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Blastocyst age, expansion, trophectoderm morphology, and number cryopreserved are variables predicting clinical implantation in single blastocyst frozen embryo transfers in freeze-only-IVF

  • Assisted Reproduction Technologies
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To determine which blastocyst assessment variables predict clinical implantations in single blastocyst frozen embryo transfers (FET) of freeze-only-IVF cycles, following improved vitrified-warmed blastocyst survival and developmental competence preservation.


In this retrospective cohort study performed at a single private IVF center, the pregnancy outcomes of 1795 single blastocyst FET cycles were analyzed, from freeze-only-IVF retrievals performed between January 2017 and January 2020. Stepwise forward logistic regressions with clinical implantation (i.e., normal gestational sac and cardiac activity) as dependent variable were performed to identify the significant predictors. All blastocysts were vitrified using Cryotop technology, with before transfer (post-warming) blastocyst morphology scores used in all analyses.


The 1795 blastocysts transferred were vitrifıed on embryo days 4 (1057), 5 (716), and 6 (22). The overall clinical implantation rate was 50.9%; however, using blastocyst age and blastocyst morphological score the clinical implantation rates increased from 49.0% (day-4 1 and 2) and 25.2% (day-5 1 and 2) to 71.2% (day-4 4AA) and 64.3% (day-5 4AA), respectively. Whereas full (≥3) blastocysts with scores of AA and BA had similar clinical implantation rates (66.2 vs. 66.7%), the rate of full blastocysts with scores of AB was lower (58.9%). In stepwise forward logistic regressions, female age, blastocyst age, blastocyst expansion score, blastocyst trophectoderm score, and number of blastocysts vitrified were significant predictors of clinical implantation.


Using blastocyst age and before transfer blastocyst expansion and trophectoderm morphology scores to select blastocysts, clinical implantation rates greater than 70% could be achieved for top-scoring blastocysts.

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  1. 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;26:1270–83.

    Article  Google Scholar 

  2. Van Royen E, Mangelschots K, De Neubourg D, Valkenburg M, Van de Meerssche M, Ryckaert G, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod. 1999;14:2345–9.

    Article  Google Scholar 

  3. Arce JC, Ziebe S, Lundin K, Janssens R, Helmgaard L, Sorensen P. Interobserver agreement and intraobserver reproducibility of embryo quality assessments. Hum Reprod. 2006;21:2141–8.

    Article  Google Scholar 

  4. Storr A, Venetis CA, Cooke S, Kilani S, Ledger W. Inter-observer and intra-observer agreement between embryologists during selection of a single day 5 embryo for transfer: a multicenter study. Hum Reprod. 2017;32:307–14.

    Article  Google Scholar 

  5. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000;73:1155–8.

    Article  CAS  Google Scholar 

  6. Fragouli E, Lenzi M, Ross R, Katz-Jaffe M, Schoolcraft WB, Wells D. Comprehensive molecular cytogenetic analysis of the human blastocyst stage. Hum Reprod. 2008;23:2596–608.

    Article  CAS  Google Scholar 

  7. Demko ZP, Simon AL, McCoy RC, Petrov DA, Rabinowitz M. Effects of maternal age on euploidy rates in a large cohort of embryos analyzed with 24-chromosome single-nucleotide polymorphism-based preimplantation genetic screening. Fertil Steril. 2016;105:1307–13.

    Article  CAS  Google Scholar 

  8. Gardner DK, Schoolcraft WB. In vitro culture of human blastocyst. In: Mortimer JR, editor. Toward Reproductive Certainty: Infertility and Genetics Beyond 1999. Carnforth: Parthenon Press; 1999. p. 378–88.

    Google Scholar 

  9. Kovacic B, Vlaisavljevic V, Reljic M, Cizek-Sajko M. Developmental capacity of different morphological types of day 5 human morulae and blastocysts. Reprod BioMed Online. 2004;8:687–94.

    Article  Google Scholar 

  10. Thompson SM, Onwubalili N, Brown K, Jindal SK, McGovern PG. Blastocyst expansion score and trophectoderm morphology strongly predict successful clinical pregnancy and live birth following elective single embryo blastocyst transfer (eSET): a national study. J Assist Reprod Genet. 2013;30:1577–81.

    Article  Google Scholar 

  11. Van den Abbeel E, Balaban B, Ziebe S, Lundin K, Cuesta MJ, Klein BM, et al. Association between blastocyst morphology and outcome of single-blastocyst transfer. Reprod BioMed Online. 2013;27:353–61.

    Article  Google Scholar 

  12. Du QY, Wang EY, Huang Y, Guo XY, Xiong YJ, Yu YP, et al. Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles. Fertil Steril. 2016;105:910–9.

    Article  Google Scholar 

  13. Bakkensen JB, Brady P, Carusi D, Romanski P, Thomas AM, Racowsky C. Association between blastocyst morphology and pregnancy and perinatal outcomes following fresh and cryopreserved embryo transfer. J Assist Reprod Genet. 2019;36:2315–24.

    Article  Google Scholar 

  14. Ahlström A, Westin C, Reismer E, Wikland M, Hardarson T. Trophectoderm morphology: an important parameter for predicting live birth after single blastocyst transfer. Hum Reprod. 2011;26:3289–96.

    Article  Google Scholar 

  15. Hill MJ, Richter KS, Heitmann RJ, Graham JR, Tucker MJ, DeCherney AH, et al. Trophectoderm grade predicts outcomes of single-blastocyst transfers. Fertil Steril. 2013;99:1283–9.

    Article  Google Scholar 

  16. Ebner T, Tritscher K, Mayer RB, Oppelt P, Duba HC, Maurer M, et al. Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender. J Assist Reprod Genet. 2016;33:49–57.

    Article  Google Scholar 

  17. Ahlström A, Westin C, Wikland M, Hardarson T. Prediction of live birth in frozen-thawed single blastocyst transfer cycles by pre-freeze and post-thaw morphology. Hum Reprod. 2013;28:1199–209.

    Article  Google Scholar 

  18. Zhao J, Yan Y, Huang X, Sun L, Li Y. Blastocoele expansion: an important parameter for predicting clinical success pregnancy after frozen-warmed blastocysts transfer. Reprod Biol Endocrinol. 2019;17:15.

    Article  Google Scholar 

  19. Honnma H, Baba T, Sasaki M, Hashiba Y, Ohno H, Fukunaga T, et al. Trophectoderm morphology significantly affects the rates of ongoing pregnancy and miscarriage in frozen-thawed single-blastocyst transfer cycle in vitro fertilization. Fertil Steril. 2012;98:361–7.

    Article  Google Scholar 

  20. Chen X, Zhang J, Wu X, Cao S, Zhou L, Wang Y, et al. Trophectoderm morphology predicts outcomes of pregnancy in vitrified-warmed single-blastocyst transfer cycle in a Chinese population. J Assist Reprod Genet. 2014;31:1475–81.

    Article  Google Scholar 

  21. Ozgur K, Bulut H, Berkkanoglu M, Humaidan P, Coetzee K. Artificial frozen embryo transfer cycle success depends on blastocyst developmental rate and progesterone timing. Reprod BioMed Online. 2018;36:269–76.

    Article  CAS  Google Scholar 

  22. Bourdon M, Pocate-Cheriet K, Finet de Bantel A, Grzegorczyk-Martin V, Amar Hoffet A, Arbo E, et al. Day 5 versus day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes. Hum Reprod. 2019;34:1948–64.

    Article  Google Scholar 

  23. Ebner T, Vanderzwalmen P, Shebl O, Urdl W, Moser M, Zech NH, et al. Morphology of vitrified/warmed day-5 embryos predicts rates of implantation, pregnancy and live birth. Reprod BioMed Online. 2009;19:72–8.

    Article  CAS  Google Scholar 

  24. Shu Y, Watt J, Gebhardt J, Dasig J, Appling J, Behr B. The value of fast blastocoele re-expansion in the selection of a viable thawed blastocyst for transfer. Fertil Steril. 2009;91:401–6.

    Article  Google Scholar 

  25. Wirleitner B, Schuff M, Stecher A, Murtinger M, Vanderzwalmen P. Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development. Hum Reprod. 2016;31:1685–95.

    Article  CAS  Google Scholar 

  26. Coello A, Meseguer M, Galán A, Alegre L, Remohí J, Cobo A. Analysis of the morphological dynamics of blastocysts after vitrification/warming: defining new predictive variables of implantation. Fertil Steril. 2017;108:659–66.

    Article  Google Scholar 

  27. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert Embryo Transf. 1985;2:59–64.

    Article  CAS  Google Scholar 

  28. Evans J, Hannan NJ, Edgell TA, Vollenhoven BJ, Lutjen PJ, Osianlis T, et al. Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence. Hum Reprod Update. 2014;20:808–21.

    Article  CAS  Google Scholar 

  29. Iwayama H, Hochi S, Yamashita M. In vitro and in vivo viability of human blastocysts collapsed by laser pulse or osmotic shock prior to vitrification. J Assist Reprod Genet. 2011;28:355–61.

    Article  Google Scholar 

  30. Ozgur K, Berkkanoglu M, Bulut H, Humaidan P, Coetzee K. Agonist depot versus OCP programming of frozen embryo transfer: a retrospective analysis of freeze-all cycles. J Assist Reprod Genet. 2016;33:207–14.

    Article  Google Scholar 

  31. Paulson RJ, Reichman DE, Zaninovic N, Goodman LR, Racowsky C. Time-lapse imaging: clearly useful to both laboratory personnel and patient outcomes versus just because we can doesn't mean we should. Fertil Steril. 2018;109:584–91.

    Article  Google Scholar 

  32. 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;15:33.

    Article  Google Scholar 

  33. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod. 2014;29:1173–81.

    Article  Google Scholar 

  34. Munné S, Kaplan B, Frattarelli JL, Child T, Nakhuda G, Shamma FN, 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;112:1071–9.

    Article  Google Scholar 

  35. Watson AJ, Barcroft LC. Regulation of blastocyst formation. Front Biosci. 2001;6:D708–30.

    Article  CAS  Google Scholar 

  36. Goto S, Kadowaki T, Tanaka S, Hashimoto H, Kokeguchi S, Shiotani M. Prediction of pregnancy rate by blastocyst morphological score and age, based on 1,488 single frozen-thawed blastocyst transfer cycles. Fertil Steril. 2011;95:948–52.

    Article  Google Scholar 

  37. Cimadomo D, Capalbo A, Levi-Setti PE, Soscia D, Orlando G, Albani E, et al. Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation. Hum Reprod. 2018;33:1992–2001.

    Article  Google Scholar 

  38. Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F, et al. Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study. Hum Reprod. 2016;31:2245–54.

    Article  Google Scholar 

  39. Consensus Group C. 'There is only one thing that is truly important in an IVF laboratory: everything’ Cairo Consensus Guidelines on IVF Culture Conditions. Reprod BioMed Online. 2020;40:33–60.

    Article  Google Scholar 

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Correspondence to Kevin Coetzee.

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Ethics approval and consent to participate

Standard and accredited procedures were perform in the treatment of all human participants, which were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki declaration and its amendments. Signed informed consent is obtained from all patients prior to the commencement of IVF treatment, which include the patients involved in this study.

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The authors declare no competing interests.

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Key message

Continuing improvement in frozen embryo transfer pregnancy rates requires blastocyst scoring and selection techniques to be re-evaluated regularly. Despite the limitations of microscopic blastocyst morphology assessment, individual blastocyst morphology scores and supplementary blastocyst variables are predictive of clinical implantation.

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Ozgur, K., Berkkanoglu, M., Bulut, H. et al. Blastocyst age, expansion, trophectoderm morphology, and number cryopreserved are variables predicting clinical implantation in single blastocyst frozen embryo transfers in freeze-only-IVF. J Assist Reprod Genet 38, 1077–1087 (2021).

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