illustrate a variety of limitations of predicting mosaicism on the basis of intermediate copy number thresholds, the number of systematic analyses is limited. Abhari and Kawwass (12Abhari S. Kawwass J.F. Pregnancy and neonatal outcomes after transfer of mosaic embryos: a review.J Clin Med. 2021; 10: 1369Crossref PubMed Scopus (6) Google Scholar) recently reviewed studies that compared clinical outcomes of “mosaic" embryo transfers with outcomes of euploid embryo transfers.. One important finding was that among more than 100 live births after the transfer of “mosaic" embryos, no abnormal phenotypes were observed. Zhang et al. (13Zhang Y.X. Chen J.J. Nabu S. Yeung Q.S.Y. Li Y. Tan J.H. et al.The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis.Genes. 2020; 11: 973Crossref Scopus (18) Google Scholar) recently performed a meta-analysis that included 137 “mosaic" and 476 euploid embryo transfers. A similar observation that all mosaic embryo transfers led to zero abnormalities was made. These systematic analyses were limited specifically to studies that compared the outcomes of euploid and “mosaic" embryo transfers. Another important analysis may involve evaluating outcomes of all “mosaic" embryo transfers reported to date, independently of whether there was a comparison with outcomes from euploid embryo transfers. In this review, a systematic analysis was performed of studies that evaluated clinical outcomes after the transfer of embryos designated as “mosaic.” All studies involved the use of intermediate chromosome copy number thresholds after PGT-A for all 24 chromosomes on trophectoderm biopsies. Studies were included after a standard PubMed search using the term “mosaic embryo” and after systematic reviews of individual reports, references cited, and personal communications. Ongoing pregnancies or live births were considered as “success.” A total of 25 relevant studies were identified (4Greco 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 (312) Google Scholar, 5Maxwell S.M. Colls P. Hodes-Wertz B. McCulloh D.H. McCaffrey C. Wells D. et al.Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing.Fertil Steril. 2016; 106: 1414-1419.e5Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Capalbo A. Poli M. Rienzi L. Girardi L. Cimadomo D. Benini F. et al.A prospective double-blinded non-selection trial of reproductive outcomes and chromosomal normalcy of newborns derived from putative low/moderate-degree mosaic IVF embryos.medRxiv. 2021; (2021.02.07.21251201)Google Scholar, 13Zhang Y.X. Chen J.J. Nabu S. Yeung Q.S.Y. Li Y. Tan J.H. et al.The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis.Genes. 2020; 11: 973Crossref Scopus (18) Google Scholar, 14Fragouli E. Alfarawati S. Spath K. Babariya D. Tarozzi N. Borini A. et al.Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid–aneuploid blastocysts.Hum Genet. 2017; 136: 805-819Crossref PubMed Scopus (137) Google Scholar, 15Munné S. Blazek J. Large M. Martinez-Ortiz P.A. Nisson H. Liu E. et al.Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.Fertil Steril. 2017; 108: 62-71.e8Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 16Inoue N. Lopez R. Delgado A. Nuñez D. Portella J. Noriega-Hoces L. et al.Mosaic embryo transfer after oocyte in vitro maturation in combination with non-invasive prenatal testing (NIPT)-first report of a euploid live birth.J Assist Reprod Genet. 2017; 34: 1199-1205Crossref PubMed Scopus (6) Google Scholar, 17Lledó B. Morales R. Ortiz J.A. Blanca H. Ten J. Llácer J. et al.Implantation potential of mosaic embryos.Syst Biol Reprod Med. 2017; 63: 206-208Crossref PubMed Scopus (27) Google Scholar, 18Spinella F. Fiorentino F. Biricik A. Bono S. Ruberti A. Cotroneo E. et al.Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments.Fertil Steril. 2018; 109: 77-83Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 19Zore T. Kroener L.L. Wang C. Liu L. Buyalos R. Hubert G. et al.Transfer of embryos with segmental mosaicism is associated with a significant reduction in live-birth rate.Fertil Steril. 2019; 111: 69-76Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 20Zhang L. Wei D. Zhu Y. Gao Y. Yan J. Chen Z.J. Rates of live birth after mosaic embryo transfer compared with euploid embryo transfer.J Assist Reprod Genet. 2019; 36: 165-172Crossref PubMed Scopus (34) Google Scholar, 21Victor A.R. Tyndall J.C. Brake A.J. Lepkowsky L.T. Murphy A.E. Griffin D.K. et al.One hundred mosaic embryos transferred prospectively in a single clinic: exploring when and why they result in healthy pregnancies.Fertil Steril. 2019; 111: 280-293Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Liu Y.L. Yu T.N. Chen C.H. Wang P.H. Chen C.H. Tzeng C.R. Healthy live births after mosaic blastocyst transfers with the use of next-generation sequencing.Taiwan J Obstet Gynecol. 2019; 58: 872-876Crossref PubMed Scopus (5) Google Scholar, 23Besser A.G. McCulloh D.H. Grifo J.A. What are patients doing with their mosaic embryos? Decision making after genetic counseling.Fertil Steril. 2019; 111: 132-137.e1Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 24Hong B. Hao Y. The outcome of human mosaic aneuploid blastocysts after intrauterine transfer: A retrospective study.Medicine (Baltimore). 2020; 99e18768Crossref PubMed Scopus (8) Google Scholar, 25Munné S. Spinella F. Grifo J. Zhang J. Beltran M.P. Fragouli E. et al.Clinical outcomes after the transfer of blastocysts characterized as mosaic by high resolution next generation sequencing - further insights.Eur J Med Genet. 2020; 63103741Crossref PubMed Scopus (49) Google Scholar, 26Kahraman S. Cetinkaya M. Yuksel B. Yesil M. Pirkevi Cetinkaya C. The birth of a baby with mosaicism resulting from a known mosaic embryo transfer: a case report.Hum Reprod. 2020; 35: 727-733Crossref PubMed Scopus (32) Google Scholar, 27Tiegs A.W. Tao X. Zhan Y. Whitehead C. Kim J. Hanson B. et al.A multicenter, prospective, blinded, nonselection study evaluating the predictive value of an aneuploid diagnosis using a targeted next-generation sequencing–based preimplantation genetic testing for aneuploidy assay and impact of biopsy.Fertil Steril. 2021; 115: 627-637Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28Lee C.I. Cheng E.H. Lee M.S. Lin P.Y. Chen Y.C. Chen C.H. et al.Healthy live births from transfer of low-mosaicism embryos after preimplantation genetic testing for aneuploidy.J Assist Reprod Genet. 2020; 37: 2305-2313Crossref PubMed Scopus (11) Google Scholar, 29Lin P.Y. Lee C.I. Cheng E.H. Huang C.C. Lee T.H. Shih H.H. et al.Clinical outcomes of single mosaic embryo transfer: high-level or low-level mosaic embryo, does it matter?.J Clin Med. 2020; 9: 1695Crossref Scopus (18) Google Scholar, 30Chuang T.H. Chang Y.P. Lee M.J. Wang H.L. Lai H.H. Chen S.U. The incidence of mosaicism for individual chromosome in human blastocysts is correlated with chromosome length.Front Genet. 2020; 11565348Crossref PubMed Scopus (3) Google Scholar, 31Viotti M. Victor A.R. Barnes F.L. Zouves C.G. Besser A.G. Grifo J.A. et al.Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use.Fertil Steril. 2021; 115: 1212-1224Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 32Yang M. Rito T. Metzger J. Naftaly J. Soman R. Hu J. et al.Depletion of aneuploid cells in human embryos and gastruloids.Nat Cell Biol. 2021; 23: 314-321Crossref PubMed Scopus (23) Google Scholar, 33Li X. Hao Y. Chen D. Ji D. Zhu W. Zhu X. et al.Non-invasive preimplantation genetic testing for putative mosaic blastocysts: a pilot study.Hum Reprod. 2021; 36: 2020-2034Crossref PubMed Scopus (7) Google Scholar, 34Alksere B. Grinfelde I. Kornejeva L. Dzalbs A. Vedmedovska N. Kovalova I. et al.The outcomes after transfers of embryos with chromosomal mosaicism: a single reproductive medicine center experience at iVF Riga clinic.Gynecol Endocrinol. 2020; 36: 53-57Crossref PubMed Scopus (1) Google Scholar) with outcomes for 2,759 “mosaic" embryos. This is the largest collection of “mosaic" embryo transfer outcomes evaluated to date. Among 2,759 “mosaic" embryo transfers, only 1 PGT-A result (0.04%) was confirmed in the resulting ongoing pregnancy. Although it was not reported, the investigators indicated that a total of 44 “mosaic" embryo transfers were performed before observing this case (personal communication). In addition, the child born was reported as healthy, with no abnormal phenotypes observed. Overall, this analysis demonstrates a remarkable lack of confirmation of PGT-A predictions of “mosaicism” in the preimplantation embryo and reflects the inaccuracy of using preimplantation embryo biopsy intermediate copy number thresholds to predict mosaicism in an ongoing pregnancy or delivery. Although several publications have provided reviews to guide the management of embryos designated as “mosaic" (12Abhari S. Kawwass J.F. Pregnancy and neonatal outcomes after transfer of mosaic embryos: a review.J Clin Med. 2021; 10: 1369Crossref PubMed Scopus (6) Google Scholar, 35Chen K. Darcy D. Boyd A. Pregnancy from mosaic embryo transfer: genetic counseling considerations.Curr Opin Obstet Gynecol. 2021; 33: 100-105Crossref PubMed Scopus (1) Google Scholar, 36Practice Committee and Genetic Counseling Professional Group (GCPG) of the American Society for Reproductive Medicine. Electronic address: [email protected]Clinical management of mosaic results from preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts: a committee opinion.Fertil Steril. 2020; 114: 246-254Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 37Zwingerman R. Langlois S. Committee Opinion No. 406: Prenatal testing after IVF with preimplantation genetic testing for aneuploidy.J Obstet Gynaecol Can. 2020; 42: 1437-1443.e1Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 38Levy B. Hoffmann E.R. McCoy R.C. Grati F.R. Chromosomal mosaicism: origins and clinical implications in preimplantation and prenatal diagnosis.Prenat Diagn. 2021; 41: 631-641Crossref PubMed Scopus (5) Google Scholar, 39Sachdev N.M. Maxwell S.M. Besser A.G. Grifo J.A. Diagnosis and clinical management of embryonic mosaicism.Fertil Steril. 2017; 107: 6-11Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the lack of clinical predictive value demonstrated by the analyses described in this systematic review should be further emphasized. One might argue that the rate of mosaicism expected in this setting is low to begin with. However, the prevalence of mosaicism in ongoing pregnancies after natural conception has been estimated as 1% to 2% (38Levy B. Hoffmann E.R. McCoy R.C. Grati F.R. Chromosomal mosaicism: origins and clinical implications in preimplantation and prenatal diagnosis.Prenat Diagn. 2021; 41: 631-641Crossref PubMed Scopus (5) Google Scholar, 40Huang A. Adusumalli J. Patel S. Liem J. Williams III , J. Pisarska M.D. Prevalence of chromosomal mosaicism in pregnancies from couples with infertility.Fertil Steril. 2009; 91: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It is unlikely that transferring more than 2,500 “mosaic" embryos would result in a rate of mosaicism that was more than 1,000 times lower than that resulting from natural conception, if in fact 100% of these embryos were truly mosaic to begin with. Despite increasing evidence that copy number thresholds are inaccurate predictors of true mosaicism, the concept that PGT-A is fully capable of providing a definitive diagnosis of mosaicism from a trophectoderm biopsy continues to be inappropriately conveyed. Beginning with a study by Greco et al.(4Greco 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 (312) Google Scholar), several reports have used the term “mosaic embryo” when describing clinical outcomes. For example, “Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use” (31Viotti M. Victor A.R. Barnes F.L. Zouves C.G. Besser A.G. Grifo J.A. et al.Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use.Fertil Steril. 2021; 115: 1212-1224Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), “The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis” (13Zhang Y.X. Chen J.J. Nabu S. Yeung Q.S.Y. Li Y. Tan J.H. et al.The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis.Genes. 2020; 11: 973Crossref Scopus (18) Google Scholar), and “Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes after transfer of mosaic embryos” (41Kushnir V.A. Darmon S.K. Barad D.H. Gleicher N. Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes following transfer of mosaic embryos.Reprod Biol Endocrinol. 2018; 16: 6Crossref PubMed Scopus (37) Google Scholar). In virtually every case, differences in clinical outcomes can be better explained by simply considering that many of the embryos deemed mosaic were never actually mosaic (i.e., false positives). For example, many embryos designated as mosaic by copy number threshold analysis are actually uniform aneuploid. A systematic review (8Marin D. Xu J. Treff N.R. Preimplantation genetic testing for aneuploidy: a review of published blastocyst reanalysis concordance data.Prenat Diagn. 2021; 41: 545-553Crossref PubMed Scopus (10) Google Scholar) demonstrated that 28% of embryos deemed mosaic by a single embryo biopsy were found to be uniform aneuploid when multiple rebiopsies were evaluated. In addition, karyomapping analysis demonstrated that 64% of chromosomes with copy numbers in the mosaic range were actually meiotic in origin (uniform aneuploid) (9Handyside A.H. McCollin A. Summers M.C. Ottolini C.S. Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos.Prenat Diagn. 2021; 41: 525-535Crossref PubMed Scopus (4) Google Scholar). Moreover, single nucleotide polymorphism genotyping coupled with chromosome copy number analysis for PGT-A revealed that chromosomes with intermediate copy numbers may additionally originate from aneuploidies in triploid embryos (42Marin D. Scott Jr., R.T. Treff N.R. Preimplantation embryonic mosaicism: origin, consequences and the reliability of comprehensive chromosome screening.Curr Opin Obstet Gynecol. 2017; 29: 168-174Crossref PubMed Scopus (17) Google Scholar). Transfer of embryos with this type of false positive diagnosis of mosaicism (false negative uniform aneuploidy) would indeed lead to reduced success rates (as observed in some studies), given that embryos with uniform aneuploidy possess near 0% reproductive potential (27Tiegs A.W. Tao X. Zhan Y. Whitehead C. Kim J. Hanson B. et al.A multicenter, prospective, blinded, nonselection study evaluating the predictive value of an aneuploid diagnosis using a targeted next-generation sequencing–based preimplantation genetic testing for aneuploidy assay and impact of biopsy.Fertil Steril. 2021; 115: 627-637Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Yang M. Rito T. Metzger J. Naftaly J. Soman R. Hu J. et al.Depletion of aneuploid cells in human embryos and gastruloids.Nat Cell Biol. 2021; 23: 314-321Crossref PubMed Scopus (23) Google Scholar, 43Scott Jr., R.T. Ferry K. Su J. Tao X. Scott K. Treff N.R. Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study.Fertil Steril. 2012; 97: 870-875Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). These studies demonstrate that embryos with uniformly aneuploid trophectoderm fail to sustain implantation and additionally contradict the notion that a trophectoderm biopsy is unable to represent the chromosomal status of the remaining embryo. Proponents of the use of mosaicism diagnosis often argue that it is worthwhile, given the observed reduction in reproductive potential of “mosaic" embryos. However, the alternative explanation that many embryos designated as mosaic aneuploid are actually uniform aneuploid provides a more well-supported interpretation of reduced success rates. From a molecular biology perspective, this type of diagnostic error could arise when a suboptimal number of trophectoderm cells are analyzed. DNA amplification occurs in an exponential manner. During the linear phase, DNA doubles in quantity after each cycle of the polymerase chain reaction. When the components of the polymerase chain reaction become limited (saturation), the DNA is no longer able to double in quantity after each cycle. This would result in a decrease in the observed relative difference between disomic and trisomic chromosomes, leading to an intermediate copy number and a false positive diagnosis of mosaic aneuploidy (i.e., false negative uniform aneuploidy) (Fig. 2). Similarly, with too few cells, amplification may not reach the linear phase before quantitation and lead to observation of an intermediate copy number and false positive mosaicism. Conversely, many embryos designated as mosaic by copy number threshold analysis are in addition completely euploid. A systematic review (8Marin D. Xu J. Treff N.R. Preimplantation genetic testing for aneuploidy: a review of published blastocyst reanalysis concordance data.Prenat Diagn. 2021; 41: 545-553Crossref PubMed Scopus (10) Google Scholar) demonstrated that 29% of embryos deemed mosaic were actually uniform euploid on rebiopsy. In addition, karyomapping analysis demonstrated that 36% of embryos with chromosome copy numbers in the intermediate mosaic range were actually euploid (9Handyside A.H. McCollin A. Summers M.C. Ottolini C.S. Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos.Prenat Diagn. 2021; 41: 525-535Crossref PubMed Scopus (4) Google Scholar). In addition, embryos with “low-level” mosaicism have been shown to possess equivalent reproductive potential to embryos designated as euploid (10Capalbo A. Poli M. Rienzi L. Girardi L. Cimadomo D. Benini F. et al.A prospective double-blinded non-selection trial of reproductive outcomes and chromosomal normalcy of newborns derived from putative low/moderate-degree mosaic IVF embryos.medRxiv. 2021; (2021.02.07.21251201)Google Scholar, 11Rubino P. Li X. Ruiz De Assin Alonso R. Mazmanian K. Guan L. Dearden L. et al.Embryos classified as low-grade mosaic (<50%) after preimplantation genetic screening (PGS) by means of high resolution next-generation screening (hr-NGS), can have the same competence of producing healthy newborns as euploid embryos.Fertil Steril. 2018; 109: e46-e47Abstract Full Text Full Text PDF Google Scholar). As shown in Table 1, several studies have reported successful outcomes after the transfer of “mosaic" embryos, and, coincidently, with an overall success rate of 38%.Table 1Rate of confirmation of mosaicism in pregnancies after transfer of embryos with intermediate copy numbers (“mosaic").StudyPGT-A platform“Mosaic” embryos transferredSuccess rate (%)Confirmed mosaicMosaic confirmation rate (%)Greco et al. (4Greco 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 (312) Google Scholar)aCGH183300Maxwell et al. (5Maxwell S.M. Colls P. Hodes-Wertz B. McCulloh D.H. McCaffrey C. Wells D. et al.Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing.Fertil Steril. 2016; 106: 1414-1419.e5Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar)NGS183300Fragouli et al. (14Fragouli E. Alfarawati S. Spath K. Babariya D. Tarozzi N. Borini A. et al.Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid–aneuploid blastocysts.Hum Genet. 2017; 136: 805-819Crossref PubMed Scopus (137) Google Scholar)NGS442700Munne et al. (15Munné S. Blazek J. Large M. Martinez-Ortiz P.A. Nisson H. Liu E. et al.Detailed investigation into the cytogenet
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