1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo and 2 CREST, Japan Science and Technology, Kawaguchi, Japan
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Abstract |
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Key words: glucose incorporation/implantation/mouse/preimplantation embryo/slow controlled-rate cryopreservation/ultrarapid cryopreservation
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Introduction |
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Vitrification involves the addition of high concentrations of cryoprotectants which at extremely low temperatures are in an amorphous state without crystallizing. The original vitrification solution consisted of permeating compounds [dimethylsulphoxide (DMSO), acetamide and propylene glycol] and a macromolecular compound (polyethylene glycol) (Rall et al., 1987). Later, a new cryoprotectant solution was developed (Ishida et al., 1997
) containing 40% ethylene glycol, 18% Ficoll and 0.3 mol/l trehalose, modifying an earlier one (Kasai et al., 1990
) containing ethylene glycol, Ficoll and sucrose. Ethylene glycol, a permeating compound, has an important role in stabilizing the cellular membrane during freezing, though it also has some harmful effects on embryo development (Kasai et al., 1990
). Ficoll is used as a low osmotic effect macromolecule to increase the viscosity of the medium. Trehalose, providing a non-permeating solution with significant osmotic effects, is a natural cryoprotectant that can be found in yeast, fungal spores, brine shrimp cysts and some soil-dwelling nematodes (Sussman and Lingappa, 1959
). It seems to prevent alteration to the cellular membrane during reduced water states but the mechanism is still not well understood (Rudolph and Crowe, 1985
).
Research comparing of the effect of conventional slow controlled-rate freezing and vitrification has been focused on embryo cleavage and implantation capacity and the results were controversial. Some studies have reported no statistical difference between the two procedures in blastocyst formation and implantation capacities of mouse (Rall and Wood, 1994) or bovine (Van Wagtendonk-De Leeuw et al., 1995
) embryos, while Dinnyes et al. (1995) reported that vitrification yielded significantly higher rates of implantation than those achieved after slow freezing using mouse embryos. However, little consideration has been devoted to functional or metabolic aspects of the embryo following cooling. We have recently reported that the slow controlled-rate freezingthawing procedure of mouse embryos decreases not only development rate to blastocyst stage, but also decreases glucose incorporation of the developed blastocysts due to decreased expression of GLUT1, suggesting that cryopreservation may have ulterior consequences on the functional development of embryos (Uechi et al., 1997
). We therefore decided to compare the efficacy of slow controlled-rate freezing and vitrification by assessing developmental potential of cryopreservedthawed 2-cell mouse embryos into blastocysts in vitro and their ability to incorporate glucose as well as morphological features and implantation rate in recipient mice.
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Materials and methods |
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Slow controlled-rate freezing and thawing procedures
Slow controlled-rate freezing and thawing of 2-cell embryos were carried out (Fugger et al., 1988). The embryos were placed in l.5 mol/l propanediol (PROH; Sigma, St Louis, MO, USA) in mBWW containing 0.3% bovine serum albumin (BSA) for 15 min at room temperature. The embryos were transferred to the same medium with 0.1 mol/l sucrose (Sigma) for 15 min and then loaded into 0.25 ml plastic straws filled with the same medium. Freezing was carried out in a programmed freezer (Cryoembryo-HP, Hoxan, Tokyo, Japan). Straws were cooled from room temperature down to 7°C at a rate of 2°C/min. Seeding was automatically induced during 15 min at this temperature. The straws were then slowly cooled down to 30°C at 0.3°C/min and then at 50°C/min to 140°C. After holding at 140°C for 5 min, they were plunged into liquid nitrogen for storage. A few days later, embryos were thawed by removing the straws from liquid nitrogen and keeping them at room temperature for 40 s. They were then hand-held until totally thawed. Cryoprotectants were removed stepwise at room temperature by transferring embryos successively (every 5 min) into mBWW supplemented with 0.3% BSA containing l.5 mol/l PROH + 0.1 mol/l sucrose, 1.0 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l PROH + 0.2 mol/l sucrose, and then into 0.2 mol/l sucrose. Sucrose was finally removed by placing the embryos in mBWW.
Vitrification and thawing procedures
Vitrification and thawing of 2-cell embryos were carried out (Ishida et al., 1997). The embryos were placed in 40% ethylene glycol, 18% Ficoll, 0.3 mol/l trehalose in phosphate-buffered saline (PBS) containing 0.3% BSA for 5 min at 4°C. The embryos were then loaded into 0.25 ml plastic straws filled with the same medium. Straws were plunged directly into liquid nitrogen for storage. A few days later, embryos were thawed by removing the straws from liquid nitrogen and keeping them at room temperature for 40 s. They were then hand-held until totally thawed. Cryoprotectants were removed stepwise at room temperature by transferring embryos successively (every 5 min) into mBWW supplemented with 0.3% BSA containing 0.35 mol/l trehalose and then into 0.2 mol/l trehalose. Trehalose was finally removed by placing the embryos in mBWW.
Cell number of blastocysts
Blastocysts were added to Hoechst 33258 (Bisbenzimide H33258 Fluorochrome; Wako, Osaka, Japan) and left for 15 min at room temperature (Tarkowski, 1966). Observation was carried out under ultraviolet light using fluoroscein microscopy (Model BX50; Olympus, Tokyo, Japan) and the number of nuclei in each blastocyst was counted (Tsutsumi et al., 1998
).
Trophoblast spreading of blastocysts
Trophoblast spreading of cultured blastocysts was quantitatively analysed as described previously (Suenaga et al., 1996). Blastocysts developed in vitro either from fresh, frozenthawed, or vitrifiedthawed 2-cell embryos were transferred to F0-CMRL medium (Suenaga et al., 1996
) supplemented with fetal bovine serum at a concentration of 20% (v/v), and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 96 h. The surface areas of the trophoblast spreads were quantitatively evaluated using a digitizer tablet (Model DT1000; Watanabe Sokki, Tokyo, Japan) connected to a personal computer (Model 9801, NEC, Tokyo, Japan).
2-Deoxyglucose uptake
Measurement of [3H]2-deoxyglucose (2-DG, Amersham, Little Chalfont, Bucks, UK, 17 Ci/mmol) uptake was performed as described (Morita et al., 1992). Fresh, frozenthawed, and vitrifiedthawed 2-cell embryos and blastocysts developed in vitro were incubated in 15 µl of mBWW solution containing 25 µmol/l 2-DG instead of glucose. They were incubated for 60 min at 37°C under an atmosphere of 95% air and 5% CO2 with 100% moisture. Each embryo was then washed five times with 100 µl of glucose-free mBWW solution. The uptake of 2-DG into each embryo was counted in a Beckman scintillation counter with 1 ml of Aquasol solution.
Embryo donation model
Seven blastocysts of each experimental group, i.e. blastocysts developed in vitro either from fresh, frozenthawed, or vitrifiedthawed 2-cell embryos were transferred surgically to the tip of one or the other uterine horn in the recipient mice on day 3 of pseudopregnancy as described previously (Morita et al., 1994). On day 9 of gestation, the recipients were killed and autopsied and the implantation rate was calculated as the ratio of implanted embryos to transferred blastocysts.
Statistical analysis
Statistical analysis was performed using Student's t-test and the 2-test. Statistical significance was established at the P < 0.05 level.
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Results |
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Discussion |
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In contrast with our present study, there have been many studies so far reporting good results with vitrification. Two main reasons may explain our obviously poor rates of blastocyst development, i.e. mouse strain and the developmental stage at which mouse embryos undergo cryopreservation. The Crj; CD-1 (ICR) mouse strain we used is inbred, showing a developmental rate of ~50% as shown in our present study and recent publication (Tsutsumi et al., 1998). Our vitrification protocol is almost the same as that previously reported (Ishida et al., 1997
), but they used a B6C3F1 hybrid mouse strain. Ethylene glycol-based cryoprotectant solution similar to that in our present study had previously been used (Kasai et al., 1990
) but with morula stage embryos. Using a later developmental stage such as 8-cell and morula caused a higher proportion of vitrified embryos to develop to blastocysts (Miyake et al., 1993
). 8-cell embryos of an ICR mouse strain were used (Mukaida et al., 1998
) and they reported that ethylene glycol-based cryoprotectant solution was more suitable for vitrification than PROH-, DMSO-, acetamide-, or glycerol-based solution. The reason that we used 2-cell embryos of ICR mouse strain is that we assumed that any difference in detrimental effect between the two cryopreservation procedures may be emphasized if `suboptimal' developmental stage embryos of a `suboptimal' strain is used instead of optimal stage embryos of an optimal strain, as previously shown (Kasai et al., 1990
; Ishida et al., 1997
). Moreover, we believe that our data obtained using 2-cell embryos of ICR mice are helpful for the improvement of the freezingthawing procedure for human embryos in clinical practice which sometimes do not have optimal quality.
These data concerning the rate of embryonic development to blastocysts also raise a question whether or not slow controlled-rate freezing and vitrification procedures exert an `all or nothing' effect on these mouse embryos. Therefore, we focused on the comparative effects of these cryopreservation procedures on the quality and viability of those embryos that survived and developed into blastocysts. However, blastocysts developed in vitro from slowly frozenthawed or vitrifiedthawed 2-cell embryos could not be morphologically distinguished from blastocysts developed from fresh 2-cell embryos and the number of cells in the blastocysts did not differ significantly between the three groups (Table I). It has been reported that ultrarapid freezing of mouse oocytes lowers the cell number in the inner cell mass of day 5 blastocysts (96 h after 2-cell embryos) (Van der Elst et al., 1998
). Similarly, in the present study, there was a slight decrease in the surface area of trophoblast spreading after 96 h culture (120 h after 2-cell embryos) of blastocysts developed from cryopreserved 2-cell embryos compared with fresh 2-cell embryos. However, there was no significant difference between the two cryopreservation procedures (Figure 2
and Table II
). Thus, we attempted to assess the viability of embryos by measuring their glucose uptake because current experimental data indicate that there is an alteration in the uptake or metabolism of glucose in early stage embryos (Leese and Barton, 1984
; Khurana and Wales, 1987
; Butler et al., 1988
; Brison and Leese, 1991
; Morita et al., 1992
). Indeed, it has been shown that glucose uptake can be used to select prospectively viable blastocysts immediately after thawing (Gardner et al., 1996
).
In the present study, it is of interest to note that 2-DG uptake in 2-cell embryos was significantly decreased by vitrification compared with those of fresh or slowly frozenthawed 2-cell embryos (Figure 3). It is postulated that vitrification itself causes an alteration in embryonic quality by affecting functional integrity of the 2-cell embryos. Cellular glucose uptake is dependent upon a family of glucose transporter proteins that contain multiple membrane-spanning domains (Birnbaum et al., 1986
; James et al., 1989
; Orci et al., 1989
). A more plausible explanation for this decrease in glucose incorporation activity is that during the vitrificationthawing procedure, glucose transporters in the membrane of the blastomeres of embryos are damaged, resulting in a decreased 2-DG uptake in the embryos.
The 2-DG uptake in blastocysts was significantly higher than the respective 2-cell embryos in all three groups. However, 2-DG uptake in the blastocysts developed in vitro from cryopreservedthawed 2-cell embryos was significantly lower than that of the blastocysts developed in vitro from fresh 2-cell embryos (Figure 3). As we have already reported (Uechi et al., 1997
), this decrease in 2-DG uptake of morphologically normal embryos may reflect a delayed effect of cryopreservation, suggesting that it may have ulterior consequences on the functional development of embryos. Moreover, 2-DG uptake of the blastocysts developed from vitrifiedthawed 2-cell embryos was significantly lower than that of the blastocysts developed from slowly frozenthawed 2-cell embryos. These data indicate that different types of freezingthawing procedure have different degrees of delayed effect as detected by 2-DG uptake, and that it is not an `all-or-nothing' type of effect that is assessed by 2-DG uptake assay. Since glucose incorporation activity is dependent on glucose transporter GLUT1 expression in early embryos (Morita et al., 1994
) and impaired GLUT1 expression is reported in slowly frozenthawed embryos (Uechi et al., 1997
), further investigation into mechanisms responsible for the gene expression may help to understand the impact of cryopreservation on the metabolic activity of embryos.
The viability of each group of embryos was assessed also by an embryo donation model that provides a way of determining whether a developmental failure occurs due to a defect in the embryo or in the environment. The implantation rate of blastocysts developed from vitrifiedthawed 2-cell embryos was significantly diminished compared with that of blastocysts developed from fresh and slowly frozenthawed 2-cell embryos (Table III). This suggests that the quality of the blastocysts developed after vitrification might be impaired as a result of delayed effects or consequences and thus implantation capacity might be reduced although they were morphologically indistinguishable from those obtained after slow controlled-rate freezing in terms of cell number and trophoblast spreading. This has implications for the application of cryopreservation technology as well as for cryobiology. Empirical studies are necessary for continued development of techniques that will maximize success rates and minimize time and expense of cryopreservation procedures and thus mechanisms of cryoinjury and its prevention may be understood.
Studies on perinatal outcome and follow-up of babies conceived from cryopreserved embryos have shown no pathological features (Wada et al., 1994; Olivennes et al., 1996
). However, embryos that survive to blastocysts following vitrification may have some cryoinjury not in an `all or nothing' way since they appear to be normal in morphology and are capable of further development. It was reported recently that slow controlled-rate freezing is more efficient than ultrarapid cooling, not vitrification, for human embryos (Van den Abbeel et al., 1997
). It remains to be determined whether offspring from vitrified embryos are phenotypically and genetically normal in all regards. Before vitrification is used routinely in clinical in-vitro fertilization programmes, its safety must be convincingly demonstrated.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 9, 1999; accepted on July 22, 1999.