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Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes following transfer of mosaic embryos

Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis... Background: Preimplantation genetic screening (PGS) is increasingly utilized as an adjunct procedure to IVF. Recently healthy euploid live birth were reported following transfer of mosaic embryos. Several recent publications have surmised that the degree of trophectoderm (TE) mosaicism in transferred embryos is predictive of ongoing pregnancy and miscarriage rates. Methods: This is a corrected analysis of previously published retrospective data on vitro fertilization (IVF) cycle outcomes involving replacement of 143 mosaic and 1045 euploid embryos tested by PGS, utilizing high-resolution next-generation sequencing (NGS) of TE and determination of percentages of mosaicism. Receiver operating curves (ROCs) and measurement of area under the curve (AUC) were used to evaluated the accuracy of the predictor variable, proportion of aneuploid cells in a TE biopsy specimen, with IVF outcomes, ongoing pregnancy and miscarriage rates. Results: Confirming findings of the previously published report we also found higher ongoing pregnancy rates (63.3% vs. 39.2%) and lower miscarriage rates (10.2% vs. 24.3%) with euploid embryo transfers than with mosaic embryo transfer. There, however, were no significant differences in ongoing pregnancy or miscarriage rates among mosaic embryo transfers at any threshold of aneuploidy. Based on AUC, TE biopsies predicted ongoing pregnancy for euploid, as well as mosaic embryos, in a range of 0.50 to 0.59 and miscarriage in a range from 0.50 to 0.66 Conclusions: Degree of TE mosaicism was a poor predictor of ongoing pregnancy and miscarriage. Keywords: In vitro fertilization, Preimplantation genetic diagnosis, Preimplantation genetic screening, Aneuploidy, Embryo selection, Next-generation sequencing Background trophectoderm (TE) mosaicism is a common finding [2], Preimplantation genetic screening (PGS), now renamed the Preimplantation Genetic Diagnosis International Soci- by some as preimplantation genetic testing for aneu- ety (PGDIS) recently recommended a radical overhaul of ploidy (PGT-A), is increasingly utilized as ad-on to vitro testing methodologies and reporting of test results [3]. fertilization (IVF). The original hypothesis behind PGS Since diagnostic platforms, like array comparative gen- utilization was “aneuploidy screening as a means to in- ome hybridization (aCGH), single-nucleotide polymorph- crease pregnancy rates, decrease loss rates, and establish ism array, and quantitative polymerase chain reaction transfer order” [1]. Because of growing recognition that (qPCR) lack capacity to detect mosaicism in a single TE biopsy, next-generation sequencing (NGS), which cur- * Correspondence: vkushnir@thechr.com rently detects mosaicism in excess of 20%, is the only Center for Human Reproduction, 21 East 69th Street, New York, NY 10021, technique recommended by PGDIS [3]. Utilizing NGS, USA embryos with less than 20% aneuploidy in the TE sample Department of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC, USA are, therefore, considered euploid; while those between 20 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 2 of 6 and 80% are reported as mosaic, and those over 80% as aneuploidy in its TE as aneuploid and, therefore, as not truly aneuploid. Moreover, PGDIS guidelines suggest that transferrable [4]. embryos designated as euploid can be freely transferred, Both sets of guidelines, however, were lacking robust while embryos designated as aneuploid should not be published clinical data in support, as are usually required transferred and, therefore, discarded [3]. for clinical diagnostic testing [5, 6]. This why a recent Finally, mosaic embryos, at 20–80% range aneuploidy publication by a multi-center conglomerate of PGS refer- in the TE sample, may be potentially transferred, though ence laboratories and referring IVF centers, offering a first the PGDIS notes that such transfers be performed with large data sets of clinical outcomes following transfer of caution and only in absence of euploid embryos. More- mosaic blastocyst in accordance with PGDIS criteria and over, the society suggested an empirical hierarchy for utilizing NGS [7], has to be viewed as a defining moment. such transfers, based on the specific aneuploidies re- Until these recent publications, only three groups have re- ported in embryos [3]. ported IVF cycle outcomes after transfers of mosaic em- A group of investigators at the 2016 World Congress on bryos in a small number of cases [8–10]. Controversies in Preconception, Preimplantation, Prenatal Since the manuscripts by (Munné et al. 2017; Fragouli Genetic Diagnosis Meeting in Barcelona reached similar et al. 2017) did not use standard statistical methods to as- conclusions, advising to prioritize transfer of mosaic sess predictive values for different degrees of mosaicism embryos with lower levels (20–40%) of aneuploidy in detected in TE biopsies, we reanalyzed the raw data re- the TE sample over those with higher levels (40–70%), ported by the authors. As this study will demonstrate, our and defining any embryo biopsy with more than 70% analysis contradicts the conclusions of Munné et al. that Table 1 Predictive ability of NGS trophectoderm biopsy for ongoing pregnancy based on proportion of abnormal cells in the specimen Euploid < 20% Abnormal Mosaic ≥20% to ≤80% Abnormal p-value AUC Ongoing Pregnancy 661/1045 63.3% 56/143 39.2% < 0.0001 0.55 Mosaic ≥20% to < 30% Abnormal Mosaic ≥30% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 12/27 44.4% 22/61 36.1% 0.46 0.54 ≥ 38 1/8 12.5% 21/46 45.7% 0.08 0.59 All Ages 13/35 37.1% 43/108 39.9% 0.78 0.51 Mosaic ≥20% to < 40% Abnormal Mosaic ≥40% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 22/61 36.1% 12/27 44.4% 0.46 0.54 ≥ 38 10/25 40.0% 12/29 41.4% 0.92 0.51 All Ages 32/87 36.8% 24/56 42.9% 0.47 0.53 Mosaic ≥20% to < 50% Mosaic Mosaic ≥50% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 30/78 38.5% 4/10 40.0% 0.93 0.50 ≥ 38 17/38 44.7% 5/16 31.3% 0.36 0.56 All Ages 47/117 40.2% 9/26 34.6% 0.60 0.52 Mosaic ≥20% to < 60% Abnormal Mosaic ≥60% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 34/86 39.5% 0/2 0.0% 0.26 0.52 ≥ 38 18/44 40.9% 4/10 40.0% 0.96 0.50 All Ages 52/131 39.7% 4/12 33.3% 0.67 0.51 Mosaic ≥20% to < 70% Abnormal Mosaic ≥70% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 34/87 39.1% 0/1 0.0% 0.42 0.51 ≥ 38 19/46 41.3% 3/8 37.5% 0.84 0.51 All Ages 53/134 39.6% 3/9 33.3% 0.71 0.51 AUC: area under the curve Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 3 of 6 40% mosaicism represents a significant differentiation point. Indeed, our analysis did not find significant predict- ability at any level of mosaicism between 20 and 80%. Methods A detailed description of patient factors and molecular methods is available in the original publications, which served as data sources for our study [7, 11]. The data for 143 NGS-tested mosaic embryos that were transferred, were extracted from Additional file 1: Table S3 in the publi- cation by Munné et al. [7]. To assess whether the de- gree of mosaicism at different ages affected ongoing pregnancy rates, these data were stratified for female ages < 38 and ≥38 years. Data for the comparison group came from for female age well matched controls from the same publication, who had undergone transfers of 1045 euploid embryos, as determined by NGS with < 20% aneuploid cells in the TE sample [7]. This control group could not be age- stratified since only aggregate data (rather than embryo level data) were provided in the source publication. Receiver operating characteristic curves (ROCs) and measurements of area under the curve (AUC) were used to evaluated accuracy of the predictor variable, proportion of abnormal cells in a TE biopsy specimen (i.e., percentage mosaicism) with IVF outcomes, including ongoing preg- nancy and miscarriage rates. The analysis was then performed for binary variables eu- ploid (< 20% aneuploid cells) vs. mosaic embryos, and for Fig. 1 Receiver operating characteristic (ROC) curves for ongoing various thresholds of aneuploidy, ranging from 20% to pregnancy and miscarriage. a Analysis for binary classification euploid 80% in 10% increments, as detailed in Table 1 and Fig. 1. (< 20% aneuploid cells) vs. mosaic embryos (20% to 80% aneuploid Comparisons between groups were made using chi- cells) based on trophectoderm sample. b Various thresholds of square tests. Statistical analyses were preformed using SAS aneuploidy within the cohort of mosaic embryos ranging from 20% to 80% in 10% increments. Predictor variable: proportion of abnormal version 9.4. A p-value < 0.05 was considered statistically cells in a trophectoderm biopsy specimen. Outcomes: ongoing significant. pregnancy and miscarriage. Area under the curve (AUC) of 0.50 Since all here addressed data were already published, denotes the screening test as having no predictive ability while an publicly available, and cannot be utilized to identify indi- AUC of 1.0 denotes an ideal screening test vidual patients, this study qualified for exemption from IRB approval. threshold of aneuploidy. Moreover, the table demon- Results strates the AUC for all comparisons ranges from 0.50 to Table 1 shows the ability of TE biopsies, utilizing NGS, to 0.59, indicating poor predictive ability of TE biopsies for predict ongoing pregnancy based on proportion of abnor- ongoing pregnancies. mal cells in each biopsy specimen. Here we confirm the Table 2 shows the predictive ability of TE biopsies, util- findings of Munné et al. [7] that ongoing pregnancy rates izing NGS, to predict miscarriages based on proportion of were, seemingly, significantly higher following euploid em- abnormal cells in a single biopsy sample. Once again we bryo transfer (i.e., biopsies with < 20% aneuploidy) than confirm the findings of Munné et al. [7] that miscarriage mosaic embryo transfer (biopsies with 20–80% aneu- rates were significantly lower following euploid embryo ploidy). Our calculation yielded a slightly lower ongoing transfer than with mosaic embryo transfer. However, the pregnancy rate in the mosaic group than Munné et al., table also demonstrates that the AUC is only 0.56, again who reported 57 ongoing and delivered pregnancies but indicting very poor predictive validity of a TE biopsy for presented embryo level data for only 56. miscarriages. Moreover, analysis of miscarriage risk among Table 1 demonstrates no significant differences in on- mosaic embryo transfers, did not find increased risk based going pregnancy rates among mosaic transfers at any on any threshold of aneuploidy. Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 4 of 6 Table 2 Predictive ability of NGS trophectoderm biopsy for miscarriage based on proportion of abnormal cells in the specimen Euploid < 20% Abnormal Mosaic ≥20% to ≤80% Abnormal p-value AUC Miscarriage 75/736 10.2% 18/74 24.3% 0.0003 0.56 Mosaic ≥20% to < 30% Abnormal Mosaic ≥30% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 5/17 29.4% 9/31 29.0% 0.69 0.50 ≥ 38 0/1 0.0% 4/25 16.0% 0.66 0.52 All Ages 5/18 27.8% 13/56 23.2% 0.69 0.52 Mosaic ≥20% to < 40% Abnormal Mosaic ≥40% to ≤80% Abnormal p-value AUC Miscarriage < 38 11/33 33.3% 3/15 20.0% 0.35 0.57 ≥ 38 2/12 16.7% 2/14 14.3% 0.87 0.52 All Ages 13/45 28.9% 5/29 17.2% 0.25 0.58 Mosaic ≥20% to < 50% Mosaic Mosaic ≥50% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 13/43 30.2% 1/5 20.0% 0.63 0.52 ≥ 38 2/19 10.5% 2/7 28.6% 0.26 0.64 All Ages 15/62 24.2% 3/12 25.0% 0.95 0.50 Mosaic ≥20% to < 60% Abnormal Mosaic ≥60% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 14/48 29.2% 0/0 0.0% –– ≥ 38 2/20 10.0% 2/6 33.3% 0.16 0.66 All Ages 16/68 23.5% 2/6 33.3% 0.59 0.59 Mosaic ≥20% to < 70% Abnormal Mosaic ≥70% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 14/48 29.2% 0/0 0.0% –– ≥ 38 3/22 13.6% 1/4 25.0% 0.56 0.56 All Ages 17/70 24.3% 1/4 25.0% 0.97 0.50 AUC: area under the curve Figure 1a demonstrates ROC curves for ongoing preg- single TE biopsy as determined by NGS, has poor pre- nancies and miscarriages based on binary classification dictive value as a screening test for an individual em- of embryos as either euploid or mosaic. Figure 1b shows bryo’s ability to establish ongoing pregnancy or lead to ROC curves for ongoing pregnancies and miscarriages miscarriage. Moreover, given the observed ongoing preg- based on various thresholds of aneuploidy (in 10% incre- nancy and miscarriage rates it appears likely that in ments, ranging from 20% to 80%) within the group of many cases aneuploidy within the TE of embryos repre- mosaic embryos. Both figures show a null line for refer- sents a physiologic variant rather than a pathological ence with an AUC of 0.50 which denotes when a screen- condition. This conclusion is supported by a recent in ing test has no predictive ability. depth literature review: “Mosaicism in Preimplantation Human Embryos: When Chromosomal Abnormalities Discussion Are the Norm” [13]. Our analysis confirms the previously published report Commonly utilized screening tests in reproductive medi- which found statistically higher ongoing pregnancy rates cine, such as mammograms and PAP smears for cancer (63.3% vs. 39.2%) and lower miscarriage rates (10.2% vs. screening [14], while themselves not ideal screening tests, 24.3%) with euploid embryo transfers than with mosaic still demonstrate far more robust clinical performance than embryo transfer. These findings are also consistent with here presented PGS data. This study reinforces previously a recent report by Spinella et al. [12]. However, our ana- raised concerns about the biological premise and clinical lysis demonstrates with ROC curves that PGS, at any effectiveness of PGS [15–18]. It is our view that to ensure threshold level of mosaicism, up to 80% aneuploidy in a optimal outcomes for patients the reproductive medicine Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 5 of 6 community should hold commercial PGS tests to the same Administration; IVF: In vitro fertilization; LDTs: Laboratory-developed tests; NGS: Next-generation sequencing; PGDIS: Preimplantation Genetic Diagnosis standards as other screening tests commonly utilized in International Society; PGS: Preimplantation genetic screening; the field. Therefore, in our opinion commercial laborator- qPCR: Quantitative polymerase chain reaction; ROCs: Receiver operating ies which market PGS tests should seek regulatory ap- curves; TE: Trophectoderm proval for these tests [19]. Acknowledgements Based on the original data set published by Munné et al. Not applicable [7], our analysis may, indeed, actually still overestimate the benefits of PGS in this patient population. In the original Authors’ contribution VAK, DHB, and NG developed the concept of the study; All authors contributed data set, the authors indicated that 133 out of 143 mosaic to data accumulation; SKD, DHB and VAK contributed to data analysis; All embryo transfers were single embryo transfers, while 10 authors contributed to data interpretation. VAK and NG wrote the manuscript. were double embryo transfers (such data were not pro- All authors contributed to revisions of the manuscript, and approved of the final submission. VAK takes responsibility for the accuracy of the data analysis. vided for the control group of patients undergoing euploid embryo transfer). A higher proportion of double embryo Funding transfers in the euploid embryo group, could, therefore, Intramural funds from the CHR and by grants from The Foundation for hypothetically explain the higher ongoing pregnancy rate Reproductive Medicine. and lower miscarriage rates observed in this group. Availability of data and materials It is also likely that patients who produce euploid em- Data were obtained from the referenced publications. Further information is bryos have better prognoses and/or less severe under- available by contacting Dr. Vitaly A. Kushnir at vkushnir@thechr.com. lying infertility than those who only produce mosaic Ethics approval and consent to participate embryos. This point is consistent with several recent Since all here addressed data were already published, publicly available, and studies which have found a correlation between propor- cannot be utilized to identify individual patients, this study qualified for tion of aneuploid blastocysts and diminished ovarian re- exemption from IRB approval. serve while controlling for patient age [20–22]. Finally, Consent for publication the original dataset does not specify the number of Not applicable treated patients nor the number of tested embryos per patient in either group. It reflects only patients who Competing interests V.A.K previously served as a consultant to the CDC. N.G., D.H.B., and V.A.K. are reached embryo transfer, thereby excluding poorer prog- listed as co-owners of several already awarded and still pending U.S. patents, nosis patients who started IVF with intention of PGS but none related to the topic of this manuscript. N.G. is a shareholder in Fertility did not have any transferrable embryos following extended Nutraceuticals, LLC and owner of the CHR. N.G. and D.H.B. receive patent royalties from Fertility Nutraceuticals, LLC. embryo culture, TE biopsy, cryopreservation, and thawing. The patient selection is, therefore, biased toward better prognosis patients, as previously documented in national Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in registry data in patients undergoing IVF with PGS [23]. published maps and institutional affiliations. Additional limitations include reporting of ongoing preg- nancy rates rather than live birth rates and lack of data on Author details Center for Human Reproduction, 21 East 69th Street, New York, NY 10021, the health of infants conceived from mosaic embryos. USA. Department of Obstetrics and Gynecology, Wake Forest School of We conclude that the degree of TE mosaicism is a Medicine, Winston-Salem, NC, USA. Foundation for Reproductive Medicine, poor predictor of ongoing pregnancy and miscarriage. New York, NY, USA. Stem Cell Biology and Molecular Embryology Laboratory, The Rockefeller University, New York, NY, USA. Department of Moreover, in our ROC analysis PGS demonstrates poor Obstetrics and Gynecology, University of Vienna School of Medicine, Vienna, clinical effectiveness for a routine screening test and, Austria. therefore, should not be routinely offered as an ad-on to Received: 14 September 2017 Accepted: 10 January 2018 IVF. Until efficacy, safety, and cost effectiveness of PGS are established it, therefore, should only be offered as an experimental test under study conditions and with ap- References propriate informed consent. 1. Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, Scott RT. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with Additional file comprehensive chromosomal screening. Fertil Steril. 2014;101:656–663.e1. 2. Sachdev NM, Maxwell SM, Besser AG, Grifo JA. Diagnosis and clinical management of embryonic mosaicism. Fertil Steril. 2017;107:6–11. Additional file 1: Data for 143 NGS-tested mosaic embryos that were 3. PGDIS position statement on chromosome mosaicism and preimplantation transferred; extracted from Table S3 in the publication by Munné et al. [7]. aneuploidy testing at the blastocyst stage. [http://www.pgdis.org/docs/ (XLSX 17 kb) newsletter_071816.html]. 4. Controversies in Preconception, Preimplantation, and Prenatal Genetic Abbreviations Diagnosis. CoGen position statement on chromosomal mosaicism detected aCGH: Array comparative genome hybridization; AUC: Area under the curve; inpreimplantation blastocyst biopsies. [https://www.ivf-worldwide.com/ CLIA: Clinical Laboratory Improvement Amendments; FDA: Food and Drug index.php?option=com_content&view=article&id=733&Itemid=464]. Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 6 of 6 5. Standards for Guideline Development - US Preventive Services Task Force [https://www.uspreventiveservicestaskforce.org/Page/Name/standards-for- guideline-development]. 6. Standards for Developing Trustworthy Clinical Practice Guidelines : Health and Medicine Division. [http://www.nationalacademies.org/hmd/Reports/ 2011/Clinical-Practice-Guidelines-We-Can-Trust/Standards.aspx]. 7. Munné S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, Tarozzi N, Borini A, Becker A, Zhang J, Maxwell S, Grifo J, Babariya D, Wells D, Fragouli E. 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(1):62-71.e8. 8. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic Aneuploid blastocysts. N Engl J Med. 2015;373:2089–90. 9. Gleicher N, Vidali A, Braverman J, Kushnir VA, Barad DH, Hudson C, Wu YG, Wang Q, Zhang L, Albertini DF. International PGS consortium study group: accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos. Reprod Biol Endocrinol. 2016;14:54. 10. Lledó B, Morales R, Ortiz JA, Blanca H, Ten J, Llácer J, Bernabeu R. Implantation potential of mosaic embryos. Syst Biol Reprod Med. 2017;63:206–8. 11. Fragouli E, Alfarawati S, Spath K, Babariya D, Tarozzi N, Borini A, Wells D. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts. Hum Genet. 2017;136:805–19. 12. Spinella F, Fiorentino F, Biricik A, Bono S, Ruberti A, Cotroneo E, Baldi M, Cursio E, Minasi MG, Greco E. Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments. Fertil Steril. 2017; 13. McCoy RC. Mosaicism in preimplantation human embryos: when chromosomal abnormalities are the norm. Trends Genet. 2017;33:448–63. 14. Maxim LD, Niebo R, Utell MJ. Screening tests: a review with examples. Inhal Toxicol. 2014;26:811–28. 15. Gleicher N, Kushnir VA, Barad DH. Preimplantation genetic screening (PGS) still in search of a clinical application: a systematic review. Reprod Biol Endocrinol. 2014;12:22. 16. Mastenbroek S, Repping S. Preimplantation genetic screening: back to the future. Hum Reprod. 2014;29:1846–50. 17. Orvieto R. Preimplantation genetic screening- the required RCT that has not yet been carried out. Reprod Biol Endocrinol. 2016;14(35). 18. Gleicher N, Metzger J, Croft G, Kushnir VA, Albertini DF, Barad DH. A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use. Reprod Biol Endocrinol. 2017;15. 19. ThePublic HealthEvidencefor FDA Oversight of Laboratory Developed Tests: 20 Case Studies. [https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ InVitroDiagnostics/LaboratoryDevelopedTests/default.htm]. 20. Katz-Jaffe MG, Surrey ES, Minjarez DA, Gustofson RL, Stevens JM, Schoolcraft WB. Association of abnormal ovarian reserve parameters with a higher incidence of aneuploid blastocysts. Obstet Gynecol. 2013;121:71–7. 21. La Marca A, Minasi MG, Sighinolfi G, Greco P, Argento C, Grisendi V, Fiorentino F, Greco E. Female age, serum antimüllerian hormone level, and number of oocytes affect the rate and number of euploid blastocysts in in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril. 2017; 108:777–783.e2. 22. Shahine LK, Marshall L, Lamb JD, Hickok LR. Higher rates of aneuploidy in blastocysts and higher risk of no embryo transfer in recurrent pregnancy loss patients with diminished ovarian reserve undergoing in vitro fertilization. Fertil Steril. 2016;106:1124–8. 23. Kushnir VA, Darmon SK, Albertini DF, Barad DH, Gleicher N. Effectiveness of in vitro fertilization with preimplantation genetic screening: a reanalysis of United States assisted reproductive technology data 2011-2012. Fertil Steril. 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Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes following transfer of mosaic embryos

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Springer Journals
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2018 The Author(s).
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1477-7827
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10.1186/s12958-018-0322-5
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Abstract

Background: Preimplantation genetic screening (PGS) is increasingly utilized as an adjunct procedure to IVF. Recently healthy euploid live birth were reported following transfer of mosaic embryos. Several recent publications have surmised that the degree of trophectoderm (TE) mosaicism in transferred embryos is predictive of ongoing pregnancy and miscarriage rates. Methods: This is a corrected analysis of previously published retrospective data on vitro fertilization (IVF) cycle outcomes involving replacement of 143 mosaic and 1045 euploid embryos tested by PGS, utilizing high-resolution next-generation sequencing (NGS) of TE and determination of percentages of mosaicism. Receiver operating curves (ROCs) and measurement of area under the curve (AUC) were used to evaluated the accuracy of the predictor variable, proportion of aneuploid cells in a TE biopsy specimen, with IVF outcomes, ongoing pregnancy and miscarriage rates. Results: Confirming findings of the previously published report we also found higher ongoing pregnancy rates (63.3% vs. 39.2%) and lower miscarriage rates (10.2% vs. 24.3%) with euploid embryo transfers than with mosaic embryo transfer. There, however, were no significant differences in ongoing pregnancy or miscarriage rates among mosaic embryo transfers at any threshold of aneuploidy. Based on AUC, TE biopsies predicted ongoing pregnancy for euploid, as well as mosaic embryos, in a range of 0.50 to 0.59 and miscarriage in a range from 0.50 to 0.66 Conclusions: Degree of TE mosaicism was a poor predictor of ongoing pregnancy and miscarriage. Keywords: In vitro fertilization, Preimplantation genetic diagnosis, Preimplantation genetic screening, Aneuploidy, Embryo selection, Next-generation sequencing Background trophectoderm (TE) mosaicism is a common finding [2], Preimplantation genetic screening (PGS), now renamed the Preimplantation Genetic Diagnosis International Soci- by some as preimplantation genetic testing for aneu- ety (PGDIS) recently recommended a radical overhaul of ploidy (PGT-A), is increasingly utilized as ad-on to vitro testing methodologies and reporting of test results [3]. fertilization (IVF). The original hypothesis behind PGS Since diagnostic platforms, like array comparative gen- utilization was “aneuploidy screening as a means to in- ome hybridization (aCGH), single-nucleotide polymorph- crease pregnancy rates, decrease loss rates, and establish ism array, and quantitative polymerase chain reaction transfer order” [1]. Because of growing recognition that (qPCR) lack capacity to detect mosaicism in a single TE biopsy, next-generation sequencing (NGS), which cur- * Correspondence: vkushnir@thechr.com rently detects mosaicism in excess of 20%, is the only Center for Human Reproduction, 21 East 69th Street, New York, NY 10021, technique recommended by PGDIS [3]. Utilizing NGS, USA embryos with less than 20% aneuploidy in the TE sample Department of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC, USA are, therefore, considered euploid; while those between 20 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 2 of 6 and 80% are reported as mosaic, and those over 80% as aneuploidy in its TE as aneuploid and, therefore, as not truly aneuploid. Moreover, PGDIS guidelines suggest that transferrable [4]. embryos designated as euploid can be freely transferred, Both sets of guidelines, however, were lacking robust while embryos designated as aneuploid should not be published clinical data in support, as are usually required transferred and, therefore, discarded [3]. for clinical diagnostic testing [5, 6]. This why a recent Finally, mosaic embryos, at 20–80% range aneuploidy publication by a multi-center conglomerate of PGS refer- in the TE sample, may be potentially transferred, though ence laboratories and referring IVF centers, offering a first the PGDIS notes that such transfers be performed with large data sets of clinical outcomes following transfer of caution and only in absence of euploid embryos. More- mosaic blastocyst in accordance with PGDIS criteria and over, the society suggested an empirical hierarchy for utilizing NGS [7], has to be viewed as a defining moment. such transfers, based on the specific aneuploidies re- Until these recent publications, only three groups have re- ported in embryos [3]. ported IVF cycle outcomes after transfers of mosaic em- A group of investigators at the 2016 World Congress on bryos in a small number of cases [8–10]. Controversies in Preconception, Preimplantation, Prenatal Since the manuscripts by (Munné et al. 2017; Fragouli Genetic Diagnosis Meeting in Barcelona reached similar et al. 2017) did not use standard statistical methods to as- conclusions, advising to prioritize transfer of mosaic sess predictive values for different degrees of mosaicism embryos with lower levels (20–40%) of aneuploidy in detected in TE biopsies, we reanalyzed the raw data re- the TE sample over those with higher levels (40–70%), ported by the authors. As this study will demonstrate, our and defining any embryo biopsy with more than 70% analysis contradicts the conclusions of Munné et al. that Table 1 Predictive ability of NGS trophectoderm biopsy for ongoing pregnancy based on proportion of abnormal cells in the specimen Euploid < 20% Abnormal Mosaic ≥20% to ≤80% Abnormal p-value AUC Ongoing Pregnancy 661/1045 63.3% 56/143 39.2% < 0.0001 0.55 Mosaic ≥20% to < 30% Abnormal Mosaic ≥30% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 12/27 44.4% 22/61 36.1% 0.46 0.54 ≥ 38 1/8 12.5% 21/46 45.7% 0.08 0.59 All Ages 13/35 37.1% 43/108 39.9% 0.78 0.51 Mosaic ≥20% to < 40% Abnormal Mosaic ≥40% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 22/61 36.1% 12/27 44.4% 0.46 0.54 ≥ 38 10/25 40.0% 12/29 41.4% 0.92 0.51 All Ages 32/87 36.8% 24/56 42.9% 0.47 0.53 Mosaic ≥20% to < 50% Mosaic Mosaic ≥50% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 30/78 38.5% 4/10 40.0% 0.93 0.50 ≥ 38 17/38 44.7% 5/16 31.3% 0.36 0.56 All Ages 47/117 40.2% 9/26 34.6% 0.60 0.52 Mosaic ≥20% to < 60% Abnormal Mosaic ≥60% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 34/86 39.5% 0/2 0.0% 0.26 0.52 ≥ 38 18/44 40.9% 4/10 40.0% 0.96 0.50 All Ages 52/131 39.7% 4/12 33.3% 0.67 0.51 Mosaic ≥20% to < 70% Abnormal Mosaic ≥70% to ≤80% Abnormal p-value AUC Age Ongoing Pregnancy < 38 34/87 39.1% 0/1 0.0% 0.42 0.51 ≥ 38 19/46 41.3% 3/8 37.5% 0.84 0.51 All Ages 53/134 39.6% 3/9 33.3% 0.71 0.51 AUC: area under the curve Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 3 of 6 40% mosaicism represents a significant differentiation point. Indeed, our analysis did not find significant predict- ability at any level of mosaicism between 20 and 80%. Methods A detailed description of patient factors and molecular methods is available in the original publications, which served as data sources for our study [7, 11]. The data for 143 NGS-tested mosaic embryos that were transferred, were extracted from Additional file 1: Table S3 in the publi- cation by Munné et al. [7]. To assess whether the de- gree of mosaicism at different ages affected ongoing pregnancy rates, these data were stratified for female ages < 38 and ≥38 years. Data for the comparison group came from for female age well matched controls from the same publication, who had undergone transfers of 1045 euploid embryos, as determined by NGS with < 20% aneuploid cells in the TE sample [7]. This control group could not be age- stratified since only aggregate data (rather than embryo level data) were provided in the source publication. Receiver operating characteristic curves (ROCs) and measurements of area under the curve (AUC) were used to evaluated accuracy of the predictor variable, proportion of abnormal cells in a TE biopsy specimen (i.e., percentage mosaicism) with IVF outcomes, including ongoing preg- nancy and miscarriage rates. The analysis was then performed for binary variables eu- ploid (< 20% aneuploid cells) vs. mosaic embryos, and for Fig. 1 Receiver operating characteristic (ROC) curves for ongoing various thresholds of aneuploidy, ranging from 20% to pregnancy and miscarriage. a Analysis for binary classification euploid 80% in 10% increments, as detailed in Table 1 and Fig. 1. (< 20% aneuploid cells) vs. mosaic embryos (20% to 80% aneuploid Comparisons between groups were made using chi- cells) based on trophectoderm sample. b Various thresholds of square tests. Statistical analyses were preformed using SAS aneuploidy within the cohort of mosaic embryos ranging from 20% to 80% in 10% increments. Predictor variable: proportion of abnormal version 9.4. A p-value < 0.05 was considered statistically cells in a trophectoderm biopsy specimen. Outcomes: ongoing significant. pregnancy and miscarriage. Area under the curve (AUC) of 0.50 Since all here addressed data were already published, denotes the screening test as having no predictive ability while an publicly available, and cannot be utilized to identify indi- AUC of 1.0 denotes an ideal screening test vidual patients, this study qualified for exemption from IRB approval. threshold of aneuploidy. Moreover, the table demon- Results strates the AUC for all comparisons ranges from 0.50 to Table 1 shows the ability of TE biopsies, utilizing NGS, to 0.59, indicating poor predictive ability of TE biopsies for predict ongoing pregnancy based on proportion of abnor- ongoing pregnancies. mal cells in each biopsy specimen. Here we confirm the Table 2 shows the predictive ability of TE biopsies, util- findings of Munné et al. [7] that ongoing pregnancy rates izing NGS, to predict miscarriages based on proportion of were, seemingly, significantly higher following euploid em- abnormal cells in a single biopsy sample. Once again we bryo transfer (i.e., biopsies with < 20% aneuploidy) than confirm the findings of Munné et al. [7] that miscarriage mosaic embryo transfer (biopsies with 20–80% aneu- rates were significantly lower following euploid embryo ploidy). Our calculation yielded a slightly lower ongoing transfer than with mosaic embryo transfer. However, the pregnancy rate in the mosaic group than Munné et al., table also demonstrates that the AUC is only 0.56, again who reported 57 ongoing and delivered pregnancies but indicting very poor predictive validity of a TE biopsy for presented embryo level data for only 56. miscarriages. Moreover, analysis of miscarriage risk among Table 1 demonstrates no significant differences in on- mosaic embryo transfers, did not find increased risk based going pregnancy rates among mosaic transfers at any on any threshold of aneuploidy. Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 4 of 6 Table 2 Predictive ability of NGS trophectoderm biopsy for miscarriage based on proportion of abnormal cells in the specimen Euploid < 20% Abnormal Mosaic ≥20% to ≤80% Abnormal p-value AUC Miscarriage 75/736 10.2% 18/74 24.3% 0.0003 0.56 Mosaic ≥20% to < 30% Abnormal Mosaic ≥30% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 5/17 29.4% 9/31 29.0% 0.69 0.50 ≥ 38 0/1 0.0% 4/25 16.0% 0.66 0.52 All Ages 5/18 27.8% 13/56 23.2% 0.69 0.52 Mosaic ≥20% to < 40% Abnormal Mosaic ≥40% to ≤80% Abnormal p-value AUC Miscarriage < 38 11/33 33.3% 3/15 20.0% 0.35 0.57 ≥ 38 2/12 16.7% 2/14 14.3% 0.87 0.52 All Ages 13/45 28.9% 5/29 17.2% 0.25 0.58 Mosaic ≥20% to < 50% Mosaic Mosaic ≥50% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 13/43 30.2% 1/5 20.0% 0.63 0.52 ≥ 38 2/19 10.5% 2/7 28.6% 0.26 0.64 All Ages 15/62 24.2% 3/12 25.0% 0.95 0.50 Mosaic ≥20% to < 60% Abnormal Mosaic ≥60% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 14/48 29.2% 0/0 0.0% –– ≥ 38 2/20 10.0% 2/6 33.3% 0.16 0.66 All Ages 16/68 23.5% 2/6 33.3% 0.59 0.59 Mosaic ≥20% to < 70% Abnormal Mosaic ≥70% to ≤80% Abnormal p-value AUC Age Miscarriage < 38 14/48 29.2% 0/0 0.0% –– ≥ 38 3/22 13.6% 1/4 25.0% 0.56 0.56 All Ages 17/70 24.3% 1/4 25.0% 0.97 0.50 AUC: area under the curve Figure 1a demonstrates ROC curves for ongoing preg- single TE biopsy as determined by NGS, has poor pre- nancies and miscarriages based on binary classification dictive value as a screening test for an individual em- of embryos as either euploid or mosaic. Figure 1b shows bryo’s ability to establish ongoing pregnancy or lead to ROC curves for ongoing pregnancies and miscarriages miscarriage. Moreover, given the observed ongoing preg- based on various thresholds of aneuploidy (in 10% incre- nancy and miscarriage rates it appears likely that in ments, ranging from 20% to 80%) within the group of many cases aneuploidy within the TE of embryos repre- mosaic embryos. Both figures show a null line for refer- sents a physiologic variant rather than a pathological ence with an AUC of 0.50 which denotes when a screen- condition. This conclusion is supported by a recent in ing test has no predictive ability. depth literature review: “Mosaicism in Preimplantation Human Embryos: When Chromosomal Abnormalities Discussion Are the Norm” [13]. Our analysis confirms the previously published report Commonly utilized screening tests in reproductive medi- which found statistically higher ongoing pregnancy rates cine, such as mammograms and PAP smears for cancer (63.3% vs. 39.2%) and lower miscarriage rates (10.2% vs. screening [14], while themselves not ideal screening tests, 24.3%) with euploid embryo transfers than with mosaic still demonstrate far more robust clinical performance than embryo transfer. These findings are also consistent with here presented PGS data. This study reinforces previously a recent report by Spinella et al. [12]. However, our ana- raised concerns about the biological premise and clinical lysis demonstrates with ROC curves that PGS, at any effectiveness of PGS [15–18]. It is our view that to ensure threshold level of mosaicism, up to 80% aneuploidy in a optimal outcomes for patients the reproductive medicine Kushnir et al. Reproductive Biology and Endocrinology (2018) 16:6 Page 5 of 6 community should hold commercial PGS tests to the same Administration; IVF: In vitro fertilization; LDTs: Laboratory-developed tests; NGS: Next-generation sequencing; PGDIS: Preimplantation Genetic Diagnosis standards as other screening tests commonly utilized in International Society; PGS: Preimplantation genetic screening; the field. Therefore, in our opinion commercial laborator- qPCR: Quantitative polymerase chain reaction; ROCs: Receiver operating ies which market PGS tests should seek regulatory ap- curves; TE: Trophectoderm proval for these tests [19]. Acknowledgements Based on the original data set published by Munné et al. Not applicable [7], our analysis may, indeed, actually still overestimate the benefits of PGS in this patient population. In the original Authors’ contribution VAK, DHB, and NG developed the concept of the study; All authors contributed data set, the authors indicated that 133 out of 143 mosaic to data accumulation; SKD, DHB and VAK contributed to data analysis; All embryo transfers were single embryo transfers, while 10 authors contributed to data interpretation. VAK and NG wrote the manuscript. were double embryo transfers (such data were not pro- All authors contributed to revisions of the manuscript, and approved of the final submission. VAK takes responsibility for the accuracy of the data analysis. vided for the control group of patients undergoing euploid embryo transfer). A higher proportion of double embryo Funding transfers in the euploid embryo group, could, therefore, Intramural funds from the CHR and by grants from The Foundation for hypothetically explain the higher ongoing pregnancy rate Reproductive Medicine. and lower miscarriage rates observed in this group. Availability of data and materials It is also likely that patients who produce euploid em- Data were obtained from the referenced publications. Further information is bryos have better prognoses and/or less severe under- available by contacting Dr. Vitaly A. Kushnir at vkushnir@thechr.com. lying infertility than those who only produce mosaic Ethics approval and consent to participate embryos. This point is consistent with several recent Since all here addressed data were already published, publicly available, and studies which have found a correlation between propor- cannot be utilized to identify individual patients, this study qualified for tion of aneuploid blastocysts and diminished ovarian re- exemption from IRB approval. serve while controlling for patient age [20–22]. Finally, Consent for publication the original dataset does not specify the number of Not applicable treated patients nor the number of tested embryos per patient in either group. It reflects only patients who Competing interests V.A.K previously served as a consultant to the CDC. N.G., D.H.B., and V.A.K. are reached embryo transfer, thereby excluding poorer prog- listed as co-owners of several already awarded and still pending U.S. patents, nosis patients who started IVF with intention of PGS but none related to the topic of this manuscript. N.G. is a shareholder in Fertility did not have any transferrable embryos following extended Nutraceuticals, LLC and owner of the CHR. N.G. and D.H.B. receive patent royalties from Fertility Nutraceuticals, LLC. embryo culture, TE biopsy, cryopreservation, and thawing. The patient selection is, therefore, biased toward better prognosis patients, as previously documented in national Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in registry data in patients undergoing IVF with PGS [23]. published maps and institutional affiliations. Additional limitations include reporting of ongoing preg- nancy rates rather than live birth rates and lack of data on Author details Center for Human Reproduction, 21 East 69th Street, New York, NY 10021, the health of infants conceived from mosaic embryos. USA. Department of Obstetrics and Gynecology, Wake Forest School of We conclude that the degree of TE mosaicism is a Medicine, Winston-Salem, NC, USA. Foundation for Reproductive Medicine, poor predictor of ongoing pregnancy and miscarriage. New York, NY, USA. Stem Cell Biology and Molecular Embryology Laboratory, The Rockefeller University, New York, NY, USA. Department of Moreover, in our ROC analysis PGS demonstrates poor Obstetrics and Gynecology, University of Vienna School of Medicine, Vienna, clinical effectiveness for a routine screening test and, Austria. therefore, should not be routinely offered as an ad-on to Received: 14 September 2017 Accepted: 10 January 2018 IVF. 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Fragouli E, Alfarawati S, Spath K, Babariya D, Tarozzi N, Borini A, Wells D. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts. Hum Genet. 2017;136:805–19. 12. Spinella F, Fiorentino F, Biricik A, Bono S, Ruberti A, Cotroneo E, Baldi M, Cursio E, Minasi MG, Greco E. Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments. Fertil Steril. 2017; 13. McCoy RC. Mosaicism in preimplantation human embryos: when chromosomal abnormalities are the norm. Trends Genet. 2017;33:448–63. 14. Maxim LD, Niebo R, Utell MJ. Screening tests: a review with examples. Inhal Toxicol. 2014;26:811–28. 15. Gleicher N, Kushnir VA, Barad DH. Preimplantation genetic screening (PGS) still in search of a clinical application: a systematic review. Reprod Biol Endocrinol. 2014;12:22. 16. Mastenbroek S, Repping S. Preimplantation genetic screening: back to the future. Hum Reprod. 2014;29:1846–50. 17. 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Journal

Reproductive Biology and EndocrinologySpringer Journals

Published: Dec 1, 2018

Keywords: reproductive medicine; endocrinology

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