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Is vitrification standard method of cryopreservation
Middle East Fertility Society Journal (2012) 17, 152–156
Middle East Fertility Society
Middle East Fertility Society Journal www.mefsjournal.org www.sciencedirect.com
OPINION ARTICLE
Is vitrification standard method of cryopreservation Safak Tavukcuoglu, Tahani Al-Azawi, Amir Afshin Khaki, Safaa Al-Hasani
*
Reproductive Medicine Unit, University of Schleswig-Holstein at Luebeck, Ratzburger Alle 160, 23538 Luebeck, Germany Received 6 July 2012; accepted 29 July 2012 Available online 26 August 2012
KEYWORDS Vitrification; Cryopreservation; Oocytes; Embryos; Blastocyts; Assisted reproduction
Abstract Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins. Vitrification is a cryopreservation technique that leads to a glass-like solidification. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Moreover, the use of vitrification technology for ovarian tissue cryopreservation to freeze eggs offers such an elderly women who sometime find more difficulty in conceiving or in maintaining pregnancy till full term because of old age compared to relatively younger women who might get better chances to get a healthy pregnancy. Furthermore, vitrification helps cancer patients who are looking to preserve their fertility later on after completing their treatment. Ó 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction Traditional cryopreservation methods allow ice to form and solute concentrations to rise during the preservation process: both ice and high solute concentrations can cause damage. Cryoprotectants are highly soluble, permeating compounds of low toxicity; they reduce the amount of ice that crystallize at any given temperature and thereby limit the solute concentration factor. Vitrification methods use cryoprotectant concentrations that are sufficient to prevent the crystallization of * Corresponding author. E-mail address: [email protected] (S. Al-Hasani). Peer review under responsibility of Middle East Fertility Society.
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ice altogether the material solidifies as an amorphous glass and both ice and solute concentration are avoided. However, the concentrations of cryoprotectant required are very high indeed and therefore are potentially, and often actually, harmful to cells. Optimization of temperature and the rate of introduction and removal of such high cryoprotectant concentrations are critical. The necessary concentration can be lowered if very rapid cooling, and even more rapid warming, are used. We discuss these basic phenomena in order to consider the important role of vitrification techniques in the cryopreservation of human gametes, embryos and gonads (1). Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins.
1110-5690 Ó 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mefs.2012.07.007
Is vitrification standard method of cryopreservation 2. Vitrification method Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. The vitrification method has advantages over the slow freezing method. Injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short time and cost effective without a programed freezer. Successful cryopreservation of human embryos was first reported in 1983 by Trounson and Mohr (2) using days 2 and 3 embryos that had been slow-cooled using dimethyl sulphoxide (DMSO). Subsequent modifications of the technique had taken place such as introducing 1,2-propanediol and sucrose as cryoprotectants (3) and slow-cooling to 30 °C prior to plunging into liquid nitrogen. All these resulted in the introduction of cryopreservation as a standard method offered by virtually every IVF program world-wide (4). Recently, lots of reproductive clinics, using embryos and blastocysts cryopreservation depend on vitrification. Vitrification is an ultra-rapid method of cryopreservation whereby the embryo is transitioned from 37 to 196 °C in <1 s, resulting in extremely fast rates of cooling. High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass-like state (5). Vitrification is a non-equilibrium method and may be regarded as a radical approach in which ice crystal formation is totally eliminated. Nevertheless, it requires an extremely high cooling rate alongside much higher concentrations of cryoprotectants when compared with slow freezing (6,7). This method does not require expensive equipment and is not time-consuming. Human embryo vitrification has been attempted with a variety of vessels such as electron microscope grids (6,7), open pulled and hemi-straws (8,9), the Flexipet (10), the Cryotop (11) and the CryoLoop (12–14). A number of innovations have been introduced to increase the rate of cooling achieved. A minimal volume of the cryoprotectant solution containing the oocyte is exposed directly to liquid nitrogen in either a thin open straw (15), which has since been modified to the Crytop (16), or on an electron microscopy grid (17), which has subsequently been modified to the Cryoloop (18). In addition, an extensive evaluation of cryoprotectant combinations for vitrification indicated that a very high concentration of ethylene glycol (EG; 5.5 M) and sucrose (1.0 M) could be applied with minimal toxicity (19). Using this cryoprotectant solution in combination with thin straws, the first birth from mature human oocyte vitrification was reported (20). The same procedure, but using grids, resulted in a pregnancy from immature human oocytes (21) and seven births from mature oocytes (22). In these studies demonstrated, vitrification of oocyte-cumulus complexes resulted in 69% survival and a fertilization rate of 72% following ICSI. Although, 95% of fertilized oocytes cleaved, a low implantation rate of 6% suggested impaired developmental potential. The same group had previously reported similar results using human immature oocytes (23–26). Resultant blastocysts that could be assessed in these studies (n = 7) all had normal karyotypes. 3. Oocyte cryopreservation The first human pregnancy from cryopreserved (by slow freezing) oocyte was reported in 1986 (27). This followed many
153 success experiments in other laboratory species that came a few years earlier, such as in mouse (28) and rat (29). Despite several decades of research since these initial reports, success is still very limited. A meta-analysis on slow freezing of human oocytes showed that clinical pregnancy rate per thawed oocyte was only 2.4% (95/4000) and only 1.9% (76/4000) resulted in live birth (30). Vitrification gained a foothold only after 2005, prior to which only ten human pregnancies resulting from vitrified oocytes were reported (30). Although, high oocyte survival rate is achieved with both methods, fertilization and embryo transfer rates are still considerably lower than when fresh oocytes are used (31). When comparing slow freezing to vitrification, higher oocyte survival rates are achieved by the latter (95%, 899/948) compared with (75%, 1275/1683) by the former but pregnancy rate per thawed/warmed oocyte is still low – in the range of 1.9–8.6% for slow freezing and 3.9–18.8% for vitrification (32). Even among females with repetitive reproductive success, the rate of live birth per oocyte retrieved was reported to be 7.3% (180/2470) among bestprognosis donors and lower than that (5.0%; 52/1044) among standard donors (33). Moreover, oocyte cryopreservation has emerged as a solution that could preserve the fertility without the need of sperms and many women consider fertility preservation as a necessary part of their plan of treatment (34). Many gynecological and non-gynecological cancers as well as their therapeutic treatment may affect the ovarian tissue and its follicles and cause gonadal toxicity. Therefore, oocyte cryopreservation at younger ages or prior to chemotherapy using vitrification had been proposed as a solution to enabling women to yield a normal pregnancy and maintain their fertility (35). 4. Embryo cryopreservation Until now, vitrification has been widely used for the cryopreservation of human oocytes (11,22,36), in vitro matured oocytes (37,38) pronuclear stage (34–39) cleavage stage (12,5,8,40,41), or blastocyst-stage (16,11,42,43). However, there are few publications that show clinical data on the basis of vitrification versus slow freezing, especially for the cleavage stage (40,16). The first reports on successful embryo cryopreservation were published by (44–46) and more than two decades after (47) reported their success trail in freezing spermatozoa. A modification to cooling rate that came a few years later (48) resulted in a basic protocol that is still in vast use today. When considered from conservation standpoint, embryo freezing has the advantage of preserving the entire genetic complement of both parents. Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For blastocysts cryopreservation, the vitrification method has advantages over the slow freezing method also. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulfoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conven-
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Various malignant and nonmalignant diseases have been successfully treated with high-dose chemotherapy or radiotherapy. Even though, many young patients receiving these treatments are at risk of developing reproductive failure, a number of fertility preservation options ranging from spermatozoa and also testicular tissue, embryo cryopreservation to ovarian tissue cryopreservation are now available (50) The sperm cryopreservation must be systematically proposed to all men (even teenagers) undergoing a treatment for cancer potentially harmful for their fertility whatever their sperm quality may be. The testicular tissue cryopreservation is also a method to be discussed for adults, teenagers in case of failure of sperm banking or for prepubertal boys. The first attempts at cryopreservation of spermatozoa were performed during the 1940s. However, it was not until Polge et al. (47) added glycerol as a cryoprotectant that recurring problems were solved. The empirical methods developed during the 1950s are still used today. The motility of cryopreserved and thawed spermatozoa normally falls to approximately 50% of the motility before freezing, wherein inter individual fluctuation can be considerable. Despite routine application, the problem of toxicity due to osmotic stress during saturation and dilution of the cryoprotectant as well as the possible negative influence on the genetic material are as yet unresolved (51–52). Stepwise saturation and dilution can minimize the negative consequences of osmotic stress. In practice, current results are acceptable, but the procedures are still altogether relatively difficult and simplification desirable. Besides the possible savings in time, it should also be considered that cryoprotectants as well as appropriate equipment are necessary. Most laboratories use programmable freezers. The entire procedure (saturation, freezing, and dilution) lasts approximately 30–60 min, and in some circumstances even longer. Compared to the slow-freezing method, vitrification has economic advantages, because no freezing instruments are needed and vitrification warming requires only a few seconds. Classical vitrification requires a high percentage of permeable cryoprotectants in medium (30–50%, compared to 5–7% with slow freezing) and is unsuitable for the vitrification of spermatozoa due to the lethal osmotic effect. Shape and size of the sperm head could be the factors that define the cryosensitivity of the cell. Comparative studies (53) on various mammalian species (boar, bull, ram, rabbit, cat, dog, horse, and human) showed a negative correlation between the size of the sperm head and cryostability.
studies regarding ovarian tissue cryopreservation resulting in live-born offspring preceded the present freezing systems in humans. On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow programmed freezing, using human serum albumin-containing medium, and propanediol, dimethylsulfoxide (DMSO) or ethylene glycol as a cryoprotectant, combined with sucrose. Vitrification is still at the experimental stage. Whole organ cryopreservation is an interesting experimental option. Transplantation of the frozen-thawed tissue is a feasible method to utilize the tissue in infertility treatment (54). Ovarian function has been restored in humans. Because one healthy child has already been born from cryopreserved tissue, the method should perhaps be offered to all young girls and women who can be predicted to undergo premature ovarian failure due to cancer treatment or genetic causes. Maturation of follicles in vitro from frozenthawed tissue is another option that is still under development. More recently, interest in vitrification of oocytes as an alternative to slow freezing has been stimulated by a number of reports. Vitrification is the process by which water is prevented from forming ice due to the viscosity of a highly concentrated cryoprotectant cooled at an extremely rapid rate (55). To reduce exposure to the toxic cocktail of cryoprotectants and prevent extreme dehydration, cells are exposed to the cryoprotectants for a very short period. In three early studies of human oocyte cryopreservation, vitrification was also assessed (56–58). Large variation in survival (4–75%) suggested that technical variations may have a major impact. (59,60) showed that brief exposure of human oocytes to a cryoprotectant cocktail had no impact on fertilization and embryo development to the morula stage. However, following vitrification using this cocktail, although 65% of oocytes survived and 45% of those fertilized, all arrested at the pronuclear stage. Effects observed following vitrification of human oocytes included damage to the zona pellucida and oolemma, disorganization of organelles, spindle disruption and formation of micronuclei (61). However, a small study of seven human oocytes vitrified at the GV stage suggested that the resulting spindle was normal (62). In animal oocytes, spindle disruption has been observed following vitrification (63–68) as have high rates of aneuploidy and polyploidy in resultant zygotes and reduced embryo development (69,17,70,71,65). Using human mature oocytes and a slightly lower concentration of EG (5.0 M) with the Cryotop, high rates of survival (90%), fertilization (90%) and development to the blastocyst stage (50%) have been reported in association with an implantation rate of 18.7% and seven births (16). A modification of the above procedure in which the concentrations of EG and sucrose have been halved, and DMSO (2.1 M) added to the cryoprotectant mix has resulted in 2 births both with normal karyotypes (72), a further 11 births (73–75) and a number of other pregnancies (76–77). No difference was observed in the fertilization rate (93 versus 97%), the proportion of cleaving embryos (97 versus 98%), the implantation rate (13 versus 10%) or the abortion rate (20 versus 18%) when compared with non-vitrified oocytes, respectively (75).
6. Ovarian tissue cryopreservation
7. Conclusions
Human ovarian tissue can be successfully cryopreserved, with good survival and function after thawing. Experimental animal
It is evident from the above discussion that wide variation exists between oocyte vitrification methodology and outcomes
tional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation (49). 5. Sperm cryopreservation
Is vitrification standard method of cryopreservation and that this has changed significantly over time. An attempt to analyze the efficiency of vitrification by meta-analysis included studies using a range of different methodologies. We have restricted our summary analysis of human oocyte vitrification to reports using the most recent protocol, which appears to be the most promising and is being used by a number of groups all over the world. It has been suggested that vitrification may be less traumatic to the meiotic spindle than slow freezing and may also have less effect on cell physiology and thus, vitrification is the standard method of cryopreservation for gametes as well as tissues.
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Conflict of interest
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There is no conflict of interest.
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References (1) Pegg DE. The role of vitrification techniques of cryopreservation in reproductive medicine. Human Fertility 2005;8(4):231–9. (2) Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983;305:707–9. (3) Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985;44:645–51. (4) Anderson AR, Wilkinson SS, Prince S, Crain JL. Reduction of high order multiples in frozen embryo transfers. Reprod Biomed Online 2005;10:402–5. (5) Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod 2008;23:1976–82. (6) Park SP, Kim EY, Oh JH, Nam HK, Leek S, Park Y, et al. Ultrarapid freezing of human multipronuclear zygotes using electron microscope grids. Hum Reprod 2000;15:1787–90. (7) Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoing twin pregnancy after vitrification of blastocysts produced by in vitro matured oocytes retrieved from a woman with polycysticovary syndrome: case report. Hum Reprod 2002;17: 2963–6. (8) El-Danasouri I, Selman H. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001;76:400–2. (9) Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Bollen N, Van Roosendal E, et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 2003;18:1504–11. (10) Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002;4:146–50. (11) Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2007;67:73–80. (12) Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online 2005; 11:434–7. (13) Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel CryoLoop container-less technique. Fertil Steril 1999;72:1073. (14) Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J, et al. Vitrification of human blastocysts using the CryoLoop method:
(21)
(22)
(23)
(24)
(25)
(26)
(27) (28)
(29)
(30) (31)
(32)
(33) (34)
(35)
successful clinical application and birth of offspring. J Assist Reprod Genet 2002;19:304–6. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–5. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11(5):608–14. Martino A, Songsasen N, Leibo S. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod (b) 1996;54:1059–69. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999;17:1234–6. Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993;99:471–7. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999;14(12):3077–9. Wu J, Zhang L, Wang X. In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes. Biol Reprod 2001;121:389–93. 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(6): 1323–6. Hong SW, Chung HM, Lim JM, et al. Improved human oocytes development after vitrification: acomparison of thawing methods. Fertil Steril 1999;72:142–6. Cha KY, Chung HM, Lim JM, Ko JJ, Han SY, Choi DH, Yoon TK. Freezing immature oocytes. Mol Cell Endocrinol 2000;169: 43–7. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, Han SY, Choi DH, Cha K. 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. Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ, Cha KY. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000;74(1):180–1. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;1(8486):884–6. Wittingham D. Fertilization in vitro and development to term of unfertilized Mouse oocytes previous stored at 196 °C. Reprod Fertil 1977;49:89–94. Kasai M, Iritani A, Chang M. Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol Reprod 1979; 21:839–44. Oktay K, Cil AP, Bong H. Efficiency of oocyte cryopreservation: meta analiysis. Fertility and Sterility 2006;86:70–80. Magli MC, Lappi M, Ferraretti AP, Capoti A, Ruberti A, Gianaroli L. Impact of oocyte cryopreservation on embryo development. Fertil Steril 2010;93:510–6. Chen S-U, Yang Y-S. Slow freezing or vitrification of oocytes: their effects on survival, and meiotic spindles and the time schedule for clinical practice. Taiwan J Obstet Gynecol 2009;48: 15–22. Martin LJ. Anticipating infertility. Gend Soc 2010;24:526–45. Soliman HH, Khaki AA, Al-Azawi T, Al-Hasani S. Oocyte cryopreservation, will be a real social choice and family solution? Middle East Fertil Soc J 2012. AL-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A. Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? Reprod Biomed Online 2007;14(3):288–93.
156 (36) Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11(3):300–8. (37) Chian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cuis J, et al. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril 2008 [Epub ahead of print]. (38) Chian RC, Gilbert L, Huang JY Demirtas E, Holzer H, Benjamin A, Buckett WM, Tulandi T, Tan SL. Live birth after vitrification of in vitro matured human oocytes. Fertil Steril 2009;91(2):372–6 [Epub 2008 Jun 2]. (39) Kumasako Y, Kumon M, Utsunomiya T, Araki Y. Successful pregnancy after the vitrification of zygotes using commercial vitrification solutions and conventional straws to protect against infections in liquid nitrogen. J Assist Reprod Genet 2005;22(1): 33–5. (40) Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998;13(1O):2874–9. (41) Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Neonatal outcome after vitrified day 3 embryo transfer: a preliminary study. Fertile steril 2008:8 [Epub ahead of print]. (42) Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004;19(12):2884–8 [Epub 2004 Sep 3]. (43) Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopre-servation. Reprod Biomed Online 2008;16(2):311–20 [Review]. (44) Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature 1971;233:125–6. (45) Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to 196 °C and 269 °C. Life Sci 1972;178: 411–4. (46) Wilmut I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development of survival of mouse embryos during freezing and thawing. Life Sci 1972;11:1071–9. (47) Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949: 164–666. (48) Willadsen SM, Polge C, Rowson LEA, Moor R. Deep freezing of sheep embryos. J Reprod Fertil 1976;46:151–4. (49) Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004;9(2):164–70. (50) So¨nmezer M, Oktay K. Assisted reproduction and fertility preservation techniques in cancer patients. Curr Opin Endocrinol Diabetes Obes 2008;15(6):514–22. (51) Critser JK, Huse-Benda AR, Aaker DV, Ameson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil Steril 1988;50:314–20. (52) Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47–58. (53) Nauk VA. Structure and function of spermatozoa from farm animal by cryopreservation. Shtiinca Kishineu 1991:1–198. (54) Ozmen B, Al-Hasani S. Techniques for ovarian tissue, whole ovary, oocytes and embryo cryopreservation. J Reprod Infertil 2010;11(1):3–11. (55) Taylor MJ. Pysico-chemical principles in low temperature biology. In: Grout BWW, Morris GJ, editors. The effects of low temperatures on biological systems. London: Edward Arnold; 1987. p. 3–71. (56) Trounson A. Preservation of human eggs and embryos. Fertil Steril 1986;46:1–12. (57) Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987;2(8):695–700.
S. Tavukcuoglu et al. (58) Pensis M, Lonmayer E, Psalti I. Screening of conditinary for rapid freezing of human oocyte preliminary study toward their cryopreservation. Fertil Steril 1989;52:787–94. (59) Rall WF, Fahy GM. Ice free cryopreservation of mouse embryo at 196 °C by vitrification. Nature 1985;313:573–5. (60) Hunter JE, Fuller BJ, Bernard A. Vitrification of human oocyte followimg minimal exposure to cryoprotectans initial studies on fertilization and embryonic development. Hum Reprod 1995;10: 1184–8. (61) Santanathan AH, Trounson A, Freeman L. Morphology and fertilizability of frozen human oocytes. Gamete Res 1987;16: 343–54. (62) Neri QV, Takeuchi T, Rosenwaks Z, Palermo GD. Imaging of the metaphase spindle in vitrified human and mouse oocytes matured in vitro. Fertil Steril 2003;80(Suppl 1):S143. (63) Chen SU, Lien YR, Chen HF, Chao KH, Ho HN, Yang YS. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000;15:2598–603. (64) Rojas C, Palomo MJ, Albarracin JL, Mogas T. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 2004;49:211–20. (65) Cai XY, Chen GA, Lian Y, Zheng XY, Peng HM. Cryoloop vitrification of rabbit oocytes. Hum Reprod 2005;20:1969–74. (66) Shi WQ, Zhu SE, Zhang D, Wang WH, Tang GL, Hou YP, Tian SJ. Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes. Biol Reprod 2006;131: 795–804. (67) Tharasanit T, Colenbrander B, Stout TA. Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Mol Reprod Dev 2006;73:627–37. (68) Wu C, Rui R, Dai J, Zhang C, Ju S, Xie B, Lu X, Zheng X. Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes. Mol Reprod Dev 2006;73:1454–62. (69) Kola I, Kirby C, Shaw J, Davey A, Trounson A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 1988;38:467–74. (70) Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004;50:685–96. (71) Rho GJ, Lee SL, Kim YS, Yeo HJ, Ock SA, Balasubramanian S, Choe S. Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development. Mol Reprod Dev 2004;68:449–55. (72) Katayama KP, Stehlik J, Kuwayama M, et al. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223–4. (73) Kyono K, Fuchinoue K, Yagi A, Nakajo Y, Yamashita A, Kumagai S. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte. Fertil Steril 2005;84:1017. (74) Okimura T, Kato K, Zhan Q, Kuwayama M, Zhang J, Kato O. Update on clinical efficiency of the vitrification method for human oocytes in an in vitro fertilization program. Fertil Steril 2005;84(Suppl 1):S174. (75) Antinori M, Licata E, Dani G, Cerusico F, Versaci C, Antinori S. Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries. Reprod Biomed Online 2007;14(1): 72–9. (76) Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006;85(1):108–11. (77) Selman H, Angelini A, Barnacchi N, Brusco GF, Pacchiarotti A, Aragona C. Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertil Steril 2006;86:997–1000.
Middle East Fertility Society
Middle East Fertility Society Journal www.mefsjournal.org www.sciencedirect.com
OPINION ARTICLE
Is vitrification standard method of cryopreservation Safak Tavukcuoglu, Tahani Al-Azawi, Amir Afshin Khaki, Safaa Al-Hasani
*
Reproductive Medicine Unit, University of Schleswig-Holstein at Luebeck, Ratzburger Alle 160, 23538 Luebeck, Germany Received 6 July 2012; accepted 29 July 2012 Available online 26 August 2012
KEYWORDS Vitrification; Cryopreservation; Oocytes; Embryos; Blastocyts; Assisted reproduction
Abstract Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins. Vitrification is a cryopreservation technique that leads to a glass-like solidification. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Moreover, the use of vitrification technology for ovarian tissue cryopreservation to freeze eggs offers such an elderly women who sometime find more difficulty in conceiving or in maintaining pregnancy till full term because of old age compared to relatively younger women who might get better chances to get a healthy pregnancy. Furthermore, vitrification helps cancer patients who are looking to preserve their fertility later on after completing their treatment. Ó 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction Traditional cryopreservation methods allow ice to form and solute concentrations to rise during the preservation process: both ice and high solute concentrations can cause damage. Cryoprotectants are highly soluble, permeating compounds of low toxicity; they reduce the amount of ice that crystallize at any given temperature and thereby limit the solute concentration factor. Vitrification methods use cryoprotectant concentrations that are sufficient to prevent the crystallization of * Corresponding author. E-mail address: [email protected] (S. Al-Hasani). Peer review under responsibility of Middle East Fertility Society.
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ice altogether the material solidifies as an amorphous glass and both ice and solute concentration are avoided. However, the concentrations of cryoprotectant required are very high indeed and therefore are potentially, and often actually, harmful to cells. Optimization of temperature and the rate of introduction and removal of such high cryoprotectant concentrations are critical. The necessary concentration can be lowered if very rapid cooling, and even more rapid warming, are used. We discuss these basic phenomena in order to consider the important role of vitrification techniques in the cryopreservation of human gametes, embryos and gonads (1). Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins.
1110-5690 Ó 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mefs.2012.07.007
Is vitrification standard method of cryopreservation 2. Vitrification method Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. The vitrification method has advantages over the slow freezing method. Injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short time and cost effective without a programed freezer. Successful cryopreservation of human embryos was first reported in 1983 by Trounson and Mohr (2) using days 2 and 3 embryos that had been slow-cooled using dimethyl sulphoxide (DMSO). Subsequent modifications of the technique had taken place such as introducing 1,2-propanediol and sucrose as cryoprotectants (3) and slow-cooling to 30 °C prior to plunging into liquid nitrogen. All these resulted in the introduction of cryopreservation as a standard method offered by virtually every IVF program world-wide (4). Recently, lots of reproductive clinics, using embryos and blastocysts cryopreservation depend on vitrification. Vitrification is an ultra-rapid method of cryopreservation whereby the embryo is transitioned from 37 to 196 °C in <1 s, resulting in extremely fast rates of cooling. High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass-like state (5). Vitrification is a non-equilibrium method and may be regarded as a radical approach in which ice crystal formation is totally eliminated. Nevertheless, it requires an extremely high cooling rate alongside much higher concentrations of cryoprotectants when compared with slow freezing (6,7). This method does not require expensive equipment and is not time-consuming. Human embryo vitrification has been attempted with a variety of vessels such as electron microscope grids (6,7), open pulled and hemi-straws (8,9), the Flexipet (10), the Cryotop (11) and the CryoLoop (12–14). A number of innovations have been introduced to increase the rate of cooling achieved. A minimal volume of the cryoprotectant solution containing the oocyte is exposed directly to liquid nitrogen in either a thin open straw (15), which has since been modified to the Crytop (16), or on an electron microscopy grid (17), which has subsequently been modified to the Cryoloop (18). In addition, an extensive evaluation of cryoprotectant combinations for vitrification indicated that a very high concentration of ethylene glycol (EG; 5.5 M) and sucrose (1.0 M) could be applied with minimal toxicity (19). Using this cryoprotectant solution in combination with thin straws, the first birth from mature human oocyte vitrification was reported (20). The same procedure, but using grids, resulted in a pregnancy from immature human oocytes (21) and seven births from mature oocytes (22). In these studies demonstrated, vitrification of oocyte-cumulus complexes resulted in 69% survival and a fertilization rate of 72% following ICSI. Although, 95% of fertilized oocytes cleaved, a low implantation rate of 6% suggested impaired developmental potential. The same group had previously reported similar results using human immature oocytes (23–26). Resultant blastocysts that could be assessed in these studies (n = 7) all had normal karyotypes. 3. Oocyte cryopreservation The first human pregnancy from cryopreserved (by slow freezing) oocyte was reported in 1986 (27). This followed many
153 success experiments in other laboratory species that came a few years earlier, such as in mouse (28) and rat (29). Despite several decades of research since these initial reports, success is still very limited. A meta-analysis on slow freezing of human oocytes showed that clinical pregnancy rate per thawed oocyte was only 2.4% (95/4000) and only 1.9% (76/4000) resulted in live birth (30). Vitrification gained a foothold only after 2005, prior to which only ten human pregnancies resulting from vitrified oocytes were reported (30). Although, high oocyte survival rate is achieved with both methods, fertilization and embryo transfer rates are still considerably lower than when fresh oocytes are used (31). When comparing slow freezing to vitrification, higher oocyte survival rates are achieved by the latter (95%, 899/948) compared with (75%, 1275/1683) by the former but pregnancy rate per thawed/warmed oocyte is still low – in the range of 1.9–8.6% for slow freezing and 3.9–18.8% for vitrification (32). Even among females with repetitive reproductive success, the rate of live birth per oocyte retrieved was reported to be 7.3% (180/2470) among bestprognosis donors and lower than that (5.0%; 52/1044) among standard donors (33). Moreover, oocyte cryopreservation has emerged as a solution that could preserve the fertility without the need of sperms and many women consider fertility preservation as a necessary part of their plan of treatment (34). Many gynecological and non-gynecological cancers as well as their therapeutic treatment may affect the ovarian tissue and its follicles and cause gonadal toxicity. Therefore, oocyte cryopreservation at younger ages or prior to chemotherapy using vitrification had been proposed as a solution to enabling women to yield a normal pregnancy and maintain their fertility (35). 4. Embryo cryopreservation Until now, vitrification has been widely used for the cryopreservation of human oocytes (11,22,36), in vitro matured oocytes (37,38) pronuclear stage (34–39) cleavage stage (12,5,8,40,41), or blastocyst-stage (16,11,42,43). However, there are few publications that show clinical data on the basis of vitrification versus slow freezing, especially for the cleavage stage (40,16). The first reports on successful embryo cryopreservation were published by (44–46) and more than two decades after (47) reported their success trail in freezing spermatozoa. A modification to cooling rate that came a few years later (48) resulted in a basic protocol that is still in vast use today. When considered from conservation standpoint, embryo freezing has the advantage of preserving the entire genetic complement of both parents. Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For blastocysts cryopreservation, the vitrification method has advantages over the slow freezing method also. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulfoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conven-
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Various malignant and nonmalignant diseases have been successfully treated with high-dose chemotherapy or radiotherapy. Even though, many young patients receiving these treatments are at risk of developing reproductive failure, a number of fertility preservation options ranging from spermatozoa and also testicular tissue, embryo cryopreservation to ovarian tissue cryopreservation are now available (50) The sperm cryopreservation must be systematically proposed to all men (even teenagers) undergoing a treatment for cancer potentially harmful for their fertility whatever their sperm quality may be. The testicular tissue cryopreservation is also a method to be discussed for adults, teenagers in case of failure of sperm banking or for prepubertal boys. The first attempts at cryopreservation of spermatozoa were performed during the 1940s. However, it was not until Polge et al. (47) added glycerol as a cryoprotectant that recurring problems were solved. The empirical methods developed during the 1950s are still used today. The motility of cryopreserved and thawed spermatozoa normally falls to approximately 50% of the motility before freezing, wherein inter individual fluctuation can be considerable. Despite routine application, the problem of toxicity due to osmotic stress during saturation and dilution of the cryoprotectant as well as the possible negative influence on the genetic material are as yet unresolved (51–52). Stepwise saturation and dilution can minimize the negative consequences of osmotic stress. In practice, current results are acceptable, but the procedures are still altogether relatively difficult and simplification desirable. Besides the possible savings in time, it should also be considered that cryoprotectants as well as appropriate equipment are necessary. Most laboratories use programmable freezers. The entire procedure (saturation, freezing, and dilution) lasts approximately 30–60 min, and in some circumstances even longer. Compared to the slow-freezing method, vitrification has economic advantages, because no freezing instruments are needed and vitrification warming requires only a few seconds. Classical vitrification requires a high percentage of permeable cryoprotectants in medium (30–50%, compared to 5–7% with slow freezing) and is unsuitable for the vitrification of spermatozoa due to the lethal osmotic effect. Shape and size of the sperm head could be the factors that define the cryosensitivity of the cell. Comparative studies (53) on various mammalian species (boar, bull, ram, rabbit, cat, dog, horse, and human) showed a negative correlation between the size of the sperm head and cryostability.
studies regarding ovarian tissue cryopreservation resulting in live-born offspring preceded the present freezing systems in humans. On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow programmed freezing, using human serum albumin-containing medium, and propanediol, dimethylsulfoxide (DMSO) or ethylene glycol as a cryoprotectant, combined with sucrose. Vitrification is still at the experimental stage. Whole organ cryopreservation is an interesting experimental option. Transplantation of the frozen-thawed tissue is a feasible method to utilize the tissue in infertility treatment (54). Ovarian function has been restored in humans. Because one healthy child has already been born from cryopreserved tissue, the method should perhaps be offered to all young girls and women who can be predicted to undergo premature ovarian failure due to cancer treatment or genetic causes. Maturation of follicles in vitro from frozenthawed tissue is another option that is still under development. More recently, interest in vitrification of oocytes as an alternative to slow freezing has been stimulated by a number of reports. Vitrification is the process by which water is prevented from forming ice due to the viscosity of a highly concentrated cryoprotectant cooled at an extremely rapid rate (55). To reduce exposure to the toxic cocktail of cryoprotectants and prevent extreme dehydration, cells are exposed to the cryoprotectants for a very short period. In three early studies of human oocyte cryopreservation, vitrification was also assessed (56–58). Large variation in survival (4–75%) suggested that technical variations may have a major impact. (59,60) showed that brief exposure of human oocytes to a cryoprotectant cocktail had no impact on fertilization and embryo development to the morula stage. However, following vitrification using this cocktail, although 65% of oocytes survived and 45% of those fertilized, all arrested at the pronuclear stage. Effects observed following vitrification of human oocytes included damage to the zona pellucida and oolemma, disorganization of organelles, spindle disruption and formation of micronuclei (61). However, a small study of seven human oocytes vitrified at the GV stage suggested that the resulting spindle was normal (62). In animal oocytes, spindle disruption has been observed following vitrification (63–68) as have high rates of aneuploidy and polyploidy in resultant zygotes and reduced embryo development (69,17,70,71,65). Using human mature oocytes and a slightly lower concentration of EG (5.0 M) with the Cryotop, high rates of survival (90%), fertilization (90%) and development to the blastocyst stage (50%) have been reported in association with an implantation rate of 18.7% and seven births (16). A modification of the above procedure in which the concentrations of EG and sucrose have been halved, and DMSO (2.1 M) added to the cryoprotectant mix has resulted in 2 births both with normal karyotypes (72), a further 11 births (73–75) and a number of other pregnancies (76–77). No difference was observed in the fertilization rate (93 versus 97%), the proportion of cleaving embryos (97 versus 98%), the implantation rate (13 versus 10%) or the abortion rate (20 versus 18%) when compared with non-vitrified oocytes, respectively (75).
6. Ovarian tissue cryopreservation
7. Conclusions
Human ovarian tissue can be successfully cryopreserved, with good survival and function after thawing. Experimental animal
It is evident from the above discussion that wide variation exists between oocyte vitrification methodology and outcomes
tional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation (49). 5. Sperm cryopreservation
Is vitrification standard method of cryopreservation and that this has changed significantly over time. An attempt to analyze the efficiency of vitrification by meta-analysis included studies using a range of different methodologies. We have restricted our summary analysis of human oocyte vitrification to reports using the most recent protocol, which appears to be the most promising and is being used by a number of groups all over the world. It has been suggested that vitrification may be less traumatic to the meiotic spindle than slow freezing and may also have less effect on cell physiology and thus, vitrification is the standard method of cryopreservation for gametes as well as tissues.
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Conflict of interest
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There is no conflict of interest.
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References (1) Pegg DE. The role of vitrification techniques of cryopreservation in reproductive medicine. Human Fertility 2005;8(4):231–9. (2) Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983;305:707–9. (3) Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985;44:645–51. (4) Anderson AR, Wilkinson SS, Prince S, Crain JL. Reduction of high order multiples in frozen embryo transfers. Reprod Biomed Online 2005;10:402–5. (5) Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod 2008;23:1976–82. (6) Park SP, Kim EY, Oh JH, Nam HK, Leek S, Park Y, et al. Ultrarapid freezing of human multipronuclear zygotes using electron microscope grids. Hum Reprod 2000;15:1787–90. (7) Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoing twin pregnancy after vitrification of blastocysts produced by in vitro matured oocytes retrieved from a woman with polycysticovary syndrome: case report. Hum Reprod 2002;17: 2963–6. (8) El-Danasouri I, Selman H. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001;76:400–2. (9) Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Bollen N, Van Roosendal E, et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 2003;18:1504–11. (10) Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002;4:146–50. (11) Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2007;67:73–80. (12) Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online 2005; 11:434–7. (13) Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel CryoLoop container-less technique. Fertil Steril 1999;72:1073. (14) Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J, et al. Vitrification of human blastocysts using the CryoLoop method:
(21)
(22)
(23)
(24)
(25)
(26)
(27) (28)
(29)
(30) (31)
(32)
(33) (34)
(35)
successful clinical application and birth of offspring. J Assist Reprod Genet 2002;19:304–6. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–5. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11(5):608–14. Martino A, Songsasen N, Leibo S. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod (b) 1996;54:1059–69. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999;17:1234–6. Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993;99:471–7. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999;14(12):3077–9. Wu J, Zhang L, Wang X. In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes. Biol Reprod 2001;121:389–93. 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(6): 1323–6. Hong SW, Chung HM, Lim JM, et al. Improved human oocytes development after vitrification: acomparison of thawing methods. Fertil Steril 1999;72:142–6. Cha KY, Chung HM, Lim JM, Ko JJ, Han SY, Choi DH, Yoon TK. Freezing immature oocytes. Mol Cell Endocrinol 2000;169: 43–7. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, Han SY, Choi DH, Cha K. 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. Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ, Cha KY. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000;74(1):180–1. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;1(8486):884–6. Wittingham D. Fertilization in vitro and development to term of unfertilized Mouse oocytes previous stored at 196 °C. Reprod Fertil 1977;49:89–94. Kasai M, Iritani A, Chang M. Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol Reprod 1979; 21:839–44. Oktay K, Cil AP, Bong H. Efficiency of oocyte cryopreservation: meta analiysis. Fertility and Sterility 2006;86:70–80. Magli MC, Lappi M, Ferraretti AP, Capoti A, Ruberti A, Gianaroli L. Impact of oocyte cryopreservation on embryo development. Fertil Steril 2010;93:510–6. Chen S-U, Yang Y-S. Slow freezing or vitrification of oocytes: their effects on survival, and meiotic spindles and the time schedule for clinical practice. Taiwan J Obstet Gynecol 2009;48: 15–22. Martin LJ. Anticipating infertility. Gend Soc 2010;24:526–45. Soliman HH, Khaki AA, Al-Azawi T, Al-Hasani S. Oocyte cryopreservation, will be a real social choice and family solution? Middle East Fertil Soc J 2012. AL-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A. Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? Reprod Biomed Online 2007;14(3):288–93.
156 (36) Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11(3):300–8. (37) Chian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cuis J, et al. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril 2008 [Epub ahead of print]. (38) Chian RC, Gilbert L, Huang JY Demirtas E, Holzer H, Benjamin A, Buckett WM, Tulandi T, Tan SL. Live birth after vitrification of in vitro matured human oocytes. Fertil Steril 2009;91(2):372–6 [Epub 2008 Jun 2]. (39) Kumasako Y, Kumon M, Utsunomiya T, Araki Y. Successful pregnancy after the vitrification of zygotes using commercial vitrification solutions and conventional straws to protect against infections in liquid nitrogen. J Assist Reprod Genet 2005;22(1): 33–5. (40) Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998;13(1O):2874–9. (41) Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Neonatal outcome after vitrified day 3 embryo transfer: a preliminary study. Fertile steril 2008:8 [Epub ahead of print]. (42) Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004;19(12):2884–8 [Epub 2004 Sep 3]. (43) Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopre-servation. Reprod Biomed Online 2008;16(2):311–20 [Review]. (44) Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature 1971;233:125–6. (45) Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to 196 °C and 269 °C. Life Sci 1972;178: 411–4. (46) Wilmut I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development of survival of mouse embryos during freezing and thawing. Life Sci 1972;11:1071–9. (47) Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949: 164–666. (48) Willadsen SM, Polge C, Rowson LEA, Moor R. Deep freezing of sheep embryos. J Reprod Fertil 1976;46:151–4. (49) Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004;9(2):164–70. (50) So¨nmezer M, Oktay K. Assisted reproduction and fertility preservation techniques in cancer patients. Curr Opin Endocrinol Diabetes Obes 2008;15(6):514–22. (51) Critser JK, Huse-Benda AR, Aaker DV, Ameson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil Steril 1988;50:314–20. (52) Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47–58. (53) Nauk VA. Structure and function of spermatozoa from farm animal by cryopreservation. Shtiinca Kishineu 1991:1–198. (54) Ozmen B, Al-Hasani S. Techniques for ovarian tissue, whole ovary, oocytes and embryo cryopreservation. J Reprod Infertil 2010;11(1):3–11. (55) Taylor MJ. Pysico-chemical principles in low temperature biology. In: Grout BWW, Morris GJ, editors. The effects of low temperatures on biological systems. London: Edward Arnold; 1987. p. 3–71. (56) Trounson A. Preservation of human eggs and embryos. Fertil Steril 1986;46:1–12. (57) Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987;2(8):695–700.
S. Tavukcuoglu et al. (58) Pensis M, Lonmayer E, Psalti I. Screening of conditinary for rapid freezing of human oocyte preliminary study toward their cryopreservation. Fertil Steril 1989;52:787–94. (59) Rall WF, Fahy GM. Ice free cryopreservation of mouse embryo at 196 °C by vitrification. Nature 1985;313:573–5. (60) Hunter JE, Fuller BJ, Bernard A. Vitrification of human oocyte followimg minimal exposure to cryoprotectans initial studies on fertilization and embryonic development. Hum Reprod 1995;10: 1184–8. (61) Santanathan AH, Trounson A, Freeman L. Morphology and fertilizability of frozen human oocytes. Gamete Res 1987;16: 343–54. (62) Neri QV, Takeuchi T, Rosenwaks Z, Palermo GD. Imaging of the metaphase spindle in vitrified human and mouse oocytes matured in vitro. Fertil Steril 2003;80(Suppl 1):S143. (63) Chen SU, Lien YR, Chen HF, Chao KH, Ho HN, Yang YS. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000;15:2598–603. (64) Rojas C, Palomo MJ, Albarracin JL, Mogas T. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 2004;49:211–20. (65) Cai XY, Chen GA, Lian Y, Zheng XY, Peng HM. Cryoloop vitrification of rabbit oocytes. Hum Reprod 2005;20:1969–74. (66) Shi WQ, Zhu SE, Zhang D, Wang WH, Tang GL, Hou YP, Tian SJ. Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes. Biol Reprod 2006;131: 795–804. (67) Tharasanit T, Colenbrander B, Stout TA. Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Mol Reprod Dev 2006;73:627–37. (68) Wu C, Rui R, Dai J, Zhang C, Ju S, Xie B, Lu X, Zheng X. Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes. Mol Reprod Dev 2006;73:1454–62. (69) Kola I, Kirby C, Shaw J, Davey A, Trounson A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 1988;38:467–74. (70) Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004;50:685–96. (71) Rho GJ, Lee SL, Kim YS, Yeo HJ, Ock SA, Balasubramanian S, Choe S. Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development. Mol Reprod Dev 2004;68:449–55. (72) Katayama KP, Stehlik J, Kuwayama M, et al. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223–4. (73) Kyono K, Fuchinoue K, Yagi A, Nakajo Y, Yamashita A, Kumagai S. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte. Fertil Steril 2005;84:1017. (74) Okimura T, Kato K, Zhan Q, Kuwayama M, Zhang J, Kato O. Update on clinical efficiency of the vitrification method for human oocytes in an in vitro fertilization program. Fertil Steril 2005;84(Suppl 1):S174. (75) Antinori M, Licata E, Dani G, Cerusico F, Versaci C, Antinori S. Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries. Reprod Biomed Online 2007;14(1): 72–9. (76) Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006;85(1):108–11. (77) Selman H, Angelini A, Barnacchi N, Brusco GF, Pacchiarotti A, Aragona C. Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertil Steril 2006;86:997–1000.