Journal of Assisted Reproduction and Genetics

, Volume 31, Issue 1, pp 55–63 | Cite as

The effect of minimal concentration of ethylene glycol (EG) combined with polyvinylpyrrolidone (PVP) on mouse oocyte survival and subsequent embryonic development following vitrification

  • Yao Wang
  • Osamu Okitsu
  • Xiao-Ming Zhao
  • Yun Sun
  • Wen Di
  • Ri-Cheng Chian
Fertility Preservation



Vitrification techniques employ a relatively high concentration of cryoprotectant in vitrification solutions. Exposure of oocytes to high concentrations of cryoprotectant is known to damage the oocytes via both cytotoxic and osmotic effects. Therefore, the key to successful vitrification of oocytes is to strike a balance between the usage of minimal concentration of cryoprotectant without compromising their cryoprotective actions.


The minimal concentration of ethylene glycol (EG) on mouse oocyte survival and subsequent embryonic development was evaluated following vitrification-warming and parthenogenetic activation. Polyvinylpyrrolidone (PVP) combined with EG on mouse oocyte survival and subsequent embryonic development as well as morphology of the spindle and chromosome alignment were also evaluated. Vitrification system was adapted with JY Straw and the cooling rate was approximately 442–500 °C/min. In contrast, the warming rate was approximately 2,210–2,652 °C/min.


Survival rate of oocytes increased significantly when 15 % EG was combined with 2 % PVP in vitrification solution (VS). The effect of combination of EG and PVP was not significant when the concentration of EG was 20 % and higher. Although there were no significant differences in embryonic development, the percentage of abnormal spindle and chromosome alignment was significantly higher in the oocytes without 2 % PVP in VS.


Our data provide a proof of principle for oocyte vitrification that may not require a high concentration of cryoprotectant. There are synergic effects of EG combined with PVP for oocyte vitrification, which may provide important information to the field in developing less cytotoxic VS.


Vitrification Cryoprotectant Concentration Oocytes Embryonic development 



We thank Dr. Bao-Zeng Xu for technical assistance for immune-fluorescent staining of tubulin and chromosomes. YW was supported by the Key Program for Basic Research of the Science and Technology Commission of Shanghai Municipality, China (Grant No. 12JC1405800).

Authors’ roles

YW performed the experiments and analyzed data. OO performed the experiment of measuring cooling and warming rates. XMZ, YS assisted the experiments and helped to analyze the data. WD designed study, analyzed the experiments. RCC designed study, analyzed the experiments, and wrote the manuscript.


This study was supported by McGill Reproductive Center, McGill University Health Center (MUHC) and a grant from China Natural Science Foundation (No. 81270746) to RCC.

Conflict of interest

YW, OO, XMZ, YS, WD have nothing declared. RCC designed and invented JY Straw.


  1. 1.
    Cai LB, Qian XQ, Wang W, Mao YD, Yan ZJ, Liu CZ, et al. Oocyte vitrification technology has made egg-sharing donation easier in China. Reprod Biomed Online. 2012;24:186–90.PubMedCrossRefGoogle Scholar
  2. 2.
    Checura CM, Seidel Jr GE. Effect of macromolecules in solutions for vitrification of mature bovine oocytes. Theriogenelogy. 2007;67:919–30.CrossRefGoogle Scholar
  3. 3.
    Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai T. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev. 2004;50:685–96.PubMedCrossRefGoogle Scholar
  4. 4.
    Chian RC, Son WY, Huang J, Cui S, Buckett WM, Tan SL. High survival rates and pregnancies of human oocytes following vitrification: preliminary report. Fertil Steril. 2005;84 Suppl 1:S36.CrossRefGoogle Scholar
  5. 5.
    Chian RC, Huang JYJ, Tan SL, Lucena E, Saa A, Rojas A, et al. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online. 2008;16:608–10.PubMedCrossRefGoogle Scholar
  6. 6.
    Chian RC, Gilbert L, Huang JYJ, Demirtas E, Holzer H, Benjamin A, et al. Live birth after vitrification of in vitro matured human oocytes. Fertil Steril. 2009;91:372–6.PubMedCrossRefGoogle Scholar
  7. 7.
    Chian RC, Gilbert L, Huang JYJ, Son WY, Holzer H, Cui SJ, et al. Obstetrical outcomes following vitrification of oocytes matured in-vivo or in-vitro. Fertil Steril. 2009;91:2391–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Chung JT, Tosca L, Huang TH, Niwa K, Chian RC. The effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development. Reprod Biomed Online. 2007;15:198–207.PubMedCrossRefGoogle Scholar
  9. 9.
    Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum Reprod. 2010;25:2239–46.PubMedCrossRefGoogle Scholar
  10. 10.
    Cobo A, Remohí J, Chang CC, Nagy ZP. Oocyte cryopreservation for donor egg banking. Reprod Biomed Online. 2010;23:341–6.CrossRefGoogle Scholar
  11. 11.
    Dumoulin JC, Bergers-Janssen JM, Pieters MH, Enginsu ME, Geraedts JP, Evers JL. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril. 1994;62:793–8.PubMedGoogle Scholar
  12. 12.
    Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update. 2012;18:536–54.PubMedCrossRefGoogle Scholar
  13. 13.
    Fahy GM, Levy DI, Ali SE. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology. 1987;24:196–213.PubMedCrossRefGoogle Scholar
  14. 14.
    Fuller BJ. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Letters. 2004;25:375–88.PubMedGoogle Scholar
  15. 15.
    Galeati G, Spinaci M, Vallorani C, Bucci D, Porcu E, Tamanini C. Pig oocyte vitrification by cryotop method: effects on viability, spindle and chromosome configuration and in vitro fertilization. Anim Reprod Sci. 2011;127:43–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Gook DA. History of oocyte cryopreservation. Reprod Biomed Online. 2011;23:281–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Gook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod Update. 2007;13:591–605.PubMedCrossRefGoogle Scholar
  18. 18.
    Huang J, Tan SL, Chian RC. Fertility preservation for female. J Reprod Contracep. 2006;17:109–28.Google Scholar
  19. 19.
    Huang JY, Chen HY, Tan SL, Chian RC. Effect of choline-supplemented sodium-depleted slow freezing versus vitrification on mouse oocyte meiotic spindles and chromosome abnormalities. Fertil Steril. 2007;88:1093–100.PubMedCrossRefGoogle Scholar
  20. 20.
    Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril. 2003;80:223–4.PubMedCrossRefGoogle Scholar
  21. 21.
    Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online. 2005;11:300–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril. 1999;72:1073–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Lawitts JA, Biggers JD. Culture of preimplantation embryos. Methods Enzymol. 1993;225:153–64.PubMedCrossRefGoogle Scholar
  24. 24.
    Leibo SP, Pool TB. The principal variables of cryopreservation: solutions, temperatures, and rate changes. Fertil Steril. 2011;96:269–76.PubMedCrossRefGoogle Scholar
  25. 25.
    Liu L, Keefe DL. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod. 2002;17:2678–85.PubMedCrossRefGoogle Scholar
  26. 26.
    Lopes CM, Felisberti MI. Mechanical behaviour and biocompatibility of poly(1-vinyl-2-pyrrolidinone)-gelatin IPN hydrogels. Biomaterials. 2003;24:1279–84.PubMedCrossRefGoogle Scholar
  27. 27.
    Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril. 2006;85:108–11.PubMedCrossRefGoogle Scholar
  28. 28.
    Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod. 1996;54:1059–69.PubMedCrossRefGoogle Scholar
  29. 29.
    Matsumoto H, Jiang JY, Tanaka T, Sasada H, Sato E. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology. 2001;42:139–44.PubMedCrossRefGoogle Scholar
  30. 30.
    Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to −196 °C at 95° to 70,000 °C/min and warmed at 610° to 118,000 °C/min: A new paradigm for cryopreservation by vitrification. Cryobiology. 2011;62:1–7.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril. 1997;68:724–6.PubMedCrossRefGoogle Scholar
  32. 32.
    Quinn P, Moinipanah R, Steinberg JM, Weathersbee P. Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions. Fertil Steril. 1995;63:922–4.PubMedGoogle Scholar
  33. 33.
    Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, et al. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod. 2012;27:1606–12.PubMedCrossRefGoogle Scholar
  34. 34.
    Seki S, Mazur P. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice. Biol Reprod. 2008;79:727–37.PubMedCrossRefGoogle Scholar
  35. 35.
    Seki S, Mazur P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology. 2009;59:75–82.PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to −196°c in dilutions of a standard vitrification solution. PLoS One. 2012;7:6058.Google Scholar
  37. 37.
    Shaw JM, Kuleshova LL, MacFarlane DR, Trouson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, ficoll, or dextran. Cryobiology. 1997;35:219–29.PubMedCrossRefGoogle Scholar
  38. 38.
    The Practice Committees of the American Society for Reproductive Medicine and the Sociaty for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013;99:37–43.CrossRefGoogle Scholar
  39. 39.
    Titterington JL, Robinson J, Killick SR, Hay DM. Synthetic and biological macromolecules: protection of mouse embryos during cryopreservation by vitrification. Hum Reprod. 1995;10:649–53.PubMedGoogle Scholar
  40. 40.
    Tucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum Reprod. 1996;11:1513–5.PubMedCrossRefGoogle Scholar
  41. 41.
    Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod. 1998;13:3156–9.PubMedCrossRefGoogle Scholar
  42. 42.
    Vajta G, Holm P, Greve T, Callesen H. Vitrification of porcine embryos using the Open Pulled Straw (OPS) method. Acta Vet Scand. 1997;38:349–52.PubMedGoogle Scholar
  43. 43.
    Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev. 1998;51:53–8.PubMedCrossRefGoogle Scholar
  44. 44.
    Wang YB, Lou Y, Luo ZF, Zhang DF, Wang YZ. Induction of apoptosis and cell cycle arrest by polyvinylpyrrolidone K-30 and protective effect of alpha-tocopherol. Biochem Biophys Res Commun. 2003;308:878–84.PubMedCrossRefGoogle Scholar
  45. 45.
    Whittingham D. Survival of mouse embryos after freezing and thawing. Nature. 1971;233:125–6.PubMedCrossRefGoogle Scholar
  46. 46.
    Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM, et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril. 2003;79:1323–6.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Center for Reproductive Medicine, Key Laboratory for Assisted Reproduction and Genetics, Department of Obstetrics and Gynecology, Ren Ji HospitalSchool of Medicine, Shanghai Jiao Tong UniversityShanghaiChina
  2. 2.Division of Reproductive Biology, Department of Obstetrics and GynecologyMcGill UniversityMontrealCanada
  3. 3.Division of Reproductive Medicine, Miyage ClinicOkayamaJapan
  4. 4.State Key Laboratory of Reproductive Medicine, Center for Clinical Reproductive MedicineThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina

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