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Freezing immature oocytes
Molecular and Cellular Endocrinology 169 (2000) 43 – 47 www.elsevier.com/locate/mce
Freezing immature oocytes K.Y. Cha a,b,*, H.M. Chung a, J.M. Lim c, J.J. Ko a, S.Y. Han a, D.H. Choi a, T.K. Yoon a a
College of Medicine, Pochon CHA Uni6ersity and Infertility Medical Center of CHA General Hospital, 606 -5 Yeoksam 1 -Dong, Seoul 135 -081, South Korea b CHA-Columbia Infertility Medical Center, Manhattan, New York, NY 10032, USA c School of Agricultural Biotechnology, Seoul National Uni6ersity, Suwon 441 -744, South Korea
Abstract The establishment of a long-term preservation system for mammalian oocytes is important for the development of both biological and medical sciences. A number of efforts have been made to develop this system. In human reproductive medicine, the development of an oocyte cryopreservation system can improve the efficacy of the current assisted reproductive technology (ART) for infertile patients with severe reproductive disorders. In this article, the technical development of cryopreservation programs for human oocytes and its biological background were reviewed. Clinical outcome after the use of this technology was further introduced. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human; Oocyte; Cryopreservation; Oocyte bank
1. History of oocyte cryopreservation As in vitro manipulation technologies, such as in vitro-maturation (IVM), in vitro-fertilization (IVF) and in vitro-culture (IVC) of mammalian gametes and zygotes, was developed in the 1980s, the significance of an oocyte cryopreservation technique was recognized. It provides the possibilities that valuable genetic resources can be preserved for a designated period without the loss of viability and that the efficacy of reproductive biotechnology is efficiently renovated. A number of efforts have been made to preserve mammalian oocytes since the first successful attempts in the bovine (Lim et al., 1991), hamster (Todorow et al., 1989), human (Trounson, 1986), mouse (Sathanathan et al., 1988), mare (Hochi et al., 1995), monkey (DeMayo et al., 1985), pig (Rubinsky et al., 1992), rabbit (Vincent et al., 1989) and rat (Kasai et al., 1979) species. In particular, scientists in the field of human reproductive medicine had a great interest in developing this technique, since an oocyte bank system could be established * Corresponding author. Present address: College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital, 606-5 Yeoksam 1-Dong, Seoul 135-081, South Korea. Tel.: +82-2-34683000; fax: +82-2-5018704.
based on it. The oocyte bank system directly contributes to establishing an oocyte donation system, which is a feasible system to treat a number of congenital infertility disorders, such as hypoplastic ovaries and premature ovarian failure. This system also provides the chances of pregnancy in patients who receive anticancer treatments. The efficacy of assisted reproductive technology (ART) program can be improved by the use of this technique. Surplus oocytes from the fresh IVFET cycle of patients can be stored and, when the patients fail their fresh cycle, the cryopreserved oocytes can be provided for the next cycle after thawing. Furthermore, the innovative family planning to support the social activity of modern women can be designed using the oocyte bank system. Considering that several European countries, such as Austria, Germany, Switzerland, Denmark and Sweden, either bans or strictly limits the research and clinical application of embryo cryopreservation, the oocyte bank system can lessen ethical and legal issues caused by human embryo freezing. Attempts to develop an oocyte cryopreservation method in the human have extensively been made after the first success of Chen (1986), who reported on the first live births from frozen human mature oocytes. As shown in Table 1, a number of pregnancies were subse-
0303-7207/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0303-7207(00)00350-6
44
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
quently established after undergoing freezing-ET programs (Al-Hasani et al., 1986, 1987; Van Uem et al., 1987; Diedrich et al., 1988; Siebzehnruebl et al., 1989; Tucker et al., 1996, 1998; Porcu et al., 1997; De Fried et al., 1998; Young et al., 1998; Nawroth and Kissing, 1999; Porcu et al., 1999a,b; Yoon et al., 2000), but very few pregnancies were established, except for the report of Porcu (1999).
2. Limitation of oocyte cryopreservation There has been solid limitation to improving the oocyte cryopreservation system. Trounson (1986) raised a number of problems which occurred during cryopreservation. Generally, the ultrastructure of oocytes is particularly sensitive to the changes of temperature and extracellular osmotic pressure during freezing and thawing. Various cellular damage, such as cytoskeleton disorganization (Johnson and Pickering, 1987), chromosome and DNA abnormality (Bouquet et al., 1993), spindle disintegration (Park et al., 1997), premature cortical granule exocytosis (Carroll et al., 1990) and its related hardening of zona pellucida (Moller and Wassarman, 1989) and plasma membrane disintegrity (Ashwood-Smith et al., 1988), are frequently found when oocytes are placed at a low temperature, below 0°C. All of these impairments negatively affect the developmental competence of frozen oocytes. Al-Hasani et al. (1987) reported that only 11% of cryopreserved oocytes from 48 patients were fertilized in vitro. Lower maturation, fertilization and cleavage rates were also found in slowly frozen oocytes than in fresh oocytes (Son et al.,
1996). Park et al. (1997) reported that the high incidence of chromosome and spindle abnormalities was detected in frozen oocytes. On the other hand, the maturation stage of oocytes at the time of freezing greatly affects the cryopreservation capacity and survival after thawing. In our preliminary study using an animal model (Lim et al., 1991), one-cell embryos has higher post-thawed survival and developmental capacities than oocytes frozen slowly at the metaphase-II (MII) stage. Furthermore, more oocytes survived, fertilized and developed after slowly freezing at the MII stage than after slowly freezing at the germinal vesicle (GV) stage (Lim et al., 1991). To enhance the viability of oocytes after thawing, we should consider the type of cryopreservation programs and the stage of oocyte maturation at the time of freezing.
3. Development of vitrification method As shown in Table 1, only a limited number of patients successfully became pregnant and delivered babies after IVF-ET program, utilizing embryos developed from frozen –thawed oocytes. To overcome such limitations, we have attempted to develop a vitrification method for the cryopreservation of human oocytes, instead of slow freezing methods. High concentration of cryoprotectant and an extremely rapid cooling speed (\ 2000°C) are employed for the vitrification protocol and, as a result, the formation of intracellular ice crystals in oocyte cytoplasm can be effectively prevented. Furthermore, the equipment used in conven-
Table 1 Pregnancies of patients received embryos developed from cryopreserved oocytesa Authors/years
Type of CPAs
Freezing methods
Stage of freezing
Pregnancies/deliveries
Remarks
(Chen, 1986) (Al-Hasani et al., 1986, 1987) (Diedrich et al., 1988) (Van Uem et al., 1987) (Siebzehnruebl et al., 1989) (Tucker et al., 1996) (Porcu et al., 1997) (Tucker et al., 1998) (De Fried et al., 1998) (Young et al., 1998) (Nawroth and Kissing, 1999) (Tucker et al., 1998) (Porcu, 1999) (Porcu et al., 1999a) (Porcu et al., 1999b) (Yoon et al., 2000)
DM PR/DM DM DM PR/DM PR PR PR PR PR PR PR PR PR PR EG+S
Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Vitri
MII MII MII MII MII MII MII MII MII MII MII GV MII MII MII MII
2/2 2/? 1/? 1/1 1/? 3/0 1/1 5/2 1/1 1/? 1/0 1/1 16/11 1/ongoing 1/ongoing 3/1+ongoing
– – – – – ICSI ICSI ICSI ICSI ICSI ICSI ICSI ICSI ICSI* ICSI** ICSI
a CPAs, cryoprotective agents; DM, dimethylsulfoxide; PR, 1,2-propanedior; EG, ethylene glycol; S, sucrose; Slow, slow freezing program; Vitri, vitrification program; GV, germinal vesicle; MII, metaphase-II. * Epididymal sperm aspiration. ** Testicular sperm aspiration.
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
45
Table 2 Morphological normality, maturation and fertilization of human oocytes vitrified at different times of maturation culture Oocytes from (cycle)
Times of vitrification (h)
No. oocytes vitrified
Parameters (%) Normality
Maturation 12 (40) –
7 (37) 5 (31)
9 (53) 9 (64) –
6 (55) 5 (56) 10 (83)
Unstimulated
0 48
30 16
19 (63) 9 (56)
Stimulated
0 8–15 24–28
17 14 12
11 (65)a 9 (64)a 12 (100)b
a b
Fertilization
PB0.05 within the same column of each cycle. PB0.05 within the same column of each cycle.
tional slow freezing methods are no longer necessary for the vitrification and the required time for the cryopreservation of oocytes can be greatly decreased. In spite of these advantages, the vitrification causes another problem by the use of a high concentration of cryoprotectants. Severe osmotic effect and the cytotoxicity of cryoprotectant may adversely affect oocyte development after cryopreservation. The modification of the vitrification method is necessary to overcome such problems and we developed a new vitrification method for human oocytes. We used ethylene glycol (EG), which has higher membrane permeability and lower cytotoxicity than other cryoprotectants, as a permeable cryoprotectant for this method. Also, appropriate concentrations of sucrose were used as a nonpermeable cryoprotectant. The use of sucrose as a cryoprotectant contributes to regulating intracellular concentration of EG during equilibration and vitrification and to effectively remove EG from oocyte cytoplasm during thawing and dilution procedures. Additional modifications were made for optimizing our vitrification method, reducing the time of the equilibration procedure before vitrification and use of a copper electron microscopic (EM) grid as an oocyte vehicle (Chung et al., 2000). The short equilibration procedure is beneficial for alleviating severe damage resulting from a high concentration of the vitrification solution and use of the EM grid improves thermal conductivity to the oocyte cytoplasm, which supports membrane integrity during equilibration. As the first step of clinical application, we evaluated whether our modified vitrification method was available for cryopreserving human oocytes. In a series of experiments, we examined whether this method was effective for immature oocytes, as well as mature oocytes and evaluated the survival and fetal development of vitrified oocytes following IVF-ET program. For this study, oocytes retrieved from unstimulated and stimulated cycles were vitrified at various times after maturation culture (Chung et al., 2000) and, after vitrification and thawing, thawed oocytes were provided for IVF and in vitro culture. The total duration of maturation culture
was 48 h in unstimulated cycles and 24–28 h in stimulated cycles. In unstimulated cycles, oocytes were vitrified either at 0 or 48 h after IVM. In stimulated cycle, oocytes were vitrified at 0, 8–15 and 24–28 h. Therefore, oocytes vitrified immediately (0 h) were cultured further for 48 h after thawing in unstimulated cycle. On the other hand, oocytes vitrified immediately (0 h) and at 8–15 h after IVM were cultured for a further 24–28 and 13–16 h in maturation medium, respectively, in stimulated cycle. Oocytes vitrified at the end of maturation in both cycles were directly provided for IVF. In oocytes retrieved from an unstimulated cycle, no significant effect was found in the number of morphologically normal oocytes after vitrification (56 –63%; Table 2). Twelve oocytes (40%) vitrified immediately extruded first polar body after 48 h of maturation culture. In this group, 37% of morphologically normal oocytes developed to the normal pronuclear stage at 16–19 h after ICSI, while 31% of oocytes vitrified at the end of maturation culture developed to the pronuclear stage. There was no significant difference in the number of pronuclear stage embryos between the times of vitrification. In oocytes retrieved from stimulated cycle, more oocytes were morphologically normal following vitrification at 24–28 h (100%) of maturation culture than following vitrification immediately or at 8–15 h of the culture (64 –65%). The maturation rate of oocytes vitrified immediately or at 8–15 h after maturation culture was within the range of 53–64% and no significant effect was detected. Also, there was no significant effect of vitrification on the number of vitrified oocytes that developed to the normal pronuclear stage (55 – 83%). Thirty-two out of 33 pronuclear stage embryos (97%) developed from vitrified oocytes cleaved at 48 h postIVF, which was independent of the time of vitrification and hormonal induction. Blastocyst formation occurred with all groups of vitrified oocytes in the range of 20–43%. A total of 12 blastocysts derived from oocytes vitrified at different times of maturation culture were analyzed for their chromosome number. As shown in Table 3, seven blastocysts were successfully analyzed;
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
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Table 3 Detailed data of karyotyping of the blastocysts (BL) derived from vitrified oocytes Oocytes retrieved from (cycle)
Patients
Times* of vitrification
Stages of examination
Results of karyotyping
No. of chromosomes
Chromosome normality
Unstimulated
A B B D E
0 0 0 48 48
Expanded BL Early BL Expanded BL Expanded BL Hatched BL
46, XX Failed to analyze 46, XY Failed to analyze 46, XY
Normal – Normal – Normal
Stimulated
F G H I J K L
0 0 8–15 24–28 24–28 24–28 24–28
Early BL Expanded BL Early BL Expanded BL Early BL Hatched BL Expanded BL
46, XX Failed to analyze Failed to analyze 46, XX 46, XX Failed to analyze 46, XY
Normal – – Normal Normal – Normal
* Hours after maturation culture.
three blastocysts were derived from vitrified oocytes from unstimulated cycles and four for stimulated cycles. All of these blastocysts had normal chromosome number (23 pairs) and the sex ratio was 3:4 male:female. These results confirmed the possibility that the vitrification method, using an EM grid and short equilibration protocol, would become one of the available methods for the cryopreservation of human oocytes retrieved from both unstimulated and stimulated cycles. Vitrified oocytes could develop to the blastocyst stage and the blastocysts had normal chromosomal numbers. Furthermore, the results obtained from this study indicated that the vitrification method could be applied for the freezing of immature and maturing oocytes, as well as for the freezing of mature oocytes.
A total of 301 oocytes in 14 cycles were retrieved from the patients and means (9 S.D) of age and infertility duration were 32.19 2.7 and 5.39 2.7 years, respectively. After vitrification and thawing, 83% (249/301) of the retrieved oocytes were morphologically normal and 68% (170/249) of the normal vitrified oocytes were matured. A total of 68% (115/170) of oocytes fertilized in vitro and 90% (103/115) of the fertilized embryos cleaved. ET was performed for these patients but, up to the present, none was pregnant. These results show that GV oocytes retrieved from PCOS patients can mature, fertilize and cleave following vitrification, IVM and IVF. Further clinical trials using this system are being conducted to optimize this alternative IVF-ET program employing vitrification program.
4. Clinical application References We have clinically applied our developed vitrification method for the cryopreservation of oocytes at the GV stage. We applied the vitrification technique for the treatment of patients with polycystic ovarian syndrome (PCOS). PCOS patients often yield large number of immature oocytes, which are developmentally incompetent. So, cryopreservation of the oocytes is necessary to store surplus oocytes from the first cycle and the stored oocytes could be provided for the next IVF-ET cycle after thawing. These two IVF-ET cycles were performed in a single cycle of the retrieval of oocytes. The cryopreservation-IVM program mainly consists of: (1) retrieval of oocytes; (2) vitrification and thawing; (3) IVM; (4) IVF; (5) IVC; and (6) assisted hatching (AH) and ET with endometrial preparation. To avoid an unpredictable response to induction drugs, unstimulation program was used for this system.
Al-Hasani, S., Diedrich, K., van der Ven, H., Krebs, D., 1986. Initial results of the cryopreservation of human oocytes. Gebu¨rtshilfe Frauenheilkd 46, 643– 644. Al-Hasani, S., Diedrich, K., van der Ven, H., Reinecke, A., Hartje, M., Krebs, D., 1987. Cryopreservation of human oocytes. Hum. Reprod. 2, 695– 700. Ashwood-Smith, M.J., Morris, G.W., Flower, R., Appleton, T.C., Ashhorn, R., 1988. Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures. Hum. Reprod. 3, 795– 802. Bouquet, M., Selva, J., Aureoux, M., 1993. Cryopreservation of mouse oocytes: mutagenic effects in the embryos? Biol. Reprod. 49, 764– 769. Carroll, J., Depypere, H., Matthews, C.D., 1990. Freeze–thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen– thawed mouse oocytes. J. Reprod. Fertil. 90, 547– 553. Chen, C., 1986. Pregnancy after human oocyte cryopreservation. Lancet 1, 884– 886.
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47 Chung, H.M., Hong, S.W., Lim, J.M., Lee, S.H., Cha, W.T., Ko, J.J., Han, S.Y., Choi, D.H., Cha, K.Y., 2000. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil. Steril. 73, 545–551. De Fried, E.P., Nortrica, J., Rubinstein, M., Marazzi, A., Gonzalez, M.G., 1998. Pregnancy after human donor oocyte cryopreservation and thawing in association with intracytoplasmic sperm injection in a patient with ovarian failure. Fertil. Steril. 69, 555– 557. DeMayo, F.J., Rawlins, R.G., Dokelow, W.R., 1985. Xenogenous and in vitro fertilization of frozen thawed primate oocytes and blastomere separation of embryos. Fertil. Steril. 43, 295–300. Diedrich, K., Al-Hasani, S., Van der Ven, D., 1988. Successful in vitro fertilization of frozen–thawed rabbit and human oocytes. Ann. N.Y. Acad. Sci. 541, 562–570. Hochi, S., Fujimoto, T., Oguri, N., 1995. Viability of immature horse oocytes cryopreserved by vitrification. Theriogenology 43, 236. Johnson, M.H., Pickering, 1987. The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte. Development 100, 313– 324. Kasai, M., Iritani, A., Chang, M.C., 1979. Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol. Reprod. 21, 839– 844. Lim, J.M., Fukui, Y., Ono, H., 1991. Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization. Theriogenology 37, 351– 361. Moller, C., Wassarman, P.M., 1989. Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. Dev. Biol. 132, 103–112. Nawroth, F., Kissing, K., 1999. Pregnancy after intracytoplasmic sperm injection (ICSI) of cryopreserved human oocytes. Acta Obstet. Gynecol. Scand. 77, 462–472. Park, S.E., Lee, K.A., Son, W.Y., Ko, J.J., Lee, S.H., Cha, K.Y., 1997. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil. Steril. 68, 920–926. Porcu, E., Fabbri, R., Seracchioli, R., Ciotti, P.M., Magrini, O., Flamigni, C., 1997. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil. Steril. 68, 724– 726. Porcu, E., 1999. Freezing of oocytes. Curr. Opin. Obstet. Gynecol. 11, 297– 300. Porcu, E., Fabbri, R., Ciotti, P.M., Petracchi, S., Seracchioli, R., Flamigni, C., 1999a. Ongoing pregnancy after intracytoplasmic sperm injection of epididymal spermatozoa into cryopreserved human oocytes. J. Assist. Reprod. Genet. 16, 283–285.
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Porcu, E., Fabbri, R., Petracchi, S., Ciotti, P.M., Flamigni, C., 1999b. Ongoing pregnancy after intracytoplasmic injection of testicular spermatozoa into cryopreserved human oocytes. Am. J. Obstet. Gynecol. 180, 1044– 1045. Rubinsky, B., Arav, A., Devries, A.L., 1992. The cryoprotective effect of antifreeze glycopeptides from Antarctic fishes. Cryobiology 29, 69 – 79. Sathanathan, A.H., Ng, S.C., Trounson, A.C., Bongso, A., Ratnam, S.S., Ho, J., et al., 1988. The effect of ultrarapid freezing on meiotic spindles of mouse oocytes and embryos. Gamate. Res. 21, 385– 401. Siebzehnruebl, E.R., Todorow, S., van Uem, J., Koch, R., Wildt, L., Lang, N., 1989. Cryopreservation of human and rabbit oocytes and one-cell embryos: A comparison of DMSO and propanediol. Hum. Reprod. 4, 312– 317. Son, W.Y., Park, S.E., Lee, K.A., Lee, W.S., Ko, J.J., Yoon, T.K., 1996. Effects of 1,2-propanediol and freezing– thawing on the in vitro developmental capacity of human immature oocytes. Fertil. Steril. 66, 995– 999. Todorow, S.J., Siebzehnruebl, E.R., Koch, R., Wildt, L., Lang, N., 1989. Comparative results on survival of human and animal eggs using different cryoprotectants and freeze– thawing regimens. I. Mouse and hamster. Hum. Reprod. 4, 805– 811. Trounson, A., 1986. Preservation of human eggs and embryos. Fertil. Steril. 46, 1 – 12. Tucker, M., Wright, G., Morton, P., Shanguo, L., Massey, J., Kort, H., 1996. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum. Reprod. 11, 1513– 1515. Tucker, M.J., Wright, G., Mortin, P.C., Massey, J.B., 1998. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil. Steril. 70, 578– 579. Van Uem, J.F.H.M., Siebzehnruebl, E.R., Schuh, B., Koch, R., Trotnov, S., Lang, N., 1987. Birth after cryopreservation of unfertilized oocytes. Lancet 1, 752– 753. Vincent, C., Garnier, V., Heyman, Y., Renard, J.P., 1989. Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes. J. Reprod. Fertil. 87, 809– 820. Yoon, T.K., Chung, H.M., Lim, J.M., Han, S.Y., Ko, J.J., Cha, K.Y., 2000. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil. Steril. 74, 180– 181. Young, E., Kenny, A., Puigdomenech, E., Van Thillo, G., Tiveron, M., Piazza, A., 1998. Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocyte: case report. Fertil. Steril. 70, 360– 361.
Freezing immature oocytes K.Y. Cha a,b,*, H.M. Chung a, J.M. Lim c, J.J. Ko a, S.Y. Han a, D.H. Choi a, T.K. Yoon a a
College of Medicine, Pochon CHA Uni6ersity and Infertility Medical Center of CHA General Hospital, 606 -5 Yeoksam 1 -Dong, Seoul 135 -081, South Korea b CHA-Columbia Infertility Medical Center, Manhattan, New York, NY 10032, USA c School of Agricultural Biotechnology, Seoul National Uni6ersity, Suwon 441 -744, South Korea
Abstract The establishment of a long-term preservation system for mammalian oocytes is important for the development of both biological and medical sciences. A number of efforts have been made to develop this system. In human reproductive medicine, the development of an oocyte cryopreservation system can improve the efficacy of the current assisted reproductive technology (ART) for infertile patients with severe reproductive disorders. In this article, the technical development of cryopreservation programs for human oocytes and its biological background were reviewed. Clinical outcome after the use of this technology was further introduced. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human; Oocyte; Cryopreservation; Oocyte bank
1. History of oocyte cryopreservation As in vitro manipulation technologies, such as in vitro-maturation (IVM), in vitro-fertilization (IVF) and in vitro-culture (IVC) of mammalian gametes and zygotes, was developed in the 1980s, the significance of an oocyte cryopreservation technique was recognized. It provides the possibilities that valuable genetic resources can be preserved for a designated period without the loss of viability and that the efficacy of reproductive biotechnology is efficiently renovated. A number of efforts have been made to preserve mammalian oocytes since the first successful attempts in the bovine (Lim et al., 1991), hamster (Todorow et al., 1989), human (Trounson, 1986), mouse (Sathanathan et al., 1988), mare (Hochi et al., 1995), monkey (DeMayo et al., 1985), pig (Rubinsky et al., 1992), rabbit (Vincent et al., 1989) and rat (Kasai et al., 1979) species. In particular, scientists in the field of human reproductive medicine had a great interest in developing this technique, since an oocyte bank system could be established * Corresponding author. Present address: College of Medicine, Pochon CHA University and Infertility Medical Center of CHA General Hospital, 606-5 Yeoksam 1-Dong, Seoul 135-081, South Korea. Tel.: +82-2-34683000; fax: +82-2-5018704.
based on it. The oocyte bank system directly contributes to establishing an oocyte donation system, which is a feasible system to treat a number of congenital infertility disorders, such as hypoplastic ovaries and premature ovarian failure. This system also provides the chances of pregnancy in patients who receive anticancer treatments. The efficacy of assisted reproductive technology (ART) program can be improved by the use of this technique. Surplus oocytes from the fresh IVFET cycle of patients can be stored and, when the patients fail their fresh cycle, the cryopreserved oocytes can be provided for the next cycle after thawing. Furthermore, the innovative family planning to support the social activity of modern women can be designed using the oocyte bank system. Considering that several European countries, such as Austria, Germany, Switzerland, Denmark and Sweden, either bans or strictly limits the research and clinical application of embryo cryopreservation, the oocyte bank system can lessen ethical and legal issues caused by human embryo freezing. Attempts to develop an oocyte cryopreservation method in the human have extensively been made after the first success of Chen (1986), who reported on the first live births from frozen human mature oocytes. As shown in Table 1, a number of pregnancies were subse-
0303-7207/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0303-7207(00)00350-6
44
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
quently established after undergoing freezing-ET programs (Al-Hasani et al., 1986, 1987; Van Uem et al., 1987; Diedrich et al., 1988; Siebzehnruebl et al., 1989; Tucker et al., 1996, 1998; Porcu et al., 1997; De Fried et al., 1998; Young et al., 1998; Nawroth and Kissing, 1999; Porcu et al., 1999a,b; Yoon et al., 2000), but very few pregnancies were established, except for the report of Porcu (1999).
2. Limitation of oocyte cryopreservation There has been solid limitation to improving the oocyte cryopreservation system. Trounson (1986) raised a number of problems which occurred during cryopreservation. Generally, the ultrastructure of oocytes is particularly sensitive to the changes of temperature and extracellular osmotic pressure during freezing and thawing. Various cellular damage, such as cytoskeleton disorganization (Johnson and Pickering, 1987), chromosome and DNA abnormality (Bouquet et al., 1993), spindle disintegration (Park et al., 1997), premature cortical granule exocytosis (Carroll et al., 1990) and its related hardening of zona pellucida (Moller and Wassarman, 1989) and plasma membrane disintegrity (Ashwood-Smith et al., 1988), are frequently found when oocytes are placed at a low temperature, below 0°C. All of these impairments negatively affect the developmental competence of frozen oocytes. Al-Hasani et al. (1987) reported that only 11% of cryopreserved oocytes from 48 patients were fertilized in vitro. Lower maturation, fertilization and cleavage rates were also found in slowly frozen oocytes than in fresh oocytes (Son et al.,
1996). Park et al. (1997) reported that the high incidence of chromosome and spindle abnormalities was detected in frozen oocytes. On the other hand, the maturation stage of oocytes at the time of freezing greatly affects the cryopreservation capacity and survival after thawing. In our preliminary study using an animal model (Lim et al., 1991), one-cell embryos has higher post-thawed survival and developmental capacities than oocytes frozen slowly at the metaphase-II (MII) stage. Furthermore, more oocytes survived, fertilized and developed after slowly freezing at the MII stage than after slowly freezing at the germinal vesicle (GV) stage (Lim et al., 1991). To enhance the viability of oocytes after thawing, we should consider the type of cryopreservation programs and the stage of oocyte maturation at the time of freezing.
3. Development of vitrification method As shown in Table 1, only a limited number of patients successfully became pregnant and delivered babies after IVF-ET program, utilizing embryos developed from frozen –thawed oocytes. To overcome such limitations, we have attempted to develop a vitrification method for the cryopreservation of human oocytes, instead of slow freezing methods. High concentration of cryoprotectant and an extremely rapid cooling speed (\ 2000°C) are employed for the vitrification protocol and, as a result, the formation of intracellular ice crystals in oocyte cytoplasm can be effectively prevented. Furthermore, the equipment used in conven-
Table 1 Pregnancies of patients received embryos developed from cryopreserved oocytesa Authors/years
Type of CPAs
Freezing methods
Stage of freezing
Pregnancies/deliveries
Remarks
(Chen, 1986) (Al-Hasani et al., 1986, 1987) (Diedrich et al., 1988) (Van Uem et al., 1987) (Siebzehnruebl et al., 1989) (Tucker et al., 1996) (Porcu et al., 1997) (Tucker et al., 1998) (De Fried et al., 1998) (Young et al., 1998) (Nawroth and Kissing, 1999) (Tucker et al., 1998) (Porcu, 1999) (Porcu et al., 1999a) (Porcu et al., 1999b) (Yoon et al., 2000)
DM PR/DM DM DM PR/DM PR PR PR PR PR PR PR PR PR PR EG+S
Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Vitri
MII MII MII MII MII MII MII MII MII MII MII GV MII MII MII MII
2/2 2/? 1/? 1/1 1/? 3/0 1/1 5/2 1/1 1/? 1/0 1/1 16/11 1/ongoing 1/ongoing 3/1+ongoing
– – – – – ICSI ICSI ICSI ICSI ICSI ICSI ICSI ICSI ICSI* ICSI** ICSI
a CPAs, cryoprotective agents; DM, dimethylsulfoxide; PR, 1,2-propanedior; EG, ethylene glycol; S, sucrose; Slow, slow freezing program; Vitri, vitrification program; GV, germinal vesicle; MII, metaphase-II. * Epididymal sperm aspiration. ** Testicular sperm aspiration.
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
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Table 2 Morphological normality, maturation and fertilization of human oocytes vitrified at different times of maturation culture Oocytes from (cycle)
Times of vitrification (h)
No. oocytes vitrified
Parameters (%) Normality
Maturation 12 (40) –
7 (37) 5 (31)
9 (53) 9 (64) –
6 (55) 5 (56) 10 (83)
Unstimulated
0 48
30 16
19 (63) 9 (56)
Stimulated
0 8–15 24–28
17 14 12
11 (65)a 9 (64)a 12 (100)b
a b
Fertilization
PB0.05 within the same column of each cycle. PB0.05 within the same column of each cycle.
tional slow freezing methods are no longer necessary for the vitrification and the required time for the cryopreservation of oocytes can be greatly decreased. In spite of these advantages, the vitrification causes another problem by the use of a high concentration of cryoprotectants. Severe osmotic effect and the cytotoxicity of cryoprotectant may adversely affect oocyte development after cryopreservation. The modification of the vitrification method is necessary to overcome such problems and we developed a new vitrification method for human oocytes. We used ethylene glycol (EG), which has higher membrane permeability and lower cytotoxicity than other cryoprotectants, as a permeable cryoprotectant for this method. Also, appropriate concentrations of sucrose were used as a nonpermeable cryoprotectant. The use of sucrose as a cryoprotectant contributes to regulating intracellular concentration of EG during equilibration and vitrification and to effectively remove EG from oocyte cytoplasm during thawing and dilution procedures. Additional modifications were made for optimizing our vitrification method, reducing the time of the equilibration procedure before vitrification and use of a copper electron microscopic (EM) grid as an oocyte vehicle (Chung et al., 2000). The short equilibration procedure is beneficial for alleviating severe damage resulting from a high concentration of the vitrification solution and use of the EM grid improves thermal conductivity to the oocyte cytoplasm, which supports membrane integrity during equilibration. As the first step of clinical application, we evaluated whether our modified vitrification method was available for cryopreserving human oocytes. In a series of experiments, we examined whether this method was effective for immature oocytes, as well as mature oocytes and evaluated the survival and fetal development of vitrified oocytes following IVF-ET program. For this study, oocytes retrieved from unstimulated and stimulated cycles were vitrified at various times after maturation culture (Chung et al., 2000) and, after vitrification and thawing, thawed oocytes were provided for IVF and in vitro culture. The total duration of maturation culture
was 48 h in unstimulated cycles and 24–28 h in stimulated cycles. In unstimulated cycles, oocytes were vitrified either at 0 or 48 h after IVM. In stimulated cycle, oocytes were vitrified at 0, 8–15 and 24–28 h. Therefore, oocytes vitrified immediately (0 h) were cultured further for 48 h after thawing in unstimulated cycle. On the other hand, oocytes vitrified immediately (0 h) and at 8–15 h after IVM were cultured for a further 24–28 and 13–16 h in maturation medium, respectively, in stimulated cycle. Oocytes vitrified at the end of maturation in both cycles were directly provided for IVF. In oocytes retrieved from an unstimulated cycle, no significant effect was found in the number of morphologically normal oocytes after vitrification (56 –63%; Table 2). Twelve oocytes (40%) vitrified immediately extruded first polar body after 48 h of maturation culture. In this group, 37% of morphologically normal oocytes developed to the normal pronuclear stage at 16–19 h after ICSI, while 31% of oocytes vitrified at the end of maturation culture developed to the pronuclear stage. There was no significant difference in the number of pronuclear stage embryos between the times of vitrification. In oocytes retrieved from stimulated cycle, more oocytes were morphologically normal following vitrification at 24–28 h (100%) of maturation culture than following vitrification immediately or at 8–15 h of the culture (64 –65%). The maturation rate of oocytes vitrified immediately or at 8–15 h after maturation culture was within the range of 53–64% and no significant effect was detected. Also, there was no significant effect of vitrification on the number of vitrified oocytes that developed to the normal pronuclear stage (55 – 83%). Thirty-two out of 33 pronuclear stage embryos (97%) developed from vitrified oocytes cleaved at 48 h postIVF, which was independent of the time of vitrification and hormonal induction. Blastocyst formation occurred with all groups of vitrified oocytes in the range of 20–43%. A total of 12 blastocysts derived from oocytes vitrified at different times of maturation culture were analyzed for their chromosome number. As shown in Table 3, seven blastocysts were successfully analyzed;
K.Y. Cha et al. / Molecular and Cellular Endocrinology 169 (2000) 43–47
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Table 3 Detailed data of karyotyping of the blastocysts (BL) derived from vitrified oocytes Oocytes retrieved from (cycle)
Patients
Times* of vitrification
Stages of examination
Results of karyotyping
No. of chromosomes
Chromosome normality
Unstimulated
A B B D E
0 0 0 48 48
Expanded BL Early BL Expanded BL Expanded BL Hatched BL
46, XX Failed to analyze 46, XY Failed to analyze 46, XY
Normal – Normal – Normal
Stimulated
F G H I J K L
0 0 8–15 24–28 24–28 24–28 24–28
Early BL Expanded BL Early BL Expanded BL Early BL Hatched BL Expanded BL
46, XX Failed to analyze Failed to analyze 46, XX 46, XX Failed to analyze 46, XY
Normal – – Normal Normal – Normal
* Hours after maturation culture.
three blastocysts were derived from vitrified oocytes from unstimulated cycles and four for stimulated cycles. All of these blastocysts had normal chromosome number (23 pairs) and the sex ratio was 3:4 male:female. These results confirmed the possibility that the vitrification method, using an EM grid and short equilibration protocol, would become one of the available methods for the cryopreservation of human oocytes retrieved from both unstimulated and stimulated cycles. Vitrified oocytes could develop to the blastocyst stage and the blastocysts had normal chromosomal numbers. Furthermore, the results obtained from this study indicated that the vitrification method could be applied for the freezing of immature and maturing oocytes, as well as for the freezing of mature oocytes.
A total of 301 oocytes in 14 cycles were retrieved from the patients and means (9 S.D) of age and infertility duration were 32.19 2.7 and 5.39 2.7 years, respectively. After vitrification and thawing, 83% (249/301) of the retrieved oocytes were morphologically normal and 68% (170/249) of the normal vitrified oocytes were matured. A total of 68% (115/170) of oocytes fertilized in vitro and 90% (103/115) of the fertilized embryos cleaved. ET was performed for these patients but, up to the present, none was pregnant. These results show that GV oocytes retrieved from PCOS patients can mature, fertilize and cleave following vitrification, IVM and IVF. Further clinical trials using this system are being conducted to optimize this alternative IVF-ET program employing vitrification program.
4. Clinical application References We have clinically applied our developed vitrification method for the cryopreservation of oocytes at the GV stage. We applied the vitrification technique for the treatment of patients with polycystic ovarian syndrome (PCOS). PCOS patients often yield large number of immature oocytes, which are developmentally incompetent. So, cryopreservation of the oocytes is necessary to store surplus oocytes from the first cycle and the stored oocytes could be provided for the next IVF-ET cycle after thawing. These two IVF-ET cycles were performed in a single cycle of the retrieval of oocytes. The cryopreservation-IVM program mainly consists of: (1) retrieval of oocytes; (2) vitrification and thawing; (3) IVM; (4) IVF; (5) IVC; and (6) assisted hatching (AH) and ET with endometrial preparation. To avoid an unpredictable response to induction drugs, unstimulation program was used for this system.
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