Journal of Assisted Reproduction and Genetics

, Volume 34, Issue 10, pp 1377–1383 | Cite as

Blastocyst utilization rates after continuous culture in two commercial single-step media: a prospective randomized study with sibling oocytes

  • Ioannis A. SfontourisEmail author
  • Efstratios M. Kolibianakis
  • George T. Lainas
  • Christos A. Venetis
  • George K. Petsas
  • Basil C. Tarlatzis
  • Tryfon G. Lainas
Assisted Reproduction Technologies



The aim of this study is to determine whether blastocyst utilization rates are different after continuous culture in two different commercial single-step media.


This is a paired randomized controlled trial with sibling oocytes conducted in infertility patients, aged ≤40 years with ≥10 oocytes retrieved assigned to blastocyst culture and transfer. Retrieved oocytes were randomly allocated to continuous culture in either Sage one-step medium (Origio) or Continuous Single Culture (CSC) medium (Irvine Scientific) without medium renewal up to day 5 post oocyte retrieval. Main outcome measure was the proportion of embryos suitable for clinical use (utilization rate).


A total of 502 oocytes from 33 women were randomly allocated to continuous culture in either Sage one-step medium (n = 250) or CSC medium (n = 252). Fertilization was performed by either in vitro fertilization or intracytoplasmic sperm injection, and embryo transfers were performed on day 5. Two patients had all blastocysts frozen due to the occurrence of severe ovarian hyperstimulation syndrome. Fertilization and cleavage rates, as well as embryo quality on day 3, were similar in the two media. Blastocyst utilization rates (%, 95% CI) [55.4% (46.4–64.1) vs 54.7% (44.9–64.6), p = 0.717], blastocyst formation rates [53.6% (44.6–62.5) vs 51.9 (42.2–61.6), p = 0.755], and proportion of good quality blastocysts [36.8% (28.1–45.4) vs 36.1% (27.2–45.0), p = 0.850] were similar in Sage one-step and CSC media, respectively.


Continuous culture of embryos in Sage one-step and CSC media is associated with similar blastocyst development and utilization rates. Both single-step media appear to provide adequate support during in vitro preimplantation embryo development. Whether these observations are also valid for other continuous single medium protocols remains to be determined.

Clinical trial registration number: NCT02302638.


Embryo culture Single step Continuous culture Blastocyst formation Utilization rate 



The authors wish to thank Mrs. Maria Banti for embryology work and Mrs. G. Stavropoulou and Mrs. Ioanna Voulgaris for patient coordination.

Author contributions

IAS conceived the study, performed acquisition, analysis and interpretation of data, writing and revision of the manuscript, and embryology work. EMK, GTL, and CAV participated in the analysis and interpretation of data and writing and revision of the manuscript. GKP and BCT participated in the interpretation of data and revision of the manuscript. TGL had the general supervision of the study, participated in study design, analysis and interpretation of data, writing and revision of the manuscript, and performed clinical work. All authors read and approved the final manuscript.

Compliance with ethical standards

Study funding

Sage one-step medium was provided by Origio, and CSC medium was provided by Biocare Europe.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Biggers JD, Racowsky C. The development of fertilized human ova to the blastocyst stage in KSOM(AA) medium: is a two-step protocol necessary? Reprod BioMed Online. 2002;5:133–40.CrossRefPubMedGoogle Scholar
  2. 2.
    Biggers JD, Summers MC. Choosing a culture medium: making informed choices. Fertil Steril. 2008;90:473–83.CrossRefPubMedGoogle Scholar
  3. 3.
    Machtinger R, Racowsky C. Culture systems: single step. In: Smith GD, Swain JE, Pool TB, editors. Embryo culture. Methods in molecular biology, 912. Humana Press; 2012. p. 199–209.Google Scholar
  4. 4.
    Reed ML, Hamic A, Thompson DJ, Caperton CL. Continuous uninterrupted single medium culture without medium renewal versus sequential media culture: a sibling embryo study. Fertil Steril. 2009;92:1783–6.CrossRefPubMedGoogle Scholar
  5. 5.
    Ciray H, Aksoy T, Goktas C, Ozturk B, Bahceci M. Time-lapse evaluation of human embryo development in single versus sequential culture media—a sibling oocyte study. J Assist Reprod Genet. 2012;29:891–900.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Basile N, Morbeck D, Garcia-Velasco J, Bronet F, Meseguer M. Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes. Hum Reprod. 2013;28:634–41.CrossRefPubMedGoogle Scholar
  7. 7.
    Hardarson T, Bungum M, Conaghan J, Meintjes M, Chantilis SJ, Molnar L, et al. Noninferiority, randomized, controlled trial comparing embryo development using media developed for sequential or undisturbed culture in a time-lapse setup. Fertil Steril. 2015;104:1452–9.e4.CrossRefPubMedGoogle Scholar
  8. 8.
    Costa-Borges N, Bellés M, Meseguer M, Galliano D, Ballesteros A, Calderón G. Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator. Fertil Steril. 2016;105:707–13.CrossRefPubMedGoogle Scholar
  9. 9.
    Morbeck DE, Baumann NA, Oglesbee D. Composition of single-step media used for human embryo culture. Fertil Steril. 2017;107:1055–60.CrossRefPubMedGoogle Scholar
  10. 10.
    Durand M, Sermondade N, Herbemont C, Benard J, Gronier H, Boujenah J, et al. Développement embryonnaire dans 2 milieux de culture globale : étude prospective autocontrôlée sur 2059 ovocytes. Gynécologie Obstétrique & Fertilité. 2016;44:163–7.CrossRefGoogle Scholar
  11. 11.
    Lainas TG, Sfontouris IA, Zorzovilis IZ, Petsas GK, Lainas GT, Alexopoulou E, et al. Flexible GnRH antagonist protocol versus GnRH agonist long protocol in patients with polycystic ovary syndrome treated for IVF: a prospective randomised controlled trial (RCT). Hum Reprod. 2010;25:683–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Mannaerts B, Van Kuijk J, Griesinger G. Prediction of OHSS in patients treated with corifollitropin alfa or rFSH in a GnRH antagonist protocol. Hum Reprod. 2012;27:ii26–i8.Google Scholar
  13. 13.
    Abdalla HI, Ah-Moye M, Brinsden P, Howe DL, Okonofua F, Craft I. The effect of the dose of human chorionic gonadotropin and the type of gonadotropin stimulation on oocyte recovery rates in an in vitro fertilization program. Fertil Steril. 1987;48:958–63.CrossRefPubMedGoogle Scholar
  14. 14.
    Kolibianakis EM, Papanikolaou EG, Tournaye H, Camus M, Van Steirteghem AC, Devroey P. Triggering final oocyte maturation using different doses of human chorionic gonadotropin: a randomized pilot study in patients with polycystic ovary syndrome treated with gonadotropin-releasing hormone antagonists and recombinant follicle-stimulating hormone. Fertil Steril. 2007;88:1382–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Ciray HN, Aksoy T, Yaramanci K, Karayaka I, Bahceci M. In vitro culture under physiologic oxygen concentration improves blastocyst yield and quality: a prospective randomized survey on sibling oocytes. Fertil Steril. 2009;91:1459–61.CrossRefPubMedGoogle Scholar
  16. 16.
    Hong KH, Lee H, Forman EJ, Upham KM, Scott RT Jr. Examining the temperature of embryo culture in in vitro fertilization: a randomized controlled trial comparing traditional core temperature (37°C) to a more physiologic, cooler temperature (36°C). Fertil Steril. 2014;102:767–73.CrossRefPubMedGoogle Scholar
  17. 17.
    Werner MD, Hong KH, Franasiak JM, Forman EJ, Reda CV, Molinaro TA, et al. Sequential versus Monophasic Media Impact Trial (SuMMIT): a paired randomized controlled trial comparing a sequential media system to a monophasic medium. Fertil Steril. 2016;105:1215–21.CrossRefPubMedGoogle Scholar
  18. 18.
    Alpha Scientists in Reproductive Medicine, Eshre Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26:1270–83.CrossRefGoogle Scholar
  19. 19.
    Gardner D, Schoolcraft W. In vitro culture of the human blastocyst. In: Jansen R, Mortimer D, editors. Towards Reproductive Certainty: Infertility and Genetics beyond 1999. Carnforth: Parthenon publishing; 1999. p. 378–88.Google Scholar
  20. 20.
    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.CrossRefPubMedGoogle Scholar
  21. 21.
    Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update. 1997;3:367–82.CrossRefPubMedGoogle Scholar
  22. 22.
    Biggers JD. Reflections on the culture of the preimplantation embryo. Int J Dev Biol. 1998;42:879–84.PubMedGoogle Scholar
  23. 23.
    Gardner DK, Lane M, Schoolcraft WB. Culture and transfer of viable blastocysts: a feasible proposition for human IVF. Hum Reprod. 2000;15:9–23.CrossRefPubMedGoogle Scholar
  24. 24.
    Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril. 1996;65:349–53.CrossRefPubMedGoogle Scholar
  25. 25.
    Sfontouris IA, Kolibianakis EM, Lainas GT, Zorzovilis IZ, Petsas GK, Lainas TG. Blastocyst development in single-step versus sequential culture media: a prospective randomized study with sibling oocytes. Hum Reprod. 2014;29:i2.Google Scholar
  26. 26.
    Sepulveda S, Garcia J, Arriaga E, Diaz J, Noriega-Portella L, Noriega-Hoces L. In vitro development and pregnancy outcomes for human embryos cultured in either a single medium or in a sequential media system. Fertil Steril. 2009;91:1765–70.CrossRefPubMedGoogle Scholar
  27. 27.
    Summers MC, Bird S, Mirzai FM, Thornhill A, Biggers JD. Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media. Hum Fertil. 2013;16:278–85.CrossRefGoogle Scholar
  28. 28.
    Paternot G, Debrock S, D’Hooghe TM, Spiessens C. Early embryo development in a sequential versus single medium: a randomized study. Reprod Biol Endocrinol. 2010;8.Google Scholar
  29. 29.
    Sfontouris IA, Kolibianakis EM, Lainas GT, Petsas GK, Tarlatzis BC, Lainas TG. Blastocyst development in a single medium compared to sequential media. Reprod Sci. 2017:1933719116687653.Google Scholar
  30. 30.
    Sfontouris IA, Martins WP, Nastri CO, Viana IG, Navarro PA, Raine-Fenning N, et al. Blastocyst culture using single versus sequential media in clinical IVF: a systematic review and meta-analysis of randomized controlled trials. J Assist Reprod Genet. 2016;33:1261–72.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Biggers JD, McGinnis LK, Lawitts JA. One-step versus two-step culture of mouse preimplantation embryos: is there a difference? Hum Reprod. 2005;20:3376–84.CrossRefPubMedGoogle Scholar
  32. 32.
    Macklon NS, Pieters MHEC, Hassan MA, Jeucken PHM, Eijkemans MJC, Fauser BCJM. A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development. Hum Reprod. 2002;17:2700–5.CrossRefPubMedGoogle Scholar
  33. 33.
    Xie Y, Wang F, Puscheck EE, Rappolee DA. Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos. Mol Reprod Dev. 2007;74:1287–94.CrossRefPubMedGoogle Scholar
  34. 34.
    Morbeck DE, Krisher RL, Herrick JR, Baumann NA, Matern D, Moyer T. Composition of commercial media used for human embryo culture. Fertil Steril. 2014;102:759–66.e9.CrossRefPubMedGoogle Scholar
  35. 35.
    Lane M, Gardner D. Embryo culture medium: which is the best? Best Res Clin Obstet Gyn. 2007;21:83–100.CrossRefGoogle Scholar
  36. 36.
    Bontekoe S, Heineman MJ, Johnson N, Blake D. Adherence compounds in embryo transfer media for assisted reproductive technologies. Cochrane Database Syst Rev. 2014;2:Cd007421.Google Scholar
  37. 37.
    Gardner D, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod. 1998;13:148–59.CrossRefPubMedGoogle Scholar
  38. 38.
    Ahlstrom A, Wikland M, Rogberg L, Barnett JS, Tucker M, Hardarson T. Cross-validation and predictive value of near-infrared spectroscopy algorithms for day-5 blastocyst transfer. Reprod BioMed Online. 2011;22:477–84.CrossRefPubMedGoogle Scholar
  39. 39.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    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.e1.CrossRefPubMedGoogle Scholar
  41. 41.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception in vitro. Hum Reprod. 1997;12 Suppl 8:1742–9.Google Scholar
  43. 43.
    Hall J, Gilligan A, Schimmel T, Cecchi M, Cohen J. The origin, effects and control of air pollution in laboratories used for human embryo culture. Hum Reprod. 1998;13 Suppl 4:146–55.Google Scholar
  44. 44.
    Merton JS, Vermeulen ZL, Otter T, Mullaart E, de Ruigh L, Hasler JF. Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos. Theriogenology. 2007;67 Suppl 7:1233–8.Google Scholar
  45. 45.
    Kleijkers SH, van Montfoort AP, Bekers O, Coonen E, Derhaag JG, Evers JL, et al. Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8 °C and during incubation at 37oC. Hum Reprod. 2016;31:1192–9.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Ioannis A. Sfontouris
    • 1
    • 2
    Email author
  • Efstratios M. Kolibianakis
    • 3
  • George T. Lainas
    • 1
  • Christos A. Venetis
    • 4
  • George K. Petsas
    • 1
  • Basil C. Tarlatzis
    • 3
  • Tryfon G. Lainas
    • 1
  1. 1.Eugonia Assisted Reproduction UnitAthensGreece
  2. 2.Division of Child Health, Obstetrics and Gynaecology, School of MedicineUniversity of NottinghamNottinghamUK
  3. 3.Unit for Human Reproduction, First Department of Obstetrics and Gynecology, Papageorgiou General Hospital, Medical SchoolAristotle University of ThessalonikiThessalonikiGreece
  4. 4.Women’s and Children’s Health, St. George HospitalUniversity of New South WalesSydneyAustralia

Personalised recommendations