1 IVF Center, Hanna Women's Clinic and 2 Department of Animal Sciences, Kon-Kuk University, Seoul, Korea
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Abstract |
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Key words: ethylene glycol/permeation/pregnancy outcome/survival rate/toxicity
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Introduction |
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With slow freezing, due to the use of a low concentration of cryoprotectant, the effects of osmotic shock and toxicity of the cryoprotectant on cell damage are less severe than that of intracellular ice formation (Bryant, 1995). A vitrification method has been developed to circumvent the obstacle of slow freezing (Rall and Fahy, 1985
). Although the use of high concentrations of cryoprotectant in vitrification dramatically reduces the risk of intracellular ice formation, the toxicity of the cryoprotectant is increased as a result of long-term exposure to high concentrations of cryoprotectant (Fahy et al., 1984
; Valdez et al., 1992
). Recently, rapid vitrification techniques have been developed in which only a brief exposure to cryoprotective agents is required (Kasai et al., 1990
; Shaw et al., 1992
), though by limiting the exposure to cryoprotectants to only one step, the degree of osmotic shock is increased. Thus, in the vitrification method, osmotically induced damage is the major reason for loss of viability (Shaw et al., 1991
). Although debate persists with regard to superiority between the two protocols, recent studies have demonstrated better outcomes of embryos that were frozen using a slow-freezing protocol (Sommerfeld and Niemann, 1999
; Uechi et al., 1999
).
Ethylene glycol (EG) has been used widely with both the slow-freezing and vitrification methods for the cryopreservation of mammalian embryos such as mouse (Ali and Shelton, 1993; Zhu et al., 1993
; Shaw et al., 1995
; Emiliani et al., 2000
), rat (Jiang et al., 1999
), rabbit (Kasai et al., 1992
), sheep (Cocero et al., 1996
) and cow (Donnay et al., 1998
; Sommerfeld and Niemann, 1999
) due to its low molecular weight, high permeation ability (Oda et al., 1992
; Gilmore et al., 1995
; Zhu et al., 1996
; Newton et al., 1998
) and low toxicity (Sommerfeld and Niemann, 1999
; Emiliani et al., 2000
). Recently, EG was used for the vitrification of human oocytes (Kuleshova et al., 1999
; Chung et al., 2000
) and embryos (Vanderzwalmen et al., 1997
; Mukaida et al., 1998
), as well as for cryopreservation of human sperm (Gilmore et al., 1997
). However, to date no data are available for the use of EG in cryopreserving human embryos in a slow-freezing protocol.
In preliminary experiments, mouse zygotes exposed to 1.5 mol/l EG exhibited a severe and rapid change in size compared with embryos exposed to 1.5 mol/l propylene glycol (PROH). Two-cell stage mouse embryos exposed to EG exhibited a higher percentage of embryos which developed to blastocyst (64.4%) than did embryos exposed to PROH (53.8%). These results indicated that EG has high permeation ability and low toxicity compared with PROH.
In the light of these preliminary studies, the possibility of using EG rather than PROH for the freezing of human embryos in a slow-freezing protocol was investigated, in order to minimize any resulting cell damage and improve clinical results. Hence, the present study was carried out to evaluate the feasibility of using EG in a slow-freezing protocol, with mouse embryos and human embryos, and to investigate the efficiency of EG in the cryopreservation of human embryos.
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Materials and methods |
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Patients
In this prospective study, which was conducted between January 1999 and May 2000 (17 months), the spare embryos after embryo transfer of 62 patients were frozen using EG. Retrospective data from the frozen embryos of 60 patients (frozen using PROH between August 1997 and December 1998) were used as a control group. There was no difference in mean (±SD) patient age between the EG and PROH groups (32.7 ± 3.6 and 32.4 ± 3.8 years respectively). Patients with polycystic ovary syndrome, poor response to gonadotrophins, uterine factors or age >40 years were excluded from the study.
The women were down-regulated with GnRH agonist, buserelin acetate (Suprefact; Hoechst AG, Germany) and stimulated with FSH (Metrodin-HP; Serono, Geneva, Switzerland) and HMG (Humegon; Organon Phamaceuticals, The Netherlands). Oocytes were retrieved at 3536 h after administration of HCG (Profasi; Serono, Geneva, Switzerland) using a vaginal ultrasound-guided procedure.
Preparation of human embryos
The aspirated oocytes were inseminated using either conventional IVF or by ICSI in modified P1 medium (glucose/phosphate-free medium) supplemented with 10% human follicular fluid (HFF). Embryos with normal fertilization were cultured in modified TCM-199 medium (Gibco, Grand Island, NY, USA) supplemented with 20% HFF (Chi et al., 1998). On day 2 or 3 after oocyte retrieval, the embryos were transferred and the spare embryos after embryo transfer were cryopreserved using either EG (Sigma) or PROH (Sigma) in a controlled slow-freezing protocol. Clinical pregnancy was determined ultrasonographically by confirming the presence of an intrauterine gestation sac.
Freezing and thawing solutions
Freezing and thawing solutions were prepared in Dulbecco's phosphate-buffered saline (PBS; Gibco-BRL, Grand Island, NY, USA) supplemented with 20% HFF, 0.2 mol/l sucrose (Sigma) and 1.5 mol/l EG or PROH. For the freezing procedure, four equilibration solutions were used with each cryoprotectant: 0.5 mol/l EG or PROH, 1.0 mol/l EG or PROH, 1.5 mol/l EG or PROH, and 1.5 mol/l EG + 0.2 mol/l sucrose or 1.5 mol/l PROH + 0.2 mol/l sucrose. Four equilibration solutions were also used for the thawing procedure: 1 mol/l EG + 0.2 mol/l sucrose or 1 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l EG + 0.2 mol/l sucrose or 0.5 mol/l PROH + 0.2 mol/l sucrose, 0.2 mol/l sucrose alone, and PBS.
Slow freezing and thawing
Cryopreservation of human embryos was performed using a programmable controlled rate-freezing machine (Planer Series III Kryo 10, T.S. Scientific, Perkasie, PA, USA). The embryos were cryopreserved using PBS supplemented with 20% HFF, 0.2 mol/l sucrose and 1.5 mol/l EG or PROH. The embryos were exposed in a stepwise fashion to increasing concentrations of EG or PROH (0.5, 1.0, 1.5 mol/l EG or PROH, and 1.5 mol/l EG + 0.2 mol/l sucrose or 1.5 mol/l PROH + 0.2 mol/l sucrose, 5 min per step) in the freezing solution. The embryos were then loaded into a 0.25 ml sterile straw (Bicef, L'Aigle, France); this was loaded into the cryo-machine (which was precooled to 20°C) and kept at 20°C for 1 min for equilibration. The straw was subsequently cooled from 20°C to 7°C at a rate of 2°C/min, held at this temperature for 5 min, and then seeded manually. It was then cooled to 39°C at a rate of 0.3°C/min and plunged into liquid nitrogen. For thawing of the frozen human embryos, the straws were rewarmed by holding them in air for 15 s before plunging them into a water bath at 37°C. The cryoprotectant was then removed by reverse stepwise dilutions with thawing solutions (5 min each step). Prior to the use of EG for cryopreservation of human embryos in clinical settings, both EG and PROH were used to freeze sibling embryos obtained from the same patients over a few cycles to confirm the feasibility of EG as a cryoprotectant. The effect of equilibration temperature (room temperature and 37°C) for freezing and thawing procedures on post-thaw survival of human embryos frozen using EG was also investigated. Although freezing and thawing of human embryos using PROH was performed at room temperature, freezing and thawing using EG was carried out at 37°C in an IVF chamber.
Statistical analysis
Post-thaw survival rates and subsequent development of mouse embryos, and implantation and pregnancy rates of human embryos in the EG and PROH groups were compared by 2 analysis. Patients' age and number of embryos transferred per cycle (presented as mean ± SD) were compared using Student's t-test. A P-value < 0.05 was considered statistically significant.
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Results |
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The effect of equilibration temperature on post-thaw survival of human embryos frozen using EG in a few cycles was investigated (Table II). Fourteen patients were allocated to two groups according to the equilibration temperature for the freezing and thawing procedure. There was no difference between the overall morphological qualities of the fresh embryos in the two groups. Post-thaw survival rates of the embryos equilibrated at room temperature and at 37°C were 81.8 and 80.5% respectively.
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Discussion |
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Although the 2-cell stage mouse embryos showed a higher survival rate than the 4- or 8-cell stage embryos, the subsequent development of 2-cell stage embryos was significantly lower than that of the 4- and 8-cell stage embryos (Table I). This suggests that the developmental potency of 2-cell stage mouse embryos is more sensitive to damage attributable to freezing and thawing procedures than either 4- or 8-cell stage embryos. In mouse embryos, the 2-cell stage is known to be a genomic activation stage. Embryonic genomic activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA (Wang and Latham, 1997
, 2000
). In addition, a deleterious effect of cryopreservation on DNA synthesis in the inner cell mass of bovine embryos has been observed (Takagi et al., 1996
). Thus, the sudden shock of the freezing and thawing procedure at this important stage may critically damage the ability of the embryos to develop subsequently at the gene level.
In the present study, the high permeation, low toxicity and high efficiency of EG on cryopreservation of mouse embryos was confirmed. This result suggests that EG might be used successfully for the slow freezing of human embryos. This possibility is supported by results of other studies in which EG was used successfully for the vitrification of human oocytes (Kuleshova et al., 1999; Chung et al., 2000
) and embryos (Vanderzwalmen et al., 1997
; Mukaida et al., 1998
).
It has been reported that transient cooling to room temperature could cause irreversible disruption of the meiotic spindle in human oocytes (Pickering et al., 1990). This suggests that the cooling to room temperature may also cause damage to the mitotic spindle of human embryos. However, no detrimental effect was observed due to cooling to room temperature on the post-thaw survival of human embryos in a few cycles (Table II
). Although freezing and thawing of human embryos in the EG group was performed at 37°C in order to minimize the potential damage incurred by cooling to room temperature, the potential damage on mitotic spindle of the embryos requires further investigation.
Implantation and pregnancy rates of embryos in the EG group were significantly higher than those of the PROH group (Table III). This finding indicates that, due to its low toxicity and high permeation, EG may be used to cryopreserve human embryos and thereby significantly improve post-thaw survival and subsequent embryo development, in turn improving pregnancy outcome. In addition, the proportion of day-3 embryos was higher in the EG group (45.3%) than in the PROH group (10.4%), which may also contribute to the improvement of the pregnancy outcome in the former group. This possibility is supported by previous data which showed the pregnancy rate of a day-3 embryo transfer group to be significantly higher than that of a day-2 embryo transfer group (Koo et al., 1999
).
The post-thaw survival rate (80.6%) of human embryos that were frozen using EG in the present study was not different from that of human embryos that were vitrified using EG (80 or 78.8%) in previous studies (Vanderzwalmen et al., 1997; Mukaida et al., 1998
). However, the pregnancy rate (46.9%) of the EG group in the present study was significantly higher than that of either vitrified EG group (16.6 or 5.5%). This suggests that the vitrification protocol is more damaging to the subsequent development of human embryos than the slow freezing protocol, though there was no difference in the morphological survival rate of embryos between the two protocols. The difference may be a result of osmotic shock effected by the high concentration of EG used in the vitrification protocol.
In conclusion, it is suggested that EG is a good substitute for PROH for the cryopreservation of human embryos, and provides better results. Although more studies are required to compare the advantages between the slow-freezing and vitrification protocols using EG, it is suggested that the slow-freezing protocol is physiologically less damaging to human embryos than the vitrification protocol. The efficiency and optimal concentration of EG for the cryopreservation of mouse and human oocytes and blastocysts in slow-freezing protocols, and the effects of cooling on the mitotic spindle of embryos, are currently under investigation.
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Acknowledgements |
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Notes |
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References |
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Bryant, G. (1995) DSC measurement of cell suspensions during successive freezing runs: implications for the mechanisms of intracellular ice formation. Cryobiology, 32, 114128.[ISI][Medline]
Chi, H.J., Kim, D.H., Koo, J.J. and Chang, S.S. (1998) The suitability and efficiency of human follicular fluid as a protein supplement in human in vitro fertilization programs. Fertil. Steril., 70, 871877.[ISI][Medline]
Chung, H.M., Hong, S.W., Lim, J.M., Lee, S.H., Cha, W.T., Ko, J.J., Han, S.Y., Choi, D.H. and 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, 545551.[ISI][Medline]
Cocero, M.J., Sebastian, A.L., Barragan, M.L. and Picazo, R.A. (1996) Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology, 33, 502507.[ISI][Medline]
Donnay, I., Auquer, P., Kaidi, S., Carolan, C., Lonergan, P., Mermillod, P. and Massip, A. (1998) Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity. Anim. Reprod. Sci., 52, 93104.[ISI][Medline]
Emiliani, S., Bergh, M.V.D., Vannin, A.S., Biramane, J. and Englert, Y. (2000) Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum. Reprod., 15, 905910.
Fahy, G.M., MacFarlane, D.R., Angell, C.A. and Meryman, H.T. (1984) Vitrification as an approach to cryopreservation. Cryobiology, 21, 407426.[ISI][Medline]
Gilmore, J.A., McGann, L.E., Liu, J., Gao, D.Y., Peter, A.T., Kleinhans, F.W. and Crister, J.K. (1995) Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol. Reprod., 53, 985995.[Abstract]
Gilmore, J.A., Liu, J., Gao, D.Y. and Crister, J.K. (1997) Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum. Reprod., 12, 112118.[ISI][Medline]
Jiang, J.Y., Umezu, M. and Sato, E. (1999) Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions. Cryobiology, 38, 160164.[ISI][Medline]
Kasai, M.., Komi, J.H., Takakamo, A., Tsudera, H., Sakurai, T. and Machida, T. (1990) A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil., 89, 9197.[Abstract]
Kasai, M., Hamaguchi, Y., Zhu, S.E., Miyake, T., Sakurai, T. and Machida, T. (1992) High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method. Biol. Reprod., 46, 10421046.[Abstract]
Koo, J.J., Chi, H.J., Kim, M.Y., Joo, J.Y., Kim, J.Y., Sung, H.R. and Chang, S.S. (1999) Comparison between embryos transferred on day 2 or day 3 in human IVF or IVF/ICSI programs: a prospective, randomized study. Fertil. Steril. (Abstract Book, O-086, ASRM 99), 33.
Kuleshova, L., Gianaroli, L., Magli, C., Ferraretti, A. and Trounson, A. (1999) Birth following vitrification of a small number of human oocytes: case report. Hum. Reprod., 14, 30773079.
Mukaida, T., Wada, S., Takahashi, K., Pedro, P.B., An, T.Z. and Kasai, M. (1998) Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum. Reprod., 13, 28742879.
Newton, H., Fisher, J., Arnold, J.R.P., Pegg, D.E., Faddy, M.J. and Gosden, R.G. (1998) Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum. Reprod., 13, 376380.[ISI][Medline]
Oda, K., Gibbons, W.E. and Leibo, S.P. (1992) Osmotic shock of fertilized mouse ova. J. Reprod. Fertil., 95, 737747.[Abstract]
Pickering, S.J., Braude, P.R., Cant, A., Currie, J. and Johnson, M.H. (1990) Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril., 54, 102108.[ISI][Medline]
Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at 196 degrees C by vitrification. Nature, 313, 573575.[ISI][Medline]
Shaw, J.M., Ward, C. and Trounson, A.O. (1995) Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow cooled mouse pronuclear and 4-cell embryos. Hum. Reprod., 10, 396402.[Abstract]
Shaw, P.W., Fuller, B.J., Bernard, A. and Shaw, R.W. (1991) Vitrification of mouse oocytes: improved rates of survival, fertilization, and development to blastocysts. Mol. Reprod. Dev., 29, 373378.[ISI][Medline]
Shaw, P.W., Fuller, B.J., Bernard, A.G. and Shaw, R.W. (1992) Vitrification of mouse oocytes using short cryoprotectant exposure: effect of varying exposure times on survival. Mol. Reprod. Dev., 33, 210214.[ISI][Medline]
Sommerfeld, V. and Niemann, H. (1999) Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology, 38, 95105.[ISI][Medline]
Stachecki, J.J., Cohen, J. and Willadsen, S.M. (1998) Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology, 37, 346354.[ISI][Medline]
Takagi, M., Sakonju, I. and Suzuki, T. (1996) Effects of cryopreservation of DNA synthesis in the inner cell mass of in vitro matured/in vitro fertilized bovine embryos frozen in various cryoprotectants. J. Vet. Med. Sci., 58, 12371238.[ISI][Medline]
Uechi, H., Tsutsumi, O., Morita, Y., Takai, Y. and Taketani, Y. (1999) Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development. Hum. Reprod., 14, 28272832.
Valdez, C.A., Abas Mazni, O., Takahashi, Y., Fujikawa, S. and Kanagawa, H. (1992) Successful cryopreservation of mouse blastocysts using a new vitrification solution. J. Reprod. Fertil., 96, 793802.[Abstract]
Vanderzwalmen, P., Delval, A., Chatziparasidou, A., Bertin, G., Ectors, F., Lejeune, B., Nijs, M., Prapas, N., Prapas, Y., Van Damme, B. et al. (1997) Pregnancies after vitrification of human day 5 embryos. Presented at the 13th Annual Meeting of ESHRE Edinburgh (abstract O-198). Hum. Reprod., 12 (Abstract Book 1), 98.
Wang, Q. and Latham, K.E. (1997) Requirement for protein synthesis during embryonic genome activation in mice. Mol. Reprod. Dev., 47, 265270.[ISI][Medline]
Wang, Q. and Latham, K.E. (2000) Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. Biol. Reprod., 62, 969978.
Zhu, S.E., Kasai, M., Otoge, H., Sakurai, T. and Machida, T. (1993) Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J. Reprod. Fertil., 98, 139145.[Abstract]
Zhu, S.E., Sakurai, T., Edashige, K., Machida, T. and Kasai, M. (1996) Cryopreservation of zona-hatched mouse blastocysts. J. Reprod. Fertil., 107, 3742.[Abstract]
Submitted on September 3, 2001; resubmitted on January 18, 2002; accepted on April 3, 2002.