Skip to main content
SpringerLink
Log in

Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid

  • Assisted Reproduction Technologies
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

This preclinical study aimed to evaluate whether using transferred mosaic embryos (primarily selected by embryonic morphology assessment (EMA) and compared by the noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) on cell-free DNA in blastocoel fluid (BF)) increases the rates of clinical pregnancies (CPs) and healthy live births (HLBs) and to investigate whether niPGT-A could provide valuable genetic information for the EMA-selected transferred mosaic embryos.

Methods

This study collected 215 blastocyst culture samples and 182 BF samples. Cell-free DNA from the BF was amplified and examined by next-generation sequencing–based niPGT-A. All 182 patients underwent EMA. However, only 147 underwent in vitro fertilization and embryo transfer, and only 113 clinical outcomes were followed up. Comprehensive chromosome screening for the chorionic villus sampling of spontaneous miscarriages and noninvasive prenatal testing for ongoing pregnancies were also performed.

Results

The implantation rate was 77.55% in 147 transferred high-quality embryos selected by EMA. Among 113 CPs, 16 led to spontaneous miscarriage (14.16%), and 97 resulted in HLBs (85.84%). According to the niPGT-A results for 113 patients with clinical outcomes, 80.4% had CP (euploid, 20.54%; single aneuploid, 1.79%; mosaic chromosome aneuploid and/or segmental aneuploid, 58.04%). Of all the mosaic aneuploids, 90.76% were false positive, transforming to euploid.

Conclusions

Transferred EMA-selected embryos showed higher implantation rates. The niPGT-A of BF provided valuable genetic status (“-ploid”) information, which helped reduce aneuploid-induced implantation failure and miscarriage, thereby increasing the CP and HLB rates. Additionally, majority of the transferred embryos with complex/chaotic mosaic aneuploid would likely develop HLBs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Verlinsky Y, Kuliev A. Preimplantation diagnosis of common aneuploidies in infertile couples of advanced maternal age. Hum Reprod. 1996;11(10):2076–7.

    Article  CAS  Google Scholar 

  2. Gleicher N, Orvieto R. Is the hypothesis of preimplantation genetic screening (PGS) still supportable? A review. J Ovarian Res. 2017;10(1):21.

    Article  Google Scholar 

  3. Victor AR, Griffin DK, Brake AJ, Tyndall JC, Murphy AE, Lepkowsky LT, Lal A, Zouves CG, Barnes FL, McCoy RC, et al. Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst. Hum Reprod. 2019;34(1):181–92.

    Article  CAS  Google Scholar 

  4. Lawrenz B, El Khatib I, Linan A, Bayram A, Arnanz A, Chopra R, De Munck N, Fatemi HM. The clinicians dilemma with mosaicism-an insight from inner cell mass biopsies. Hum Reprod. 2019;34(6):998–1010.

    Article  CAS  Google Scholar 

  5. Sermon K, Capalbo A, Cohen J, Coonen E, De Rycke M, De Vos A, Delhanty J, Fiorentino F, Gleicher N, Griesinger G, et al. The why, the how and the when of PGS 2.0: current practices and expert opinions of fertility specialists, molecular biologists, and embryologists. Mol Hum Reprod. 2016;22(8):845–57.

    Article  Google Scholar 

  6. Gleicher N, Patrizio P, Brivanlou A. Preimplantation genetic testing for aneuploidy - a castle built on sand. Trends Mol Med. 2021;27(8):731–42.

    Article  CAS  Google Scholar 

  7. Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, Brivanlou AH. Self-organization of the in vitro attached human embryo. Nature. 2016;533(7602):251–4.

    Article  CAS  Google Scholar 

  8. Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013;100(3):624–30.

    Article  Google Scholar 

  9. 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. 2018;109(1):77–83.

    Article  Google Scholar 

  10. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med. 2015;373(21):2089–90.

    Article  Google Scholar 

  11. Viotti M, Victor AR, Barnes FL, Zouves CG, Besser AG, Grifo JA, Cheng EH, Lee MS, Horcajadas JA, Corti L, et al. Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use. Fertil Steril. 2021;115(5):1212–24.

    Article  CAS  Google Scholar 

  12. Treff NR, Marin D. The, “mosaic” embryo: misconceptions and misinterpretations in preimplantation genetic testing for aneuploidy. Fertil Steril. 2021;116(5):1205–11.

    Article  Google Scholar 

  13. Gazzo E, Pena F, Valdez F, Chung A, Bonomini C, Ascenzo M, Velit M, Escudero E. The Kidscore™ D5 algorithm as an additional tool to morphological assessment and PGT-A in embryo selection: a time-lapse study. JBRA Assist Reprod. 2020;24(1):55–60.

    Google Scholar 

  14. Sturmey RG, Bermejo-Alvarez P, Gutierrez-Adan A, Rizos D, Leese HJ, Lonergan P. Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability. Mol Reprod Dev. 2010;77(3):285–96.

    Article  CAS  Google Scholar 

  15. Pallinger E, Bognar Z, Bodis J, Csabai T, Farkas N, Godony K, Varnagy A, Buzas E, Szekeres-Bartho J. A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer. Sci Rep. 2017;7:39927.

    Article  CAS  Google Scholar 

  16. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, Bulletti C. Genomic DNA in human blastocoele fluid. Reprod Biomed Online. 2013;26(6):603–10.

    Article  CAS  Google Scholar 

  17. Leaver M, Wells D. Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics? Hum Reprod Update. 2020;26(1):16–42.

    Article  CAS  Google Scholar 

  18. Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, Wang H, Song X, Ma T, Bo S, 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(42):11907–12.

    Article  CAS  Google Scholar 

  19. Khalife D, Abu-Musa A, Khalil A, Ghazeeri G. Towards the selection of embryos with the greatest implantation potential. J Obstet Gynaecol. 2021;41(7):1010–5.

    Article  Google Scholar 

  20. Wang J, Wei Y, Diao F, Cui Y, Mao Y, Wang W, Liu J. The association between polycystic ovary syndrome and ectopic pregnancy after in vitro fertilization and embryo transfer. Am J Obstet Gynecol. 2013;209(2):e31-139.

    Article  Google Scholar 

  21. Cai LB, Qian XQ, Wang W, Mao YD, Yan ZJ, Liu CZ, Ding W, Huang J, Chai DC, Chian RC, et al. Oocyte vitrification technology has made egg-sharing donation easier in China. Reprod Biomed Online. 2012;24(2):186–90.

    Article  Google Scholar 

  22. Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11(3):307–11.

    Article  CAS  Google Scholar 

  23. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270–83.

    Article  Google Scholar 

  24. Zhang H, Zhao YY, Song J, Zhu QY, Yang H, Zheng ML, Xuan ZL, Wei Y, Chen Y, Yuan PB, et al. Statistical approach to decreasing the error rate of noninvasive prenatal aneuploid detection caused by maternal copy number variation. Sci Rep. 2015;5:16106.

    Article  CAS  Google Scholar 

  25. Shen J, Wu W, Gao C, Ochin H, Qu D, Xie J, Gao L, Zhou Y, Cui Y, Liu J. Chromosomal copy number analysis on chorionic villus samples from early spontaneous miscarriages by high throughput genetic technology. Mol Cytogenet. 2016;9:7.

    Article  Google Scholar 

  26. Hou Y, Fan W, Yan L, Li R, Lian Y, Huang J, Li J, Xu L, Tang F, Xie XS, et al. Genome analyses of single human oocytes. Cell. 2013;155(7):1492–506.

    Article  CAS  Google Scholar 

  27. Liu W, Liu J, Du H, Ling J, Sun X, Chen D. Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium. Ann Med. 2017;49(4):319–28.

    Article  CAS  Google Scholar 

  28. Vera-Rodriguez M, Diez-Juan A, Jimenez-Almazan J, Martinez S, Navarro R, Peinado V, Mercader A, Meseguer M, Blesa D, Moreno I, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod. 2018;33(4):745–56.

    Article  CAS  Google Scholar 

  29. Kuznyetsov V, Madjunkova S, Antes R, Abramov R, Motamedi G, Ibarrientos Z, Librach C. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS ONE. 2018;13(5):e0197262.

    Article  Google Scholar 

  30. Li P, Song Z, Yao Y, Huang T, Mao R, Huang J, Ma Y, Dong X, Huang W, Huang J, et al. Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization. Sci Rep. 2018;8(1):9275.

    Article  Google Scholar 

  31. Rubio C, Rienzi L, Navarro-Sanchez L, Cimadomo D, Garcia-Pascual CM, Albricci L, Soscia D, Valbuena D, Capalbo A, Ubaldi F, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications. Fertil Steril. 2019;112(3):510–9.

    Article  CAS  Google Scholar 

  32. Yeung QSY, Zhang YX, Chung JPW, Lui WT, Kwok YKY, Gui B, Kong GWS, Cao Y, Li TC, Choy KW. 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(8):1609–21.

    Article  Google Scholar 

  33. Kuznyetsov V, Madjunkova S, Abramov R, Antes R, Ibarrientos Z, Motamedi G, Zaman A, Kuznyetsova I, Librach CL. 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):7244.

    Article  CAS  Google Scholar 

  34. Tsuiko O, Zhigalina DI, Jatsenko T, Skryabin NA, Kanbekova OR, Artyukhova VG, Svetlakov AV, Teearu K, Trosin A, Salumets A, et al. Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass. Fertil Steril. 2018;109(6):1127-1134 e1121.

    Article  Google Scholar 

  35. Huang L, Bogale B, Tang Y, Lu S, Xie XS, 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(28):14105–12.

    Article  CAS  Google Scholar 

  36. Jiao J, Shi B, Sagnelli M, Yang D, Yao Y, Li W, Shao L, Lu S, Li D, Wang X. Minimally invasive preimplantation genetic testing using blastocyst culture medium. Hum Reprod. 2019;34(7):1369–79.

    Article  CAS  Google Scholar 

  37. Tiegs AW, Tao X, Zhan Y, Whitehead C, Kim J, Hanson B, Osman E, Kim TJ, Patounakis G, Gutmann J, 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(3):627–37.

    Article  CAS  Google Scholar 

  38. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, Ferraretti AP. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102(6):1692-1699 e1696.

    Article  CAS  Google Scholar 

  39. Wu H, Ding C, Shen X, Wang J, Li R, Cai B, Xu Y, Zhong Y, Zhou C. Medium-based noninvasive preimplantation genetic diagnosis for human alpha-thalassemias-SEA. Medicine (Baltimore). 2015;94(12):e669.

    Article  CAS  Google Scholar 

  40. Cohen J, Grudzinskas G, Johnson MH. Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD? Reprod Biomed Online. 2013;26(6):520–1.

    Article  CAS  Google Scholar 

  41. Orvieto R, Shimon C, Rienstein S, Jonish-Grossman A, Shani H, Aizer A. Do human embryos have the ability of self-correction? Reprod Biol Endocrinol: RB&E. 2020;18(1):98.

    Article  CAS  Google Scholar 

  42. Scott RT Jr, Galliano D. The challenge of embryonic mosaicism in preimplantation genetic screening. Fertil Steril. 2016;105(5):1150–2.

    Article  Google Scholar 

  43. Popovic M, Dhaenens L, Boel A, Menten B, Heindryckx B. Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma. Hum Reprod Update. 2020;26(3):313–34.

    Article  CAS  Google Scholar 

  44. Munne S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, Tarozzi N, Borini A, Becker A, Zhang J, 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(1):62-71 e68.

    Article  CAS  Google Scholar 

  45. Victor AR, Tyndall JC, Brake AJ, Lepkowsky LT, Murphy AE, Griffin DK, McCoy RC, Barnes FL, Zouves CG, Viotti M. One hundred mosaic embryos transferred prospectively in a single clinic: exploring when and why they result in healthy pregnancies. Fertil Steril. 2019;111(2):280–93.

    Article  Google Scholar 

  46. Capalbo A, Poli M, Rienzi L, Girardi L, Patassini C, Fabiani M, Cimadomo D, Benini F, Farcomeni A, Cuzzi J, et al. Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial. Am J Hum Genet. 2021;108(12):2238–47.

    Article  CAS  Google Scholar 

  47. 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(7):805–19.

    Article  CAS  Google Scholar 

  48. Lin PY, Lee CI, Cheng EH, Huang CC, Lee TH, Shih HH, Pai YP, Chen YC, Lee MS. Clinical outcomes of single mosaic embryo transfer: high-level or low-level mosaic embryo, does it matter? J Clin Med. 2020;9(6):1695.

    Article  Google Scholar 

  49. Munne S, Kaplan B, Frattarelli JL, Child T, Nakhuda G, Shamma FN, Silverberg K, Kalista T, Handyside AH, Katz-Jaffe M, 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(6):1071-1079 e1077.

    Article  CAS  Google Scholar 

  50. Ozgur K, Berkkanoglu M, Bulut H, Yoruk GDA, Candurmaz NN, Coetzee K. Single best euploid versus single best unknown-ploidy blastocyst frozen embryo transfers: a randomized controlled trial. J Assist Reprod Genet. 2019;36(4):629–36.

    Article  Google Scholar 

  51. Practice Committee and Genetic Counseling Professional Group (GCPG) of the American Society for Reproductive Medicine. Clinical management of mosaic results from preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts: a committee opinion. Fertil Steril. 2020;114(2):246–54.

    Article  Google Scholar 

  52. Sachdev NM, Maxwell SM, Besser AG, Grifo JA. Diagnosis and clinical management of embryonic mosaicism. Fertil Steril. 2017;107(1):6–11.

    Article  Google Scholar 

  53. Cram DS, Leigh D, Handyside A, Rechitsky L, Xu K, Harton G, Grifo J, Rubio C, Fragouli E, Kahraman S, et al. PGDIS position statement on the transfer of mosaic embryos 2019. Reprod Biomed Online. 2019;39(Suppl 1):e1–4.

    Google Scholar 

  54. Leigh D, Cram DS, Rechitsky S, Handyside A, Wells D, Munne S, Kahraman S, Grifo J, Katz-Jaffe M, Rubio C, et al. PGDIS position statement on the transfer of mosaic embryos 2021. Reprod Biomed Online. 2022;45(1):19–25.

    Article  CAS  Google Scholar 

  55. Kahraman S, Cetinkaya M, Yuksel B, Yesil M, Pirkevi CC. The birth of a baby with mosaicism resulting from a known mosaic embryo transfer: a case report. Hum Reprod. 2020;35(3):727–33.

    Article  Google Scholar 

  56. Orvieto R, Gleicher N. Should preimplantation genetic screening (PGS) be implemented to routine IVF practice? J Assist Reprod Genet. 2016;33(11):1445–8.

    Article  Google Scholar 

  57. Li X, Hao Y, Chen D, Ji D, Zhu W, Zhu X, Wei Z, Cao Y, Zhang Z, Zhou P. Non-invasive preimplantation genetic testing for putative mosaic blastocysts: a pilot study. Hum Reprod. 2021;36(7):2020–34.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Mr. Anthony Liu from the State Key Laboratory of Reproductive Medicine, The Center for Clinical Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, for the critical readings and valuable comments.

Funding

This work was supported by the National Nature and Science Foundation of China (81730041 to JY Liu, 82071719 to W Wu), National Key R&D Program of China (2016YFC1000207 to DW Wang), National Key Research and Development Program of China (2017YFC1001301 to W Wu), Jiangsu Provincial Project (BL20122009 and YXZXB2016001 to JY Liu), and Fudan Scholar Grant to B-L Wu.

Author information

Authors and Affiliations

  1. State Key Laboratory of Reproductive Medicine, The Center for Clinical Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, China

    Lingbo Cai, Qiao Zeng, Chao Gao, Wei Wu, Jiandong Shen, Yugui Cui & Jiayin Liu

  2. Institute of Biomedical Science, Fudan University, Children’s Hospital and Shanghai Medical College, Fudan University, Shanghai, 2100032, China

    Bai-Lin Wu

  3. Departments of Pathology and Laboratory Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA, 02115, USA

    Bai-Lin Wu

  4. State Key Laboratory of Reproductive Medicine, Genetic Laboratory, The Center for Clinical Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, China

    Dao Wu Wang

Authors
  1. Lingbo Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiao Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chao Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiandong Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bai-Lin Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dao Wu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yugui Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jiayin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wei Wu, Dao Wu Wang or Jiayin Liu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Drs. Jiayin Liu and Bai-Lin Wu are the senior authors for this collaborative study.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cai, L., Zeng, Q., Gao, C. et al. Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid. J Assist Reprod Genet 39, 2483–2504 (2022). https://doi.org/10.1007/s10815-022-02651-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-022-02651-5

Keywords

Search

Navigation