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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Key words: cryopreservation/ethylene glycol/IVF/mouse blasto-cyst/mouse embryos
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Introduction |
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Materials and methods |
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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 (280285 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.
Freezingthawing procedure
The same freezingthawing 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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Discussion |
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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 freezingthawing 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 freezingthawing 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 5253 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 frozenthawed 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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References |
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Submitted on August 13, 1999; accepted on January 5, 2000.