116 J. Mamm. Ova Res. Vol.31 (4), ,422 4, ,024 * 95,764 22, , ,473 5,415 80, ,659 32, , * ) AR
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1 J. Mamm. Ova Res. Vol.31 (4), , 2014 The Safety of Cryopreservation Devices in Protecting Against Liquid Nitrogen Yoko Kumasako, Kyoko Kido, Eiko Otsu, Takafumi Utsunomiya St. Luke Clinic, Higashi-Omichi, Oita city, Oita , Japan 0.25 ml 0.25 ml Rapid-i Rapid-i Abstract: Currently, in Japan, the most commonly used embryo vitrification method is ultra-rapid vitrifi cation which is designed to achieve the maximum cooling rate by exposing embryos directly to LN2 in a tiny droplet of cryoprotectant. Though this method has yielded acceptable embryo viability rates, questions have been raised by various countries regulatory agencies regarding the effects of LN2 exposure. The 0.25 ml plastic straw has long been used as an entirely closed vitrification system. However, the cooling rate is slower using the 0.25 ml straw than when using the ultra-rapid cooling method, resulting in injury to embryos exposed to a high concentration of cryoprotectant. Recently, a new system has been developed which addresses both the issues of rates of cooling and safeguards against LN2. This paper compares the clinical results of blastocyst vitrifi cation and warming using the conventional 0.25 ml straw method with those of the Rapid-i method. The survival rate after warming the group cryopreserved using Rapid-i was significantly higher than that of the group of frozen-thawed cryopreserved using the 0.25 ml straw. Moreover, the on-going pregnancy rate after transfer blastocysts into recipients uteri was higher after cryopreservation with the Rapid-i method. These results suggest that the new closed system is safe to use in cryopreservation. Key words: liquid nitrogen, contamination, vitrifi cation, safety, straw lukelab@oct-net.ne.jp ,050,698 32, )
2 116 J. Mamm. Ova Res. Vol.31 (4), ,422 4, ,024 * 95,764 22, , ,473 5,415 80, ,659 32, , * ) ART ) , 4) 1972 Whittingham 5) 6, 7) DMSO 1983 Trounson 8) 1985 Rall 9) 85 Rall 1990 Gordts 10) Yokota 11) 0.25 ml Mukaida 12, 13) Kuwayama 14) Cryoloop Cryotop
3 ml 2,500 OPS open pulled straw Cryoloop Cryotop 20, , ml Rapid-i 0.25 ml DMSO Rapid-i 10 μl 0.05 μl DMSO Yokota 11) 0.25 ml 15) 16) ) ml Rapid-i Vitrolife Sweden Cryotop SC-N CVM CryoLogic, Austraria Cryopette Origio, Denmark 0.25 ml 0.25 ml Rapid-i ml 10 μl Rapid-i
4 118 J. Mamm. Ova Res. Vol.31 (4), ml 10 ES % 12.5 DMSO 1/2VSED 1 25% 25 DMSO VSED M μl Rapid-i DMSO 0.25 ml 3 1-step Rapid-i ml Rapid-i 0.25 ml Rapid-i P= , 19) 30% ART mm mm χ ml 1 Ishimori 20) Yokota 11) 20 SSS Serum Substitute Supplement: Irvine Scientific America m-hff 10 ES5 20 SSS m-hff DMSO Sigma America 2:1:1 VSED 5 ES 1/2 VSED 1 VSED SSS m-hff 0.5 mol/l Sigma, America load 10 μl load load Rapid-i TM
5 119 2 Rapid-i RapidVit Blast Vitri 1 5~20 Vitri 2 2 Vitri 3 45Rapid-i Vitri3 RapidStraw Rapid-i RapidStraw RapidWarm Blast Warm 1 2 Warm 2 3 Warm 3 5~10 1 Vitrolife RapidVit Blast 5 20RapidVit Blast 2 RapidVit Blast 45 Rapid-i Rapid- 2 Rapid-i RapidWarm Blast Rapid-i 2 RapidWarm Blast 3 RapidWarm Blast ml Rapid-i 0.25 ml 79.5% 174/219 Rapid-i 90.6% 174/ ml 40.7% 66/162 Rapid-i 41.6% 67/161 3 Rapid-i
6 120 J. Mamm. Ova Res. Vol.31 (4), Rapid-i 5 Rapid-i % 34/ % 42/165 5 Rapid-i Rapid-i 0.25 ml Rapid-i 0.25 ml Rapid-i 2 12,21-23 RapidVit Blast Rapid-i 0.25ml 4 5 Rapid-i ) Bielanski
7 μm ART EUTCD EU Tissue and Cells Directives 2004 CE UV Scaravelli 27) Bielanski 28) FDA ) 1) 24 2) Wen, J., Jiang, J., Ding, C., Dai, J., Liu, Y., Xia, Y., Liu, J. and Hu, Z. (2012): Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertil. Steril., 97, ) Ethics Committee of the American Society for Reproductive Medicine (2005): Fertility preservation and reproduction in cancer patients. Fertil. Steril., 83, ) von Wolff, M., Montag, M., Dittrich, R., Denschlag, D., Nawroth, F. and Lawrenz, B. (2011): Fertility preservation in women a practical guide to preservation techniques and therapeutic strategies in breast canser, Hopkin s lymphoma and borderline ovarian tumors by the fertility preservation network FertiPROTEKT. Arch. Gynecol. Obstet., 284, ) Whittingham, D, G., Leibo, S, P. and Mazur, P. (1972): Survival of mouse embryos frozen to -196 degrees and -269 degrees C. Science, 27, ) Kasai, M., Niwa, K. and Iritani, A. (1981): Effects of various cryoprotective agents on the survival of unfrozen and frozen mouse embryos. J. Reprod. Fertil., 63, ) Kasai, M., Niwa, K. and Iritani, A. (1982): Survival of rat embryos after freezing. J. Reprod. Fertil., 66, ) Trounson, A. and Mohr, L. (1983): Human pregnancy following cryopreservation, thawing and transfer of an eight cell embryo. Nature, 35, ) Rall, W.F. and Fahy, G.M. (1985): Ice free cryopreservation of mouse embryos by vitrification. Nature, 313, ) Gordts, S., Roziers, P., Campo, R. and Noto, V. (1990): Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil. Steril., 53, ) Yokota, Y., Sato, S., Yokota, M., Ishikawa, Y., Makita, M., Asada, T. and Araki, Y. (2000): Successful pregnancy following blastocyst vitrification. Hum. Reprod., 15, ) Mukaida, T., Wada, S., Takahashi, K., Pedro, P, B., An, T, Z. and Kasai, M. (1998): Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum. Reprod., 13, ) Mukaida, T., Nakamura, S., Tomiyama, T., Wada, S., Kasai, M. and Takahashi, K. (2001): Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil. Steril., 76, ) Kuwayama, M. and Kato, O. (2000): All round vitrification of human oocytes and embryos. J. Assist. Reprod. Genetic., 17, ) Kumon, M., Kumasako, Y., Utsunomiya, T. and Araki, Y. (2003): A vitrification method by means of a straw to prevent infections in mouse pronuclear embryos. J. Mamm. Ova Res., 20, ) Kumasako, Y., Kumon, M., Utsunomiya, T. and Araki, Y. (2005): Successful pregnancy after the vitrificaion of zygotes using commercial vitrification solutions and conventional straws to protect against infections in liquid nitrogen. J. Assist. Reprod. Genetic., 22, ) 2005 Straw ART pp ) Utsunomiya, T., Naitou, T. and Nagaki, M. (2002): A prospective trial of blastocyst culture and transfer. Hum. Reprod., 17,
8 122 J. Mamm. Ova Res. Vol.31 (4), ) Utsunomiya, T., Ito, H., Hirai, K., Otsu, E., Watanabe, H. and Mori, T. (2006): Developmentally retarded frozen blastocysts can be rescued by synchronizing culture prior to transfer. Reprod. Biomed. Online, 12, ) Ishimori, H., Saeki, K., Nagao, Y., Itasaka, J., Miki, Y., Seike, N. and Kainuma, H. (1993): Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology, 40, ) Zhu, S, E., Kasai, M., Otoge, H., Sakurai, T. and Machida, T. (1993): Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycolbased solutions. J. Raprod. Fertil., 98, ) Kasai, M. and Mukaida, T. (2004): Cryopreservation of animal and human embryos by vitrification. Reprod. Biomed. Online, 9, ) Jin, B., Mochida, K., Ogura, A., Koshimoto, C., Matsukawa, K., Kasai, M. and Egashige, K. (2012): Equilibrium vitrification of mouse embryos at various developmental stages. Mol. Reprod. Dev., 79, ) Kuleshova, L.L. and Shaw, J.M. (2000): A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during strage in liquid nitrogen. Hum. Reprod., 15, ) Letur-Konirsch, H., Collin, G., Sifer, C., Devaux, A., Kuttenn, F., Madelenat, P., Brun-Vezinet., F., Feldmann, G. and Benifla, J.L. (2000): Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions. Hum. Reprod., 18, ) Bielanski, A., Bergeron, H., Lau, P, C. and Devenish, J. (2003): Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology, 46, ) Scaravelli, G. (2011): Benefit and risk of application of Europian tissue management regulation in ART. Placenta, 32, S243 S ) Bielanski, A. and Vajta, G. (2009): Risk of contamination of germplasm during cryobanking in IVF units. Hum. Reprod., 24, ) 2005 ART pp
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