생쥐 전핵기 배아 냉동보존에서 완만동결과 유리화동결의 비교

Comparison of Vitrification and Slow Freezing for the Cryopreservation of Mouse Pronuclear Stage Embryos

  • 김미영 (전남대학교 의과대학 산부인과학교실) ;
  • 이여일 (전남대학교 의과대학 산부인과학교실)
  • Kim, Mi-Young (Department of Obstetrics and Gynecology, Chonnam National University Medical School) ;
  • Lee, Yu-Il (Department of Obstetrics and Gynecology, Chonnam National University Medical School)
  • 발행 : 2007.06.30

초록

목 적: 본 연구는 생쥐 전핵시기 배아를 완만동결법과 유리화동결법으로 동결-융해 후 배아의 생존율과 성장률을 비교하고자 시행하였다. 연구방법: 과배란을 유도한 생쥐로부터 전핵시기 배아를 획득하여 10% SSS가 첨가된 HTF 배양액으로 약 1시간 동안 배양한 후 두 개의 전핵이 관찰되는 정상적인 형태의 배아만 선별하여 동결하였다. 동결방법으로는 1.5 M PROH에 0.1 M sucrose가 함유된 완만동결법과 40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 혼합된 EFS40 용액과 EM grid를 이용하여 이용한 유리화동결법을 실시하였다. 동결-융해 후 전핵시기 배아의 회수율, 생존을 및 부화 포배기로의 성장률과 부화율을 비교하였다. 결 과: 각각의 방법으로 동결-융해 후 24시간 동안 배양하였을 때 2-세포기까지의 성장률은 완만동결군이 59.1%이었고 유리화동결군이 77.0%로 두 군간에 유의한 차이를 보였고 (p<0.003), 48시간 동안의 배양에서도 완만동결군이 53.3%이고 유리동결군이 72.6%로 유의하게 유리화동결군에서 높은 수세포기까지의 성장률을 보였으며 (p<0.003), 72시간 배양하였을 때의 상실배로의 성장률 역시 완만동결군이 46.7%이고 유리화동결군이 67.3%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.001). 융해 후 144시간 동안 배양하였을 때의 부화포배기로의 성장률은 완만동결군이 26.3%이고 유리화동결군이 43.4%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.005). 결 론: 생쥐 전핵시기 배아의 동결보존에서 유리화동결법은 완만동결법 보다 시간이 단축되고 비싼 장비가 필요없어 경제적이고 간단했을 뿐 아니라 동결-응해 후 전반적으로 높은 생존율과 성장률을 나타내었다.

Objective: The aim of this study was to compare the efficacy of slow freezing with vitrification method for cryopreservation of mouse pronuclear stage embryos. Methods: Mouse pronuclear embryos obtained from superovulated mice and classified into 2 groups of slow freezing and vitrification. Slow freezing solution consisted of 1.5 M PROH, 0.1 M sucrose, while vitrification solution consisted of 40% ethylene glycol, 18% Ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. Recovery and survival rates after thawing and development rates to hatching balstocyst stage were compared between two groups. Results: After freezing and thawing, recovery rate of slow freezing group was 93.8%, whereas vitrification group was 66.5% (p<0.01). Survival rate of recovered embryos were similar between two groups as 83.2% in slow freezing and 87.6% in vitrification. Embryo development rates to 2-cell stage after 24 hrs (77.0% vs 59.1%), 4-cell after 48 hrs (72.6% vs 53.3%), blastocyst after 96 hrs (53.1% vs 40.1%) of thawing were significantly higher in vitrification group than those of slow freezing group, respectively. Conclusion: The vitrification method may provide better developmental competence of frozen-thawed embryos than that of slow freezing method for cryopreservation of mouse pronuclear stage embryos.

키워드

참고문헌

  1. Kerin JF, Warnes GM, Quinn PJ, Jeffery R, Kirby C, Matthews CD, et al. Incidence of multiple pregnancy after in-vitro fertilization and embryo transfer. Lancet 1983; 2: 537-40
  2. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to -196$^{\circ}C$ and -296$^{\circ}C$. Science 1972; 178: 411-4 https://doi.org/10.1126/science.178.4059.411
  3. Rall WF, Polge C. Effect of warming rate on mouse embryos frozen and thawed in glycerol. J Reprod Fertil 1984; 70: 285 -92 https://doi.org/10.1530/jrf.0.0700285
  4. Rall WF, Reid DS, Polge C. Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physicochemical methods. Cryobiology 1984; 21: 106-21 https://doi.org/10.1016/0011-2240(84)90027-0
  5. Leibo SP. Cryobiology: Preservation of mammalian embryos. Basic Life Sci 1986; 37: 251-72
  6. Kahn JA, von During V, Sunde A, Sordal T, Moine K. The efficacy and efficiency of an in-vitro fertilization programme including embryo cryopreservation: a cohort study. Hum Reprod 1993; 8: 247-52 https://doi.org/10.1093/oxfordjournals.humrep.a138032
  7. Van Voorhis BJ, Syrop CH, Allen BD, Sparks AE, Stovall DW. The efficacy and cost effectiveness of embryo cryopreservation compared with other assisted reproductive techniques. Fertil Steril 1995; 64: 647-50 https://doi.org/10.1016/S0015-0282(16)57808-X
  8. Kasai M, Ito K, Edashige K. Morphological appearance of the cryopreserved mouse blastocysts as a tool to identify the type of cryoinjury. Hum Reprod 2002; 17: 1863-74 https://doi.org/10.1093/humrep/17.7.1863
  9. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos a.-196$^{\circ}C$ by vitrification. Nature 1985; 313: 573-5 https://doi.org/10.1038/313573a0
  10. Pickering SJ, Braude PR, Johnson M, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102-8 https://doi.org/10.1016/S0015-0282(16)53644-9
  11. Mandelbaum J, Belaisch-Allart J, Junka AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocyte. Hum Reprod 1998; Suppl 13: 161-74 https://doi.org/10.1093/humrep/13.1.161
  12. Damario MA, Hammit DG, Galantis TM, Session DR, Dumesic DA. Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Ferril Steril 1999; 72: 1049-54 https://doi.org/10.1016/S0015-0282(99)00444-6
  13. Veeck LL, Amundson CH, Brothman LJ, DeScisciolo C, Maloney MK, Muasher SJ, et al. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil Steril 1993; 59: 1202-7 https://doi.org/10.1016/S0015-0282(16)55977-9
  14. Cohen J, DeVane GW, Elsner CW, Fehilly CB, Kort HI, Massey JB, et al. Cryopreservation of zygotes and early cleaved human embryos. Fertil Steril 1988; 49: 283-9 https://doi.org/10.1016/S0015-0282(16)59717-9
  15. Fugger EF, BustilloM, Katz LP, Dorfmam AD, Bender SD, Schulman JD. Embryonic development and pregnancy from fresh and cryopreservation sibling pronucleate human zygotes. Fertil Steril 1988; 50: 273-8 https://doi.org/10.1016/S0015-0282(16)60072-9
  16. Testart J, Lassalle B, Belaisch-Allart J, Hazoult A, Forman R, Rainbom JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986; 46: 268-72 https://doi.org/10.1016/S0015-0282(16)49524-5
  17. Schatten G, Simerly C, Schatten H. Micrituble configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtuble-mediated motility during mammalian fertilization. Proc Natl Acad Sci USA 1985; 82: 4152-6 https://doi.org/10.1073/pnas.82.12.4152
  18. Wright G, Wiker S, Elsner C, Kort H, Massey J, Mitchell D, et al. Observations on the morphology of pronuclei and nucleoli in human zygote and implications for cryopreservation. Hum Reprod 1990; 5: 109-15 https://doi.org/10.1093/oxfordjournals.humrep.a137029
  19. Bafrani HR, Salsabili N, Pasbakhsh P, Hassani H, Movahedin M, Al-tarihi T, et al. Comparison of 1,2-propanediol and ethylene glycol for cryopreservation of slow-cooled mouse zygote and their subsequent development. J Assist Reprod Genet 2003; 20: 234-40 https://doi.org/10.1023/A:1024159512353
  20. Isachenko V. Modified vitrification of human pronuclear oocyte: efficacy and effect on ultrastructure. Reprod Biomed Online 2003; 7: 211-6 https://doi.org/10.1016/S1472-6483(10)61754-3
  21. Kim EY, Yi BK, Nam HK, Lee KS, Yoon SH, Park SP, et al. Cryopreservation of human multi-pronuclear (PN) zygote by ultra-rapid freezing. Kor J Fertil Steril 1998; 25: 129-40
  22. Trounson A, Mohr L. Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo. Nature 1983; 305: 707-9 https://doi.org/10.1038/305707a0
  23. Miyake T, Kasai M, Zhu SE, Sakurai T, Machida T. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene-glycol based solution by a simple method. Theriogenology 1993; 40: 121-34 https://doi.org/10.1016/0093-691X(93)90346-7
  24. Rail WF, Wood MJ, Kirby C, Whittimgham DO. Development of mouse embryos cryopreserved by vitrification. J Reprod Fert 1987; 80: 499-504 https://doi.org/10.1530/jrf.0.0800499
  25. Bautista JA, Dela Pena EC, Katagiri S, Takahashi Y, Kanagawa H. In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution, Jpn J Vet Res 1998; 46: 13-8
  26. Zeilmaker GH, Alberda AT, van Getn I, Rijkmans CMPM, Drogenkijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984; 42: 293-6 https://doi.org/10.1016/S0015-0282(16)48029-5
  27. Kasai M, Komi JH, Takamo A, Tsudera HM, Sakurai T, Machida T. A simple methods for mouse embryo cryopreservation in a low toxic vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91-7 https://doi.org/10.1530/jrf.0.0890091
  28. Zhu SE, Kasai M, Otoge H, Sakurai T, Machida T. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J Reprod Fertil 1993; 98: 139-45 https://doi.org/10.1530/jrf.0.0980139
  29. Kim MK, Lee SJ, Uhm EY, Yoon SH, Park SP, Chung KS, et al. Cryopreservation of mouse IVF zygotes by vitrification. Korean J Animal Re prod 1996; 20: 119-26
  30. Kim MK, Yi SH, Yoon SH, Park SP, Chung KS, Lim JH. In Vitro/In Vivo Development of mouse oocytes vitrified by EFS. Korean J Animal Reprod 1998; 25: 87-92
  31. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059-69 https://doi.org/10.1095/biolreprod54.5.1059
  32. Isachenko V, Montag M, Isachenko E, Nawroth F, Dessole S, van der Ven H. Developmental rate and ultrastructure of vitrified human pronuclear oocyte after step-wise versus direct rehydration. Hum Reprod 2004; 19: 660-5 https://doi.org/10.1093/humrep/deh151