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The influence of morphologic grading and COS protocol on the outcomes of Day 5 versus Day 6 single fresh blastocyst transfers: a retrospective analysis of clinical outcomes from one center experience



The purpose of this study was to evaluate whether the blastocyst morphologic grading and the protocol of controlled ovarian stimulation (COS) would influence pregnancy outcomes, aiming to provide guidance when choosing blastocyst transfer.


The clinical data of 612 patients who received single fresh blastocyst transfer for first cycle, as well as the data of 253 patients who had already delivered were analyzed retrospectively. The patients were divided into two groups according to blastocyst formation time (D5 or D6). The following subgroup analyses were performed: (i) the morphologic grading of blastocyst and (ii) the protocol of COS.


We observed that D5 single embryo transfer (SET) were associated with higher clinical pregnancy rate (CPR, 59.04% vs. 31.73%, P < 0.001) and live birth rate (LBR, 43.90% vs. 24.04%, P < 0.001) than D6 SET following fresh cycle. Patients in D5 group experienced more good blastocysts transfer (45.47%vs. 13.46%, P < 0.001) and less poor blastocysts transfer (9.64%vs. 45.19%, P < 0.001) than patients in D6 group. As to early stage and good quality blastocysts, the CPR and LBR were similar between D5 and D6 group. GnRH antagonist protocol had a demonstrable inferiority comparing with the early-follicular-phase long-acting GnRH-agonist long protocol (EFLL) or the mid-luteal-phase long-acting GnRH-agonist long protocol (MLLL) with regard to the CPR and LBR in D6-SET group.


The analysis found that ovarian reserve of patients in D6-SET group was comparatively worse than that of patients in D5-SET group and D6-SET patients represented a subgroup of infertility patients usually having relatively poor embryo quality. The results should be interpreted with caution as the very low numbers in the respective group limited the use of statistical tests and the real significance values.

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Fig. 1

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.



Antral follicle counting


Anti-Müllerian hormone


Assisted reproductive technology


Body mass index


Controlled ovarian stimulation


Clinical pregnancy rate


Double blastocyst transfer


Early-follicular-phase long-acting GnRH-agonist long protocol


Embryo transfer


Frozen-embryo transfer


Gonadotropin releasing hormone agonist


In vitro fertilization


Inner cell mass


Intracytoplasmic sperm injection


Trophectoderm cells


Live birth rate


Mid-luteal-phase long-acting GnRH-agonist long protocol


Ovarian hyperstimulation syndrome




Preimplantation genetic testing for aneuploidy


Randomized controlled trials


Recombinant follicle-stimulating hormone


Single embryo transfers (SET)


Single blastocyst transfer


  1. Bhattacharya S, Kamath MS (2014) Reducing multiple births in assisted reproduction technology. Best Pract Res Clin 28:191–199

    Google Scholar 

  2. Cutting R (2018) Single embryo transfer for all. Best Pract Res Clin 53:30–37

    Google Scholar 

  3. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 69(1):84–88

    CAS  Article  Google Scholar 

  4. Glujovsky D, Farquhar C, Quinteiro RAM, Alvarez SCR, Blake D (2016) Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev 30(6):CD002118

    Google Scholar 

  5. Shapiro BS, Richter KS, Harris DC, Daneshmand ST (2001) A comparison of day 5 and day 6 blastocyst transfers. Fertil Steril 75(6):1126–1130

    CAS  Article  Google Scholar 

  6. Barrenetxea G, Larruzea AL, Ganzabal T, Jiménez R, Carbonero K, Mandiola M (2005) Blastocyst culture after repeated failure of cleavage-stage embryo transfers: a comparison of day 5 and day 6 transfers. Fertil Steril 83(1):49–53

    Article  Google Scholar 

  7. Sunkara SK, Siozos A, Bolton VN, Khalaf Y, Braude PR, El-Toukhy T (2010) The influence of delayed blastocyst formation on the outcome of frozen-thawed blastocyst transfer: a systematic review and meta-analysis. Hum Reprod 25(8):1906–1915

    Article  Google Scholar 

  8. Bourdon M, Pocate-Cheriet K, Finet de Bantel A, Grzegorczyk-Martin V, Amar Hoffet A, Arbo E, Poulain M, Santulli P (2019) Day 5 versus day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes. Hum Reprod 34(10):1948–1964

    Article  Google Scholar 

  9. Murugappan G, Kim JG, Kort JD, Hanson BM, Neal SA, Tiegs AW, Osman EK, Scott RT, Lathi RB (2020) Prognostic value of blastocyst grade after frozen euploid embryo transfer in patients with recurrent pregnancy loss. F S Rep 1(2):113–118

    PubMed  PubMed Central  Google Scholar 

  10. Peng XD, Yu M, Li L, Fu W, Chen H, Sun X, Chen J (2020) Effects of euploid blastocyst morphological development on reproductive outcomes. Reprod Biol 20(4):496–500

    Article  Google Scholar 

  11. Xiong F, Sun Q, Li GG, Yao Z, Chen P, Wan C, Zhong H, Zeng Y (2020) Association between the number of top-quality blastocysts and live births after single blastocyst transfer in the first fresh or vitrified-warmed IVF/ICSI cycle. Reprod Biomed Online 40(4):530–537

    Article  Google Scholar 

  12. Papanikolaou EG, D’haeseleer E, Verheyen G, Van de Velde H, Camus M, Van Steirteghem A, Devroey P, Tournaye H (2005) Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture A randomized prospective study. Hum Reprod 20(11):3198–3203

    Article  Google Scholar 

  13. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB (2000) Blastocyst score affects implantation and pregnancy outcome. Fertil Steril 73:1155–1158

    CAS  Article  Google Scholar 

  14. Ferreux L, Bourdon M, Sallem A, Santulli P, Barraud-Lange V, Foll NL, Maignien C, Chapron C, Ziegler D, Wolf JP, Pocate-Cheriet K (2018) Live birth rate following frozen-thawed blastocyst transfer is higher with blastocysts expanded on Day 5 than on Day 6. Hum Reprod 33:390–398

    Article  Google Scholar 

  15. Younes G, Tannus S, Son WY, Dahan MH (2019) When to do intracytoplasmic sperm injection: a prospective comparison. Arch Gynecol Obstet 300(5):1461–1471

    CAS  Article  Google Scholar 

  16. Yang HY, Yang QL, Dai SJ, Li G, Jin HX, Yao GD, Sun YP (2016) Comparison of differences in development potentials between frozen-thawed D5 and D6 blastocysts and their relationship with pregnancy outcomes. J Assist Reprod Genet 33:865–872

    Article  Google Scholar 

  17. Kroener L, Ambartsumyan G, Briton-Jones C, Dumesic D, Surrey M, Munné S, Hill D (2012) The effect of timing of embryonic progression on chromosomal abnormality. Fertil Steril 98:876–880

    Article  Google Scholar 

  18. Piccolomini MM, Nicolielo M, Bonetti TCS, Motta ELA, Serafini PC, Alegretti JR (2016) Does slow embryo development predict a high aneuploidy rate on trophectoderm biopsy? Reprod Biomed Online 33:398–403

    Article  Google Scholar 

  19. Shen X, Long H, Gao HY, Guo WY, Xie YT, Chen D, Cong YY, Wang Y, Li DY, Si JQ, Zhao LW, Lyu QF, Kuang YP, Wang L (2020) The valuable reference of live birth rate in the single vitrified-warmed BB/BC/CB blastocyst transfer: The cleavage-stage embryo quality and embryo development speed. Front Physiol 11:1102

    Article  Google Scholar 

  20. McDaniel KE, Awadalla MS, McGinnis LK, Ahmady L (2021) Transfer the best and biopsy the rest? Blastocyst euploidy rates differ by morphology and day of biopsy. Arch Gynecol Obstet 303(1):249–258

    Article  Google Scholar 

  21. Dobson SJA, Lao MT, Michael E, Varghese AC, Jayaprakasan K (2018) Effect of transfer of a poor quality embryo along with a top quality embryo on the outcome during fresh and frozen in vitro fertilization cycles. Fertil Steril 110(4):655–660

    Article  Google Scholar 

  22. Chen SP, Du HZ, Liu JQ, Liu HY, Li L, He YX (2020) Live birth rate and neonatal outcomes of different quantities and qualities of frozen transferred blastocyst in patients requiring whole embryo freezing stratified by age. BMC Pregnancy Childbirth 20:655

    CAS  Article  Google Scholar 

  23. Barash OO, Ivani KA, Willman SP, Rosenbluth EM, Wachs DS, Hinckley MD, Reid SP, Weckstein LN (2017) Association between growth dynamics, morphological parameters, the chromosomal status of the blastocysts, and clinical outcomes in IVF PGS cycles with single embryo transfer. J Assist Reprod Genet 34(8):1007–1016

    Article  Google Scholar 

  24. Xie F, Timme KA, Wood JR (2018) Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos. Sci Rep 8(1):7930

    Article  Google Scholar 

  25. Lonergan P, Rizos D, Gutiérrez-Adán A, Fair T, Boland MP (2003) Effect of culture environment on embryo quality and gene expression-experience from animal studies. Reprod Biomed Online 7(6):657–663

    CAS  Article  Google Scholar 

  26. Wrenzycki C, Herrmann D, Niemann H (2003) Timing of blastocyst expansion affects spatial messenger RNA expression patterns of genes in bovine blastocysts produced in vitro. Biol Reprod 68(6):2073–2080

    CAS  Article  Google Scholar 

  27. Xi QS, Yang QY, Wang M, Huang B, Zhang B, Li Z, Liu S, Yang L, Zhu LX, Jin L (2021) Individualized embryo selection strategy developed by stacking machine learning model for better in vitro fertilization outcomes: an application study. Reprod Biol Endocrinol 19(1):53

    Article  Google Scholar 

  28. Bouillon C, Celton N, Kassem S, Frapsauce C, Guérif F (2017) Obstetric and perinatal outcomes of singletons after single blastocyst transfer: is there any difference according to blastocyst morphology? Reprod Biomed Online 35:197–207

    Article  Google Scholar 

  29. Kalra SK, Ratcliffe SJ, Barnhart KT, Coutifaris C (2012) Extended embryo culture and an increased risk of preterm delivery. Obstet Gynecol 120(1):69–75

    Article  Google Scholar 

  30. Dar S, Librach CL, Gunby J, Bissonnette F, Cowan L (2013) Increased risk of preterm birth in singleton pregnancies after blastocyst versus Day 3 embryo transfer: Canadian ART Register (CARTR) analysis. Hum Reprod 28(4):924–928

    CAS  Article  Google Scholar 

  31. Lou H, Li N, Guan YC, Zhang YC, Hao DY, Cui SH (2021) Association between morphologic grading and implantation rate of euploid blastocyst. J Ovarian Res 14(1):18

    CAS  Article  Google Scholar 

  32. Ren JZ, Sha AG, Han DM, Li P, Geng J, Ma CH (2014) Does prolonged pituitary down-regulation with gonadotropin-releasing hormone agonist improve the live-birth rate in in vitro fertilization treatment? Fertil Steril 102:75–81

    CAS  Article  Google Scholar 

  33. Geng YD, Xun Y, Hu SQ, Lai QH, Jin L (2019) GnRH antagonist versus follicular phase single-dose GnRHa protocol in patients of normal ovarian response during controlled ovarian stimulation. Gynecol Endocrinol 35:309–313

    CAS  Article  Google Scholar 

  34. Yang JW, Zhang XD, Ding XY, Wang YT, Huang GN, Ye H (2021) Cumulative live birth rates between GnRH-agonist long and GnRH-antagonist protocol in one ART cycle when all embryos transferred: real-word data of 18,853 women from China. Reprod Biol Endocrinol 19(1):124

    CAS  Article  Google Scholar 

  35. Lambalk CB, Banga FR, Huirne JA, Toftager M, Pinborg A, Homburg R, van der Veen F, van Wely M (2017) GnRH antagonist versus long agonist protocols in IVF: a systematic review and meta-analysis accounting for patient type. Hum Reprod Update 23:560–579

    CAS  Article  Google Scholar 

  36. Xu B, Geerts D, Hu SQ, Yue J, Li Z, Zhu GJ, Jin L (2020) The depot GnRH agonist protocol improves the live birth rate per fresh embryo transfer cycle, but not the cumulative live birth rate in normal responders: a randomized controlled trial and molecular mechanism study. Hum Reprod 35(6):1306–1318

    CAS  Article  Google Scholar 

  37. Li N, Sun J, Wang J, Jian W, Lu J, Miao YH, Li YF, Chen F, Chen DJ, Ye XQ, Chen M (2021) Selective termination of the fetus in multiple pregnancies using ultrasound-guided radiofrequency ablation. BMC Pregnancy Childbirth 21(1):821

    CAS  Article  Google Scholar 

  38. Poikkeus P, Gissler M, Unkila-Kallio L, Hyden-Granskog C, Tiitinen A (2007) Obstetric and neonatal outcome after single embryo transfer. Hum Reprod 22(4):1073–1079

    CAS  Article  Google Scholar 

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The authors are grateful to all staff of the Reproductive Medicine center, Department of Obstetrics and Gynecology, Wuhan Kangjian Maternal and Infant Hospital, Wuhan, Hubei, China for making the project successful.


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ZC and WL are responsible for the concept and the study design. CX and YL performed the data collection, and WL did the statistical analysis. ZC drafted the manuscript. AG contributed to the critical discussion, interpretation and editing of the manuscript.

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Correspondence to Aidong Gong.

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Chen, Z., Li, W., Xiao, C. et al. The influence of morphologic grading and COS protocol on the outcomes of Day 5 versus Day 6 single fresh blastocyst transfers: a retrospective analysis of clinical outcomes from one center experience. Arch Gynecol Obstet 306, 1739–1752 (2022).

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  • Blastocyst
  • D5
  • D6
  • Fresh blastocyst transfer
  • GnRH antagonist protocol
  • Early-follicular-phase long-acting GnRH-agonist long protocol (EFLL)
  • Mid-luteal-phase long-acting GnRH-agonist long protocol (MLLL)
  • Pregnancy outcomes