Human Reproduction, Vol. 15, No. 4, 905-910,
April 2000
© 2000 European Society of Human Reproduction and Embryology
Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts
1 Fertility Clinic and 2 Laboratory of Biology and Psychology of Human Fertility, Hopital Erasme French Speaking Free University Brussels, Brussels, Belgium
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Abstract |
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The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.
Key words: cryopreservation/ethylene glycol/IVF/mouse blasto-cyst/mouse embryos
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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During the past decade, in-vitro fertilization (IVF) and related techniques have become reasonably efficient treatments for infertile patients. The number of replaced embryos has been reduced from four to two (Devreker et al., 1999
), and since the introduction of sequential media (Fitzgerald and Di Mattina, 1992) there has been a move towards the transfer of a single blastocyst. The number of supernumerary embryos is continuously increasing, and thus the need for highly efficient cryopreservation techniques is becoming urgent. Because of ethical implications concerning human experimentation, preliminary exploration of the properties of new cryoprotectants can be made only on animal models. Ethylene glycol (ETG) is a compound used widely for embryo cryopreservation in many domestic animals such as rabbits (Kasai et al., 1992), cattle (Donnay et al., 1998) and sheep (Cocero et al., 1996), as well in slow-freezing (Sommerfeld and Niemann, 1999) and vitrification procedures (Ali and Shelton, 1993a; Zhu et al., 1993; Sommerfeld and Niemann, 1999). The low toxicity of ETG (Zhu et al., 1993; Donnay et al., 1998; Sommerfeld and Niemann, 1999) and the normal live births achieved with frozen–thawed embryos in animals (Ali and Shelton, 1993b; Zhu et al., 1993, 1996) make this molecule a good candidate for human embryo cryopreservation. Unfortunately, none of the studies on animal models employed freezing–thawing conditions similar to those used for human embryo cryopreservation. In a previous study (Shaw et al., 1995), the cryoprotective properties of 1.5 mol/l ETG were compared with those of 1.5 mol/l 1,2-propanediol (PROH) on slowly cooled mouse zygotes and 4-cell stage embryos. Freezing and thawing conditions similar to those described in protocols used for human embryos were used. In our study, the aim was to extend the evaluation of the cryoprotective properties of 1.5 mol/l ETG to the blastocyst stage, using the same freezing protocol and thawing procedure that led to the higher embryo survival rate described previously (Shaw et al., 1995). The toxicity of ETG and its osmolar properties were determined by using a Van t'Hoff curve (Mazur and Scheider, 1986). The activity of ETG was compared with that of PROH and glycerol, the classical cryoprotectants used for freezing early human embryos and blastocysts. |
Materials and methods |
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Mouse embryos
Female (C57BL/CBAca/J6) F1 generation mice were superovulated with 10 IU of follicle stimulating hormone (FSH) (Folligon; Intervet, Lane Cove, Australia) followed 45–47 h later with 10 IU of human chorionic gonadotrophin (HCG) (Chorulon; Intervet). The females were then mated to F1 males of the same strain; the day after only females with a vaginal plug were killed to collect zygotes from the ampoules. The total mouse litters were randomly distributed to the different experimental groups. The zygotes were placed in culture 18–20 h after HCG in Earle's modified balanced salt solution (mEBSS) (Van den Bergh et al., 1996
) supplemented with 0.5% bovine serum albumin (BSA) fraction V (A 6507; Sigma, St Louis, MO, USA) in 300 µl drops under mineral oil (M8410; Sigma), at 37°C in a humidified atmosphere of 5% CO2 and 5% O2. Embryos were randomly allocated to the control, toxicity or frozen groups.
Solutions
Freezing and thawing solutions were prepared in modified HEPES buffered EBSS (mHEBSS) supplemented with 0.5% BSA, with and without sucrose (S1888; Sigma). The cryoprotectants were 1.5 mol/l ETG (E9129; Sigma), 1.5 mol/l PROH (P6209; Sigma) and 1.5 mol/l glycerol (G5516; Sigma). Three equilibration solutions were used in three different equilibration steps for the freezing procedure with each cryoprotectant. Equilibration solutions were: 0.75 and 1.5 mol/l PROH and 1.5 mol/l PROH + 0.1 mol/l sucrose; 0.75 and 1.5 mol/l ETG and 1.5 mol/l ETG + 0.1 mol/l sucrose; 0.75 and 1.5 mol/l glycerol and 1.5 mol/l glycerol + 0.1 mol/l sucrose. Three equilibration solutions were also used in three different steps for the thawing procedure with each cryoprotectant. The equilibration solutions were: 1 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l PROH + 0.2 mol/l sucrose and 0.2 mol/l sucrose; 1 mol/l ETG + 0.2 mol/l sucrose, 0.5 mol/l ETG + 0.2 mol/l sucrose and 0.2 mol/l sucrose; 1 mol/l glycerol + 0.2 mol/l sucrose, 0.5 mol/l glycerol + 0.2 mol/l sucrose and 0.2 mol/l sucrose.
Van t'Hoff curve
The osmolar properties of ETG were evaluated by exposing the zygotes to 1.5 mol/l ETG, corresponding to an osmolality of 2600 mOsm, at room temperature and measuring, by video recording, their shrinkage and the time necessary to restore their initial volume. The volume of zygotes in an isotonic solution (280–285 mOsm) was plotted as 100% in a Van t'Hoff graph of percentage relative volume versus time.
Toxicity test
The embryotoxicity of ETG was tested by exposing the zygotes to 0.75 and 1.5 mol/l ETG for 10 min at room temperature, in each solution and by washing out the cryoprotectant in three steps with 1.0, 0.5 and 0.0 mol/l ETG. The exposed embryos were then placed in culture under the conditions described above. The cleavage rate (CR) 24 h later and expanded blastocyst percentage (EBP) 120 h later were recorded and compared with such data obtained from the control, non-ETG-exposed group.
Freezing–thawing procedure
The same freezing–thawing procedure was used for the three cryoprotectants. The embryos were exposed to 0.75 mol/l cryoprotectant for 10 min, then to 1.5 mol/l cryoprotectant for 10 min, and finally to the solution containing the cryoprotectant with sucrose, again for 10 min. All passages were made at room temperature. During this last period of equilibration, the embryos were loaded in 0.25 ml Institute Medicine Veterinaire straws (Bicef, L'Aigle, France) and charged in the freezing machine. Not more than 20 embryos were charged into each straw. A Kryo 10 machine (Planer, Middlesex, UK) was used for freezing, and the embryos were cooled as follows: from 22°C to –7°C at –2°C/min, at –7°C manual seeding, from –7°C to –33°C at –0.3°C/min, from –33°C to –150°C at –50°C/min; the embryos were then plunged into liquid nitrogen.
For thawing, the straws were held in air for 15 s and then plunged into a water bath at 37°C for a few seconds until the ice melted. The cryoprotectants were removed from the embryos in three steps: 10 min in 1.0 mol/l cryoprotectant + sucrose, 10 min in 0.5 mol/l cryoprotectant + sucrose, 10 min in sucrose alone, and 10 min washed in mHEBSS. Finally, the embryos were placed in culture in a 300 µl drop of modified EBSS with 0.5% BSA under mineral oil, at 37°C in a humidified atmosphere with 5% CO2 and 5% O2. Not more than 20 embryos were placed in each drop. For each freezing experiment, some of the embryos were randomly allocated to the control group that was placed in culture without freezing. Embryos were frozen at three stages: (i) Pronuclear, with ETG and PROH; the survival rate (SR), CR and EBP were measured after thawing, after 24 h and after 120 h respectively. (ii) 4-cell, with ETG and PROH. The SR was measured after thawing (only embryos with at least 50% of intact blastomeres were considered to have survived). The EBP was evaluated after a further 72 h of culture. (iii) Blastocysts, with ETG and glycerol. The percentage of re-expanded blastocysts was checked 24 h after the moment of thawing.
Statistical analysis
Statistical analysis was carried out by means of a 
2 test with a StatCalcul software package. Statistical significance was defined as P < 0.05.
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Results |
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Van t'Hoff curve
The Van t'Hoff curve for 1.5 mol/l ETG (2600 mOsm) is shown in Figure 1
. Shrinkage to 75% of initial relative volume of the zygotes was observed within the first 2 min, and a further 3 min were necessary for the cells to be restored almost to their initial volume. Following this observation, a preliminary step of exposure of embryos to 0.75 mol/l ETG was added (1400 mOsm). This step was introduced also for PROH and glycerol to compare the cryoprotective properties on the embryos of the three cryoprotectants under the same conditions.
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Toxicity test
Results of the toxicity test for ETG are shown in Figure 2
. In the ETG-exposed group, 89 out of 101 mouse zygotes (88%) cleaved after 24 h of culture, while in the control group 85 cells out of 101 cleaved (84%). After a further 96 h, 78 embryos became expanded blastocysts in the exposed group (88%), and 65 in the control group (76%). The differences between the two groups (2 test) were not statistically significant.
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Pronuclear stage embryo freezing
The results of pronuclear stage freezing with ETG are summarized in Figure 3
. A total of 339 cells were frozen in seven different experiments, 205 survived to the thawing (60%), 169 cleaved (82%), and 75 became expanded blastocysts (44%). In the control group (not frozen embryos), 85% of the zygotes (133/156) cleaved after 120 h and 85% (113/133) became expanded blastocysts. The difference in EBP between the two groups was statistically significant (P < 0.01). When the zygotes were frozen with PROH, 92% survived after thawing (70/76), 96% cleaved (67/70), and 70% (47/67) became expanded blastocysts. The difference in SR between the ETG and PROH frozen groups was statistically significant (2; P < 0.01).
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4-cell stage embryo freezing
The percentages of damaged blastomeres in 4-cell stage embryos frozen with ETG (n = 207) and PROH (n = 61) are presented in Figure 4
. Of the 51% (n = 189) of 4-cell embryos frozen with ETG having at least 50% of blastomeres intact, 108 (57%) became expanded blastocysts. This value was significantly lower compared with the group of 133 unfrozen control embryos where 113 (85%) became expanded blastocysts (2; P < 0.01). Figure 5 shows the results of a separate experiment in which 4-cell stage embryos were frozen with ETG or PROH. In total, 87 cells were frozen with ETG, 71 survived the thaw (82%), and 47 became expanded blastocysts (66%). In addition, 93 cells were frozen with PROH, 82 survived (88%), and 48 became expanded blastocysts (59%). No significant differences (2 test) were observed in SR and EBP between the two groups.
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Blastocyst freezing
The results of freezing young blastocysts (96 h after collection of zygotes) with either ETG or glycerol are shown in Figure 6
. In total, 103 blastocysts were frozen with ETG, and 50 (49%) re-expanded after 18–20 h of culture; 115 blastocysts were frozen with glycerol and 75 (65%) re-expanded. The difference between the two groups was statistically significant (2; P < 0.05).
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The percentages of expanded blastocysts obtained after freezing mouse embryos at different stages of development with 1.5 mol/l ETG are summarized in Table I
. The higher EBP was reached with 4-cell stage embryos.
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Discussion |
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Ethylene glycol appears to have a low toxic effect on mouse embryos, confirming the results reported previously for other animal species (Ali and Shelton, 1993b
; Donnay et al., 1998; Sommerfeld and Niemann, 1999). The Van t'Hoff curve for 1.5 mol/l ETG showed that a short time interval was necessary for the exposed zygotes to restore their initial volume after shrinkage, and this observation suggested a rapid diffusion and a quick equilibration of ETG into the cell through the zona pellucida and the cellular membrane. On the other hand, the excessive shrinkage observed suggested the risk of an intense osmotic stress by exposing the embryos to this ETG concentration.
Although ETG is chemically closely related to 1,2-propanediol, only one report exists concerning its use in the human for blastocyst vitrification (Vanderzwalmen et al., 1998), and no data are available regarding its use for human embryo cryopreservation with slow-freezing protocols. This suggests that preliminary larger studies evaluating the cryoprotective properties of ETG, by employing freezing–thawing conditions similar to those used for human embryos, are necessary. It was observed recently (Shaw et al., 1995) that previous studies comparing the cryoprotective properties of ETG and PROH in animal early embryos by slow cooling (Voelkel and Hu, 1992; Takagi et al., 1994a), did not use thawing conditions which resemble those used in human embryo freezing with PROH (Lassalle et al., 1985). Different freezing–thawing protocols were employed for blastocyst cryopreservation with ETG or glycerol in animals (Takagi et al., 1994b; Cocero et al., 1996; Sommerfeld and Niemann, 1999) and with glycerol in humans (Kaufmann et al., 1995). For this reason it is difficult to evaluate the differences in properties of various cryoprotectants and to extrapolate the results from animal species to the human situation. The impact of ETG on the survival of slow-cooled mouse early embryos was evaluated earlier (Shaw et al., 1995) by comparing different thawing conditions which closely resembled those used for freezing human embryos, the temperature in the straw being measured during each thawing procedure. Interestingly, the group of Shaw observed a higher survival rate of the embryos when slow warming-up (keeping the straw in air for 15 s) was followed by a fast warming-up (plunging the straws into a water bath at 37°C), at the moment when the straw reached the temperature of –70°C; consequently, this thawing protocol was selected for the present studies. Lower survival rates were observed in thawing protocols where embryos were warmed up either too rapidly (by direct plunging into water at 37°C) or too slowly (by keeping the straw in air for longer).
Our results from freezing pronuclear stage and 4-cell stage embryos differed somewhat from those reported earlier (Shaw et al., 1995). In our experience, ETG did not seem to be a good cryoprotectant for pronuclear stage embryos, but both survival rate and expanded blastocyst rate improved when 4-cell stage embryos were frozen, both with ETG and PROH. Furthermore, compared with these earlier studies (Shaw et al., 1995), a lower survival rate for 4-cell stage embryos was observed with both cryoprotectants in our study, though this may be due to differences in genetic constitution between the two mouse strains used. Furthermore, Shaw and colleagues left the 4-cell stage embryos in the oviducts until 52–53 h after the injection of HCG; this represented a more physiological situation compared with our study, where the embryos were removed from the oviduct at the pronuclear stage. Finally, interlaboratory variations in chemical and physical factors might be a further reason for these observed differences. Dimethlysulphoxide (DMSO), the classical cryoprotectant used for 4- to 8-cell stage embryos, is more toxic than other cryoprotectants (Kasai et al., 1981; Ali and Shelton, 1993b); while its use extends the freezing procedure, because of a slow cooling phase until –80°C, it is less practical for multicellular embryo freezing. Thus, the present data concerning the survival rate of 4-cell stage embryos are quite interesting in relation to a potential use for ETG in the freezing of human embryos.
ETG has been largely used for blastocyst vitrification in animals (Ali and Shelton, 1993b; Zhu et al., 1996; Sommerfeld and Niemann 1999) and humans (Vanderzwalmen et al., 1998), but because of the previously reported better outcome of cryopreserved embryos frozen with a slow cooling procedure compared with rapid and ultrarapid freezing (Van den Abbeel et al., 1997; Sommerfeld and Niemann 1999), we were more attracted by a slow-freezing protocol. The survival of mouse blastocysts before the moment of hatching frozen with ETG observed in our study is not really satisfactory. Glycerol, the classical cryoprotectant used for blastocyst cryopreservation gave a significantly higher blastocyst survival rate (P < 0.05). Many studies have evaluated the cryoprotective properties of ETG for blastocyst freezing in slow freezing protocols (Takagi et al., 1994b; Cocero et al., 1996b; Sommerfeld and Niemann, 1999). In one of these studies (Takagi et al., 1994b), the authors did not observe any difference in bovine blastocyst percentage developed after freezing with ETG or glycerol, while in another (Cocero et al., 1996b) a higher survival rate of sheep blastocysts frozen with ETG compared to glycerol was observed, though similar birth rates of lambs were observed with both cryoprotectants. In the final study (Sommerfeld and Niemann, 1999), a high hatching rate of bovine blastocysts (81%) frozen with ETG was observed. In the present study, significantly better results for blastocyst freezing were obtained with glycerol than with ETG, though differences between the procedures could be a reason for the observed discrepancies. Secondly, the different genetic constitution between animal species can strongly influence the resistance of the embryos to freezing, as well as the interaction between embryo and cryoprotectant. For this reason, many precautions must be taken before extrapolating the observations made on freezing animal embryos to the human situation. A second phase of experimentation on human embryos will of course be necessary to confirm these findings.
Finally, our results indicate that different embryonic stages are differently affected by the freezing procedure, and for each embryonic stage optimal results can be achieved using a different cryoprotectant. It has been reported (Li and Trounson, 1991) that the exposure time of embryos to the cryoprotectant strongly influences their viability after thawing. The equilibration time in a freezing protocol should in fact be neither too short (in order to allow the substitution of water molecules in the cell), nor too long (in order to avoid toxic effects on the embryos). Different cryoprotectants with different permeability to the cellular membrane and toxicity levels can variously influence developmental embryonic stage, characterized by a progressively decreasing blastomere volume. Of course, it is not only the blastomere volume but also changes in embryonic metabolism, as a consequence of the start of its genomic expression after the 8-cell stage, which could play an important role in the interaction between the cytoplasm and the cryoprotectant. The observed decline in blastocyst survival rate (92% and 88% for early embryos with PROH compared with 65% for blastocysts with glycerol) confirms previous data (Troup et al., 1990; Nakayama et al., 1995), where a low developmental, survival rate and no pregnancies were observed after replacement of frozen–thawed human blastocysts. Reduction of embryo viability caused by a longer period of in-vitro culture as a reason for lower embryo resistance to the freezing was proposed.
In conclusion, ETG does not appear to be a good alternative to the classical 1,2-propanediol and glycerol methods for freezing of mouse pronuclear stage embryos and blastocysts. The low toxicity observed, together with the good survival and expanded blastocyst rates obtained after freezing of mouse 4-cell stage embryos, may satisfy the real need in our centre for an alternative freezing protocol for 4-cell stage human embryos.
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Notes |
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3 To whom correspondence should be addressed at: Clinic of Fertility, Department of Obstetrics and Gynaecology, Erasmus Hospital, Rout de Lennik 808, 1070 Brussels, Belgium
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References |
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Submitted on August 13, 1999; accepted on January 5, 2000.
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