Journal of Assisted Reproduction and Genetics

, Volume 35, Issue 7, pp 1161–1168 | Cite as

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos

  • Amir AravEmail author
  • Yehudit Natan
  • Dorit Kalo
  • Alisa Komsky-Elbaz
  • Zvika Roth
  • Paolo Emanuele Levi-Setti
  • Milton Leong
  • Pasquale Patrizio
Technological Innovations



This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos.


Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates.


Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos’ survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts’ rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts’ rates.


This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.


Vitrification Oocytes Embryos Automation Cryopreservation 


  1. 1.
    American Society for Reproductive Medicine. Mature oocyte cryopreservation: a guideline. Practice committees of American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. Fertil Steril. 2013;99:37–43.CrossRefGoogle Scholar
  2. 2.
    Cobo A, Garcia-Velasco JA, Domingo J, Remohi J, Pellicer A. Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients? Fertil Steril. 2013;99:1485–95.CrossRefPubMedGoogle Scholar
  3. 3.
    Rudick B, Opper N, Paulson R, Bendikson K, Chung K. The status of oocyte cryopreservation in the United States. Fertil Steril. 2010;94:2642–6.CrossRefPubMedGoogle Scholar
  4. 4.
    Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23(2):139–55.PubMedGoogle Scholar
  5. 5.
    Arav A. Cryopreservation of oocytes and embryos. Theriogenology. 2014;81(1):96–102.CrossRefPubMedGoogle Scholar
  6. 6.
    Mazur P, Schneider U, Mahowald AP. Characteristics and kinetics of subzero chilling injury in Drosophila embryos. Cryobiology. 1992;29(1):39–68.CrossRefPubMedGoogle Scholar
  7. 7.
    Arav A. Cryopreservation of oocytes and embryos, DVM thesis 1989.Google Scholar
  8. 8.
    Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011;141(1):1–19.CrossRefPubMedGoogle Scholar
  9. 9.
    Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature. 1985;313(6003):573–5.CrossRefPubMedGoogle Scholar
  10. 10.
    Arav A. Vitrification of oocytes and embryos. In: Lauria A, Gandolfi F, editors. Embryonic development and manipulation in animal reproduction. Portland press; 1992: 255–264.Google Scholar
  11. 11.
    Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology. 2007;67(1):73–80.CrossRefPubMedGoogle Scholar
  12. 12.
    Levi-Setti PE, Patrizio P, Scaravelli G. Evolution of human oocyte cryopreservation: slow freezing versus vitrification. Curr Opin Endocrinol Diabetes Obes. 2016;23(6):445–50.CrossRefPubMedGoogle Scholar
  13. 13.
    Roy TK, Brandi S, Tappe NM, Bradley CK, Vom E, Henderson C, et al. Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method. Hum Reprod. 2014;29(11):2431–8.CrossRefPubMedGoogle Scholar
  14. 14.
    Alpha Scientists in Reproductive Medicine. The alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online. 2012;25:146–67.CrossRefGoogle Scholar
  15. 15.
    Kader AA, Choi A, Orief Y, Agarwal A. Factors affecting the outcome of human blastocyst vitrification. Reprod Biol Endocrinol. 2009;7:99.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kalo D, Roth Z. Effects of mono(2-ethylhexyl)phthalate on cytoplasmic maturation of oocytes—the bovine model. Reprod Toxicol. 2015;53:141–51.CrossRefPubMedGoogle Scholar
  17. 17.
    Arav A, Natan Y, Levi-Setti PE, Menduni F, Patrizio P. New methods for cooling and storing oocytes and embryos in a clean environment of −196°C. Reprod BioMed Online. 2016;33(1):71–8.CrossRefPubMedGoogle Scholar
  18. 18.
    Cobo A, García-Velasco JA. Why all women should freeze their eggs. Curr Opin Obstet Gynecol. 2016;28(3):206–10.CrossRefPubMedGoogle Scholar
  19. 19.
    Cobo A, García-Velasco JA, Coello A, Domingo J, Pellicer A, Remohí J. Oocyte vitrification as an efficient option for elective fertility preservation. Fertil Steril. 2016;105(3):755–764.e8.CrossRefPubMedGoogle Scholar
  20. 20.
    Cobo A, Garrido N, Pellicer A, Remohí J. Six years’ experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril. 2015;104(6):1426–34.CrossRefPubMedGoogle Scholar
  21. 21.
    Nagy ZP, Nel-Themaat L, Chang CC, Shapiro DB, Berna DP. Cryopreservation of eggs. Methods Mol Biol. 2014;1154:439–54.CrossRefPubMedGoogle Scholar
  22. 22.
    Cobo A, Vajta G, Remohí J. Vitrification of human mature oocytes in clinical practice. Reprod BioMed Online. 2009;19(Suppl 4):4385.PubMedGoogle Scholar
  23. 23.
    Nagy ZP, Chang CC, Shapiro DB, Bernal DP, Kort HI, Vajta G. The efficacy and safety of human oocyte vitrification. Semin Reprod Med. 2009;27(6):450–5.CrossRefPubMedGoogle Scholar
  24. 24.
    Liebermann J. Chapter 11 human embryo Vitrification. Methods Mol Biol. 2017;1568:141–59.CrossRefPubMedGoogle Scholar
  25. 25.
    Fasano G, Fontenelle N, Vannin AS, Biramane J, Devreker F, Englert Y, et al. A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos. J Assist Reprod Genet. 2014;31(2):241–7.CrossRefPubMedGoogle Scholar
  26. 26.
    Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90(1):186–93.CrossRefPubMedGoogle Scholar
  27. 27.
    Shi Q, Xie Y, Wang Y, Li S. Vitrification versus slow freezing for human ovarian tissue cryopreservation: a systematic review and meta-anlaysis. Sci Rep. 2017;7(1):8538.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Silber S. Ovarian tissue cryopreservation and transplantation: scientific implications. J Assist Reprod Genet. 2016;33(12):1595–603.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Silber S. Chapter 13 human ovarian tissue vitrification. Methods Mol Biol. 2017;1568:177–94.CrossRefPubMedGoogle Scholar
  30. 30.
    Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic study of vitrification of immature bovine oocyte. J Reprod Fert. 1993;99:353–8.CrossRefGoogle Scholar
  31. 31.
    Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Hum Reprod. 2005;20(12):3385–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril. 2001;75(4):769–77.CrossRefPubMedGoogle Scholar
  33. 33.
    Yavin S, Arav A. Measurement of essential physical properties of vitrification solutions. Theriogenology. 2007;67(1):81–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Martino A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev. 1996;45(4):503–12.CrossRefPubMedGoogle Scholar
  35. 35.
    Morató R, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology. 2008;57(2):137–41.CrossRefPubMedGoogle Scholar
  36. 36.
    Do VH, Walton S, Catt S, Taylor-Robinson AW. A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages. Cryobiology. 2017;77:58–63.CrossRefPubMedGoogle Scholar
  37. 37.
    Massip A, Van der Zwalmen P, Leroy F. Effect of stage of development on survival of mouse embryos frozen–thawed rapidly. Cryobiology. 1984;21:574–7.CrossRefPubMedGoogle Scholar
  38. 38.
    Camus M, Van Den Abbeel E, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghem A. Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertil Steril. 1989;51(3):460–5.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Amir Arav
    • 1
    Email author
  • Yehudit Natan
    • 1
  • Dorit Kalo
    • 2
    • 3
  • Alisa Komsky-Elbaz
    • 2
    • 3
  • Zvika Roth
    • 2
    • 3
  • Paolo Emanuele Levi-Setti
    • 4
  • Milton Leong
    • 5
  • Pasquale Patrizio
    • 1
    • 6
  1. 1.FertileSafe LtdNes-ZionaIsrael
  2. 2.Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and EnvironmentThe Hebrew UniversityRehovotIsrael
  3. 3.Center of Excellence in Agriculture and Environmental HealthThe Hebrew UniversityRehovotIsrael
  4. 4.Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility CenterHumanitas Research HospitalMilanItaly
  5. 5.The Women’s ClinicHong KongHong Kong
  6. 6.Yale Fertility CenterNew HavenUSA

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