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Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing

  • Gamete Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

The cryopreservation of immature oocytes permits oocyte banking for patients at risk of losing their fertility. However, the optimum protocol for such fertility preservation remains uncertain.

Methods

The present study investigated the survival, maturation, cytoskeletal and chromosome organization of sibling immature oocytes leftover from controlled ovarian stimulation cycles, that were either slow-frozen (with choline-substitution) or vitrified. A comparison group included oocytes that were never cryopreserved.

Results

Among the three groups, comparable rates were observed for both survival (67-70%) and polar body extrusion (59-79%). Significantly more oocytes underwent spontaneous activation after IVM following slow-freezing compared with either vitrification or no cryopreservation. Likewise, the incidence of spindle abnormalities was greatest in the slow-frozen group, with no differences in spindle morphometrics or chromosome organization.

Conclusions

While the overall incidence of mature oocytes with normal bipolar spindles from warmed immature oocytes was low, the yield using Cryoleaf vitrification was slightly superior to choline-based slow-freezing.

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References

  1. Stachecki JJ, Cohen J. An overview of oocyte cryopreservation. Reprod Biomed Online. 2004;9:152–63.

    Article  PubMed  Google Scholar 

  2. Koutlaki N, Schoepper B, Maroulis G, Diedrich K, Al-Hasani S. Human oocyte cryopreservation: past, present and future. Reprod Biomed Online. 2006;13:427–36.

    Article  PubMed  CAS  Google Scholar 

  3. Cobo A, Vajta G, Remohi J. Vitrification of human mature oocytes in clinical practice. Reprod Biomed Online. 2009;19 Suppl 4:4385.

    PubMed  Google Scholar 

  4. Mandelbaum J, Anastasiou O, Levy R, Guerin JF, de Larouziere V, Antoine JM. Effects of cryopreservation on the meiotic spindle of human oocytes. Eur J Obstet Gynecol Reprod Biol. 2004;113 Suppl 1:S17–23.

    Article  PubMed  Google Scholar 

  5. Ledda S, Bogliolo L, Succu S, Ariu F, Bebbere D, Leoni GG, Naitana S. Oocyte cryopreservation: oocyte assessment and strategies for improving survival. Reprod Fertil Dev. 2007;19:13–23.

    Article  PubMed  CAS  Google Scholar 

  6. Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril. 1998;70:578–9.

    Article  PubMed  CAS  Google Scholar 

  7. Toth TL, Baka SG, Veeck LL, Jones Jr HW, Muasher S, Lanzendorf SE. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril. 1994;61:891–4.

    PubMed  CAS  Google Scholar 

  8. Baka SG, Toth TL, Veeck LL, Jones Jr HW, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod. 1995;10:1816–20.

    PubMed  CAS  Google Scholar 

  9. Son WY, Park SE, Lee KA, Lee WS, Ko JJ, Yoon TK, Cha KY. Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes. Fertil Steril. 1996;66:995–9.

    PubMed  CAS  Google Scholar 

  10. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril. 1997;68:920–6.

    Article  PubMed  CAS  Google Scholar 

  11. Goud A, Goud P, Qian C, Van der Elst J, Van Maele G, Dhont M. Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions. Fertil Steril. 2000;74:487–94.

    Article  PubMed  CAS  Google Scholar 

  12. Boiso I, Marti M, Santalo J, Ponsa M, Barri PN, Veiga A. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod. 2002;17:1885–91.

    Article  PubMed  Google Scholar 

  13. Wu J, Zhang L, Wang X. In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes. Reproduction. 2001;121:389–93.

    Article  PubMed  CAS  Google Scholar 

  14. Isachenko V, Montag M, Isachenko E, Dessole S, Nawroth F, van der Ven H. Aseptic vitrification of human germinal vesicle oocytes using dimethyl sulfoxide as a cryoprotectant. Fertil Steril. 2006;85:741–7.

    Article  PubMed  CAS  Google Scholar 

  15. Cao Y, Xing Q, Zhang ZG, Wei ZL, Zhou P, Cong L. Cryopreservation of immature and in-vitro matured human oocytes by vitrification. Reprod Biomed Online. 2009;19:369–73.

    Article  PubMed  Google Scholar 

  16. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, Han SY, Choi DH, Cha KY. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil Steril. 2000;73:545–51.

    Article  PubMed  CAS  Google Scholar 

  17. Boldt J, Tidswell N, Sayers A, Kilani R, Cline D. Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method. Reprod Biomed Online. 2006;13:96–100.

    Article  PubMed  CAS  Google Scholar 

  18. Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod. 2003;18:1250–5.

    Article  PubMed  Google Scholar 

  19. Petracco A, Azambuja R, Okada L, Michelon J, Oliani A, Badalotti M. Comparison of embryo quality between sibling embryos originating from frozen or fresh oocytes. Reprod Biomed Online. 2006;13:497–503.

    Article  PubMed  Google Scholar 

  20. Cobo A, Perez S, Santos MJ De los, Zulategui J, Domingo J, Remohi J. Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes. Reprod Biomed Online. 2008;17:350–9.

    Article  PubMed  CAS  Google Scholar 

  21. Chian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cui SJ, Buckett WM, Tulandi T, Tan SL. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril. 2009;91:2391–8.

    Article  PubMed  Google Scholar 

  22. Coticchio G, Bromfield JJ, Sciajno R, Gambardella A, Scaravelli G, Borini A, Albertini DF. Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes. Reprod Biomed Online. 2009;19 Suppl 3:29–34.

    Article  PubMed  Google Scholar 

  23. Fadini R, Brambillasca F, Renzini MM, Merola M, Comi R, De Ponti E, Dal Canto MB. Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Reprod Biomed Online. 2009;19:171–80.

    Article  PubMed  CAS  Google Scholar 

  24. Nottola SA, Coticchio G, Sciajno R, Gambardella A, Maione M, Scaravelli G, Bianchi S, Macchiarelli G, Borini A. Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop. Reprod Biomed Online. 2009;19 Suppl 3:17–27.

    Article  PubMed  Google Scholar 

  25. Fasano G, Vannin AS, Biramane J, Delbaere A, Englert Y. Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling. Cryobiology. 2010;61:243–7.

    Article  PubMed  CAS  Google Scholar 

  26. Cao YX, Xing Q, Li L, Cong L, Zhang ZG, Wei ZL, Zhou P. Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification. Fertil Steril. 2009;92:1306–11.

    Article  PubMed  Google Scholar 

  27. Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, Alegretti JR, Motta EL. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril. 2010;94:2088–95.

    Article  PubMed  Google Scholar 

  28. Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology. 1998;37:346–54.

    Article  PubMed  CAS  Google Scholar 

  29. Messinger SM, Albertini DF. Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci. 1991;100(Pt 2):289–98.

    PubMed  Google Scholar 

  30. Cekleniak NA, Combelles CM, Ganz DA, Fung J, Albertini DF, Racowsky C. A novel system for in vitro maturation of human oocytes. Fertil Steril. 2001;75:1185–93.

    Article  PubMed  CAS  Google Scholar 

  31. Toth TL, Lanzendorf SE, Sandow BA, Veeck LL, Hassen WA, Hansen K, Hodgen GD. Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil Steril. 1994;61:1077–82.

    PubMed  CAS  Google Scholar 

  32. Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner DK. 1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology. Hum Reprod. 2007;22:250–9.

    Article  PubMed  CAS  Google Scholar 

  33. Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction. 2006;131:53–61.

    Article  PubMed  CAS  Google Scholar 

  34. Kim BY, Yoon SY, Cha SK, Kwak KH, Fissore RA, Parys JB, Yoon TK, Lee DR. Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development. Pflugers Arch. 2011;461:515–26.

    Article  PubMed  CAS  Google Scholar 

  35. Jones KT. Mammalian egg activation: from Ca2+ spiking to cell cycle progression. Reproduction. 2005;130:813–23.

    Article  PubMed  CAS  Google Scholar 

  36. De Santis L, Coticchio G, Paynter S, Albertini D, Hutt K, Cino I, Iaccarino M, Gambardella A, Flamigni C, Borini A. Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation. Hum Reprod. 2007;22:2776–83.

    Article  PubMed  Google Scholar 

  37. Nottola SA, Coticchio G, De Santis L, Macchiarelli G, Maione M, Bianchi S, Iaccarino M, Flamigni C, Borini A. Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol. Reprod Biomed Online. 2008;17:368–77.

    Article  PubMed  CAS  Google Scholar 

  38. Ahn HJ, Sohn IP, Kwon HC, Jo DH, Park YD, Min CK. Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos. Mol Reprod Dev. 2002;61:466–76.

    Article  PubMed  CAS  Google Scholar 

  39. Wang X, Al Naib A, Sun DW, Lonergan P. Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol. Cryobiology. 2010;61:58–65.

    Article  PubMed  CAS  Google Scholar 

  40. Arav A, Zvi R. Do chilling injury and heat stress share the same mechanism of injury in oocytes? Mol Cell Endocrinol. 2008;282:150–2.

    Article  PubMed  CAS  Google Scholar 

  41. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril. 2001;75:1049–56.

    Article  PubMed  CAS  Google Scholar 

  42. Comizzoli P, Wildt DE, Pukazhenthi BS. In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes. Reprod Domest Anim. 2009;44 Suppl 2:269–74.

    Article  PubMed  Google Scholar 

  43. Combelles CM, Cekleniak NA, Racowsky C, Albertini DF. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod. 2002;17:1006–16.

    Article  PubMed  CAS  Google Scholar 

  44. Albertini DF. Regulation of meiotic maturation in the mammalian oocyte: interplay between exogenous cues and the microtubule cytoskeleton. Bioessays. 1992;14:97–103.

    Article  PubMed  CAS  Google Scholar 

  45. Ferreira EM, Vireque AA, Adona PR, Meirelles FV, Ferriani RA, Navarro PA. Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence. Theriogenology. 2009;71:836–48.

    Article  PubMed  CAS  Google Scholar 

  46. Men H, Monson RL, Rutledge JJ. Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation. Theriogenology. 2002;57:1095–103.

    Article  PubMed  Google Scholar 

  47. Reichman DE, Politch J, Ginsburg ES, Racowsky C. Extended in vitro maturation of immature oocytes from stimulated cycles: an analysis of fertilization potential, embryo development, and reproductive outcomes. J Assist Reprod Genet. 2010;27:347–56.

    Article  PubMed  Google Scholar 

  48. Banwell KM, Thompson JG. In vitro maturation of Mammalian oocytes: outcomes and consequences. Semin Reprod Med. 2008;26:162–74.

    Article  PubMed  CAS  Google Scholar 

  49. Imoedemhe DG, Sigue AB. Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol. J Assist Reprod Genet. 1992;9:323–7.

    Article  PubMed  CAS  Google Scholar 

  50. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod. 1993;8:1101–9.

    PubMed  CAS  Google Scholar 

  51. Isachenko EF, Nayudu PL. Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity. Hum Reprod. 1999;14:400–8.

    Article  PubMed  CAS  Google Scholar 

  52. Bogliolo L, Ariu F, Fois S, Rosati I, Zedda MT, Leoni G, Succu S, Pau S, Ledda S. Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells. Theriogenology. 2007;68:1138–49.

    Article  PubMed  CAS  Google Scholar 

  53. Tharasanit T, Colleoni S, Galli C, Colenbrander B, Stout TA. Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes. Reproduction. 2009;137:391–401.

    Article  PubMed  CAS  Google Scholar 

  54. Zhou XL, Al Naib A, Sun DW, Lonergan P. Bovine oocyte vitrification using the Cryotop method: effect of cumulus cells and vitrification protocol on survival and subsequent development. Cryobiology. 2010;61:66–72.

    Article  PubMed  CAS  Google Scholar 

  55. Hyttel P, Vajta G, Callesen H. Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. Mol Reprod Dev. 2000;56:80–8.

    Article  PubMed  CAS  Google Scholar 

  56. Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology. 2000;53:59–72.

    Article  PubMed  CAS  Google Scholar 

  57. Comizzoli P, Wildt DE, Pukazhenthi BS. Impact of anisosmotic conditions on structural and functional integrity of cumulus-oocyte complexes at the germinal vesicle stage in the domestic cat. Mol Reprod Dev. 2008;75:345–54.

    Article  PubMed  CAS  Google Scholar 

  58. Modina S, Beretta M, Lodde V, Lauria A, Luciano AM. Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells. Eur J Histochem. 2004;48:337–46.

    PubMed  CAS  Google Scholar 

  59. Luciano AM, Franciosi F, Lodde V, Perazzoli F, Slezakova M, Modina S. Cryopreservation of immature bovine oocytes to reconstruct artificial gametes by germinal vesicle transplantation. Reprod Domest Anim. 2009;44:480–8.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Biology Department, Middlebury College, Middlebury, VT, 05753, USA

    Catherine M. H. Combelles

  2. Department of Obstetrics, Gynecology, and Reproductive Biology Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    S. Temel Ceyhan, Haiyan Wang & Catherine Racowsky

  3. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, People’s Republic of China, 100191

    Haiyan Wang

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  1. Catherine M. H. Combelles
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  2. S. Temel Ceyhan
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  3. Haiyan Wang
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  4. Catherine Racowsky
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Correspondence to Catherine M. H. Combelles.

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Capsule

While the incidence of mature oocytes with normal bipolar spindles from warmed immature oocytes was low, the yield using vitrification was slightly superior to slow-freezing.

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Combelles, C.M.H., Ceyhan, S.T., Wang, H. et al. Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing. J Assist Reprod Genet 28, 1183–1192 (2011). https://doi.org/10.1007/s10815-011-9674-x

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  • DOI: https://doi.org/10.1007/s10815-011-9674-x

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