Comparison of the Efficiency between Slow Freezing and Vitrification Method for Cryopreservation of Human Embryos

인간 수정란의 완만 동결과 유리화 동결의 비교

  • 김은국 (광주 미래와희망산부인과) ;
  • 김미연 (광주 미래와희망산부인과) ;
  • 손선미 (광주 미래와희망산부인과) ;
  • 김동원 (광주 미래와희망산부인과)
  • Published : 2008.03.31

Abstract

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

Keywords

References

  1. Bafrani HH, Salsabili N, Pasbakhsh P, Hassani H, Movahedin M and Al-tarihi T. 2003. Comparison of 1,2-propanedial and ethylene glycol for cryopreservation of slow-cooled mouse zygote and their subsequent development. J. Assist. Reprod. Genet. 20: 234-240 https://doi.org/10.1023/A:1024159512353
  2. Bautista JA, Dela Pena EC, Katagiri S, Takahashi Y and Kanagawa H. 1998. In vitro viability of mouse oocytes vitrified in an ethylen glycol-based solution. Jpn. J. Vet. Res. 46:13-18
  3. Chen SU, Lien YR, Chen YY, Chen HF, Ho NH and Yang YS. 2001. Vitrification of mouse oocytes using closed pulled straw (OPS) achieves a high survival and preserves good patterns of meiotic a spindles, compared with conventional straws, open pulled straw(OPS) and grids. Hum. Reprod. 16:2350-2359 https://doi.org/10.1093/humrep/16.11.2350
  4. Cohen J, DeVane GW, Elsner CW, Rehilly CB, Kort HI, Massey JB and Turner TG Jr. 1988. Cryopreservation of zygotes and early cleaved human embryos. Fertil. Steril. 49:283-289 https://doi.org/10.1016/S0015-0282(16)59717-9
  5. Cohen J, Simons RF, Edwards RG, Fehilly CB and Fishel SB. 1985. Pregnancies following the frozen storage of expanding human blastocysts. J. In Vitro Fert. Embryo Transfer. 2:59-64 https://doi.org/10.1007/BF01139337
  6. Damario MA, Hammit DG, Galantis TM, Session DR and Dumesic DA. 1999. Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil. Sterill. 72:1049-1054 https://doi.org/10.1016/S0015-0282(99)00444-6
  7. Dobrinsky JR, Pursel VG, Long CR and Johnson LA. 2000. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol. Reprod. 62:564-570 https://doi.org/10.1095/biolreprod62.3.564
  8. Fahy GM, Macfarlane DR, Angell CA and Meryman HT. 1984. Vitrification as an approach to cryopreservation. Cryobiology 21:407-426 https://doi.org/10.1016/0011-2240(84)90079-8
  9. Fugger EF, Bustillo M, Katz LP, Dorfmam AD, Bender SD and Schulman JD. 1988. Embryonic development and pregnancy from fresh and cryopreservation sibling pronucleate human zygotes. Fertil. Steril. 50:273-278 https://doi.org/10.1016/S0015-0282(16)60072-9
  10. Gardner DK, Weissman A, Howles CM and Shoham Zeev. 2004. Textbook of Assisted Reproductive Techniques. 2nd ed, Taylor & Francis, United Kingdom, pp. 257-289
  11. Isachenko V, Montag M, Isachenko E, Nawroth F, Dessole S and van der Ven H. 2004. Developmental rate and ultrastructure of vitrified human pronuclear oocyte after step-wise versus direct rehydration. Hum. Reprod. 19:660-665 https://doi.org/10.1093/humrep/deh151
  12. Isachenko V. 2003. Modified vitrification of human promuclear oocyte: efficacy and effect on ultrastructure. Rerod. Biomed. Online. 7:211-216 https://doi.org/10.1016/S1472-6483(10)61754-3
  13. Kaidi S, Donnay I, Lambert P, Dessy F and Massip A. 2000. Osmotic behavior of in vitro produce bovine blastocyst in cryoprotectant solutions as a potential predictive test of survival. Cryobiology 41:106-115 https://doi.org/10.1006/cryo.2000.2272
  14. Kasai M, Ito K and Edashige K. 2002. Morphological appearance of the cryopreserved mouse blastocysts as a tool to identify the type of cryoinjury. Hum. Reprod. 17:1863-1874 https://doi.org/10.1093/humrep/17.7.1863
  15. Kasai M, Komi H, Takakamo A, Tsudera H, Sakurai T and Machida T. 1990. A simple method for mouse embryo cryopreservation in low toxicity vitrification solution without appreciable loss of viability. J. Reprod. Fert. 89:91-97 https://doi.org/10.1530/jrf.0.0890091
  16. Kaufmann RA, Nicollet B, Menezo Y, DuMont M, Hazout A and Servy EJ. 1995. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil. Steril. 64:1125- 1129 https://doi.org/10.1016/S0015-0282(16)57972-2
  17. Mandelbaun J, Belaisch-Allart J, Junka AM, Antoine JM, Plachot M, Alvarez S, et al. 1998. Cryopreservation in humna assisted reproduction is now routine for embryos but remains a research procedure for oocyte. Hum Reprod. Suppl. 13:161-174
  18. Menezo Y, Nicollet B, Herbaut N and Andre D. 1992. Freezing cocultured human blastocysts. Fertil. Steril. 58:977-980 https://doi.org/10.1016/S0015-0282(16)55445-4
  19. Miyake T, Kasai M, Zhu SE, Sakurai T and Machida T. 1993. Vitrification of mouse oocytes and embryos at various stage of development in an ethylene-glycol based solution by a simple method. Theriogenology 40:121-134 https://doi.org/10.1016/0093-691X(93)90346-7
  20. Ohta N, Nohara M, Kojimahara T, Ito M, Saito T, Nakahara K, Tezuka N, Saito H and Hiroi M. 1996. Ultrarapid freezing of human embryos by vitrification method: a case of delivery. Jpn. J. Fertil. Steril. 41:276-279
  21. Park JE and Ruffing H. 1992. Factors affecting low temperature survivals of mammalian oocytes. Theriogenology 37:59-73 https://doi.org/10.1016/0093-691X(92)90247-O
  22. Rall WF and Fahy GM. 1985. Ice-free cryopreservation of mouse embryos at -$196^{\circ}C$ by vitrification. Nature 313:573-575 https://doi.org/10.1038/313573a0
  23. Scheffen B, Van Der Zwalmen P and Massip A. 1986. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-Letters 7:260-269
  24. Testart J, Lassalle B, Belaisch-Allart J, Hazoult A, Forman R, Rainborn JD and Frydman R. 1986. High pregnancy rate after early human embryo freezing. Fertil. Steril. 46:268-272 https://doi.org/10.1016/S0015-0282(16)49524-5
  25. Trounson A and Mohr L. 1983. Human pregnancy following cyropreservation thawing and transfer of an eight-cell embryo. Nature 305:707-709 https://doi.org/10.1038/305707a0
  26. Vanderzwalmen P, Delval A, Chatziparasidou A, Bertin G, Ectors F, Lejeune B, Nijis M, Prapas N, Prapas Y, Van Damme B, Zech H and Schoysman R. 1997. Pregnancies after vitrification of human day 5 embryos. Hum Reprod. Abst. 12:O-198 https://doi.org/10.1093/oxfordjournals.humrep.a019553
  27. Veeck LL, Amundson CH, Brothman LJ, DeScisciolo C, Maloney MK, Muasher SJ and Jones HW Jr. 1993. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril. 59:1202-1207 https://doi.org/10.1016/S0015-0282(16)55977-9
  28. Whittingham DG, Leibo SP and Mazur P. 1972. Survival of mouse embryos frozen to $-196^{\circ}C$ and $-296^{\circ}C$. Science 178:411-414 https://doi.org/10.1126/science.178.4059.411
  29. 공일근, 조성균, 조성근. 1999. 동결보호제의 종류 및 배발달 단계가 OPP vitrification 동결 보존시 생쥐 수정란의 생존성에 미치는 영향. 한국가축번식학회지 23(1):85-92