Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan
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Abstract |
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Key words: blastocyst/cryopreservation/morphology/mouse/vitrification
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Introduction |
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For the cryopreservation of various mammalian embryos, vitrification has proven to be the preferred strategy; embryos of mice (Kasai et al., 1990), rabbits (Kasai et al., 1992a
) and cattle (Ishimori et al., 1993
; Tachikawa et al., 1993
) have been successfully vitrified at quite high survival rates by a simple method. As yet, limited application has been made in humans, but vitrification also seems to be a promising strategy in fertility centres, since successful vitrification of IVF embryos has recently been reported using cryostraws (Mukaida et al., 1998
; Yokota et al., 2001
) or cryoloops (Mukaida et al., 2001
). In vitrification, however, a slight difference in the conditions for embryo handling will lead to a great difference in the survival of cryopreserved embryos, because the time and temperature of exposure of embryos to the vitrification solution before cooling is critical. In addition, the concentration of the vitrification solution surrounding each embryo may vary depending on the skill of the handler and even on the instrument (i.e. pipettes) used.
To find optimal conditions for embryo cryopreservation, it is essential to identify the mechanism by which embryos are injured in each protocol or procedure. Embryos are at risk of various types of injuries during cryopreservation (Kasai, 1996, 2002
). The main injuries are those from intracellular ice and concentrated solutes. In slow freezing of embryos, intracellular ice is a major cause of injury (Whittingham et al., 1972
), whereas in vitrification, the effect of the chemical toxicity of a high concentration of cryoprotectant is a major obstacle (Rall and Fahy, 1985
). However, the strategies to circumvent these injuries are completely different. To prevent intracellular ice from forming, a heavier loading of cryoprotectant is necessary, whereas to prevent toxic injury, the effects of the cryoprotectant must be reduced.
The introduction of a cryoprotectant into the cell can cause a third type of injury, osmotic swelling. During the process of cryoprotectant removal, water permeates more rapidly than the cryoprotectant diffuses out, and the embryo swells and may become injured. To prevent this, non-permeating sugar (usually sucrose) is widely used for the dilution of the cryoprotectant (Kasai et al., 1980; Leibo, 1983
). However, the hypertonic sugar solution may cause injury from osmotic shrinkage (Pedro et al., 1997a
). In addition, cryopreserved cells are at risk of physical injury from the fracture plane and extracellular ice. Fracture damage is the physical dissection of embryos along the fracture plane, which is formed by a non-uniform change in the volume of the medium during a rapid phase change (Kroener and Luyet, 1966
; Rall and Meyer, 1989
). Cells may also be damaged physically by extracellular ice if the unfrozen fraction in which the cells are located becomes smaller as the amount of extracellular ice increases with the lowering of temperature (Schneider and Mazur, 1987
).
In the routine recovery and temporal culture of cryopreserved embryos, those who handle embryos must observe them under a microscope. If they could deduce the mechanism of injury of the embryos, it would be possible to adjust and optimize the protocol for embryo cryopreservation in each laboratory. In this study, the conditions under which mouse blastocysts are injured by specific mechanisms were determined. To induce a specific injury, vitrification was adopted because injuries related to ice could be excluded. To induce intracellular ice formation, embryos were vitrified after an insufficient period of exposure to a vitrification solution (EFS40) (Kasai et al., 1990), which would make the cytoplasm and blastocoele remain less concentrated. To induce toxic injury, embryos were exposed to EFS40 for excess periods. To induce osmotic swelling, embryos were suspended in a hypotonic solution or vitrified embryos were diluted at a lower temperature where diffusion of permeated cryoprotectant would be slower. To induce osmotic shrinkage, embryos were suspended in a hypertonic solution containing a high concentration of non-permeating solute (sucrose). To induce fracture damage, embryos in EFS40 were passed through the glass transition temperature rapidly and repeatedly. To induce an injury by extracellular ice, embryos were frozen in a physiological solution without cryoprotectant. In all the cases, embryos were observed during dilution in sucrose solution, just after recovery in an isotonic solution, and after culture for 1 h and 24 h, in order to characterize each type of injury in cryopreserved embryos by the appearance of recovered embryos.
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Materials and methods |
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For recovery, the straw was kept in air for 15 s, and then immersed in water at 25°C for 8 s. As soon as the crystallized sucrose solution in the straw began to melt, the straw was removed from the water and quickly wiped dry, and the contents of the straw were expelled into a watch glass by flushing the straw with 0.8 ml of 0.5 mol/l sucrose/PB1. The embryos were then pipetted into fresh 0.5 mol/l sucrose/PB1. At
5 min after being flushed out, the embryos were transferred to fresh PB1 medium, and then cultured in modified M16 medium.
Based on this control method, embryos were treated under various conditions to determine a condition by which embryos should be injured by a specific mechanism, as described below; in some cases, the condition was adopted from reported data. Then the appearance of a small number of embryos was observed after being treated by the specific conditions.
Injury by intracellular ice
To induce intracellular ice to form, embryos were vitrified based on the control protocol, with a reduced exposure time in the cryoprotectant, which would decrease the concentration of ethylene glycol in the cell. Embryos were transferred to EFS40 directly from PB1 medium or after pretreatment with 10% ethylene glycol/PB1. Then, the embryos were cooled in liquid nitrogen vapour after exposure to EFS40 for 10 or 30 s at 25°C. Embryos were recovered following the control method.
Injury by the toxicity of cryoprotectant
Fresh embryos
Embryos were directly suspended in EFS40 which had been loaded in a straw as the control at 25°C. To give the chemical toxicity of the cryoprotectant, the embryos were exposed to the vitrification solution for 2, 5, 10, 30 or 60 min, and then they were recovered by transfer into 0.5 mol/l sucrose/PB1 and then into isotonic PB1 without vitrification following the control method.
Vitrified embryos
Embryos were vitrified and recovered following the control method, except that they were exposed to EFS40 for 2 or 5 min at 25°C without pretreatment before vitrification, which should be long enough to decrease the survival by the chemical toxicity of the cryoprotectant.
Cooled embryos
Since embryos can be injured by the toxicity of cryoprotectant at various temperatures, blastocysts were exposed to the cryoprotectant for a longer time at 20°C, a temperature far above the glass transition temperature. Embryos were treated and loaded in a straw by the control method, and the straw was immersed in ethanol at 20°C. After being held for 1, 3 or 6 h, the sample was thawed by direct immersion in water at 25°C, and the embryos were recovered by the control method.
Injury by osmotic swelling
Fresh embryos
Embryos were injured by osmotic swelling as described elsewhere (Pedro et al., 1997b): when fresh mouse blastocysts were exposed to a 0.25xisotonic solution at 25°C for 30 min, the embryos swelled by permeation of water into the cell and 49% (42/86) of them were injured. The 0.25xisotonic solution (0.077 Osm/kg) was prepared by mixing one volume of PB1 medium with three volumes of distilled water. Fresh embryos were suspended in a drop of this solution under paraffin oil at 25°C. After 30 min, they were recovered in isotonic PB1 medium.
Vitrified embryos
Embryos vitrified by the control method were warmed and the contents of the straw were expelled into a small glass test tube (10x90 mm) containing 1 ml of diluent precooled at 0°C, where permeated cryoprotectant would diffuse out slowly but water would permeate easily. The diluent was 0.5 mol/l sucrose/PB1 or isotonic PB1 medium. After 5 or 30 min, 2 ml of PB1 medium (25°C) was added to the test tube, and the embryos were recovered in PB1 medium.
Injury by osmotic shrinkage
Fresh embryos
Embryos were injured by osmotic shrinkage as described elsewhere (Pedro et al., 1997a): 86% (57/66) of fresh mouse blastocysts were injured by exposure to PB1 medium containing 1.5 mol/l sucrose, a non-permeating sugar, for 30 min at 25°C. Fresh embryos were introduced into a drop of the hypertonic solution under paraffin oil at 25°C. After 30 min in the solution, the embryos were recovered in PB1 medium.
Vitrified embryos
Embryos were vitrified, warmed and diluted by the control method. Five minutes after dilution with 0.5 mol/l sucrose/PB1, embryos were transferred to PB1 medium containing 1.0 mol/l sucrose at 25°C (in 0.2 ml of medium in a culture dish covered with paraffin oil) or at 0°C (in 1 ml of medium in a small glass test tube). After 30 min, embryos treated at 25°C were recovered in PB1 medium. For embryos treated at 0°C, 23 ml of PB1 medium (25°C) was added in the test tube before recovery.
Fracture damage
Fracture damage was induced by a method described elsewhere (Kasai et al., 1996). Briefly, straw samples loaded with embryos by the control method were cooled by direct plunging in liquid nitrogen and warmed by direct immersion in 25°C water, without keeping the straws in the gas phase. By this procedure, the vitrification solution passes through the glass transition temperature (approximately 130°C) very rapidly, which would make non-uniform volume change of the solution, and thus fracture planes. Immediately after warming, the straws were plunged again in liquid nitrogen. This rapid cooling and warming was repeated 10 times before embryos were diluted and recovered. By this treatment, 75% (41/55) of embryos were physically injured (Kasai et al., 1996
).
Injury by extracellular ice
Embryos were suspended in 0.8 ml of PB1 medium without cryoprotectant in a glass test tube and placed in an alcohol bath at 3°C. After 3 min, the medium was seeded by touching the outer surface of the tube with a pair of forceps precooled in liquid nitrogen. After another
3 min, the sample was cooled at 1°C/min to 5, 10 or 15°C, which would increase the volume of the extracellular ice, and thus decrease the unfrozen fraction where embryos must be located. The sample was kept at this temperature for 2 min and then thawed by adding 23 ml of PB1 medium at 25°C. The contents were transferred into a watch glass, and embryos were recovered in fresh PB1 medium.
Assessment of survival
Embryos treated to determine the conditions to induce cryoinjury were cultured in modified M16 medium in a CO2 incubator. They were examined at 1016 h intervals and at 48 h. Survival of blastocysts was assessed by the re-expansion of the blastocoele during 48 h of culture. Survival rates were calculated as the proportion of recovered embryos, since a small percentage of embryos were not recovered at random. The survival rate after each treatment was compared by
2-tests unless the expected frequency was <5, in which case Fisher's exact probability test was used.
Observation of the appearance of embryos
For morphological observation, five embryos were treated in each sample under a specific set of conditions (except for fracture damage experiments in which 10 embryos were vitrified). After dilution with 0.5 mol/l sucrose/PB1, recovered embryos were placed in 50 µl of fresh 0.5 mol/l sucrose/PB1 on a clean glass slide at room temperature. The embryos were photographed under a differential interference contrast microscope at 35 min after dilution. The embryos were then transferred to PB1 medium under paraffin oil at 25°C. After
5 min, they were placed in
50 µl of PB1 medium on a glass slide and photographed. The embryos were cultured in modified M16 medium in a CO2 incubator, and at 1 and 24 h after culture were transferred to PB1 medium on a glass slide and photographed.
The same embryos were photographed at four stages, but in experiments on osmotic swelling of fresh embryos and on extracellular ice, a different group of treated embryos were suspended in 0.5 mol/l sucrose/PB1 to take a photograph in the sucrose solution, since the treated embryos were directly recovered in PB1 medium.
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Results |
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As shown in Figure 2B, the embryos recovered in 0.5 mol/l sucrose/PB1 shrank with a clear cytoplasmic outline and looked quite normal. However, in PB1 medium, the cytoplasm, and thus each blastomere, swelled. After 1 h of culture, all the embryos had a shrunken cytoplasm, but some seemed to have intact blastomeres, which probably formed a small blastocoele after 24 h of culture.
Toxicity of cryoprotectant
Fresh embryos 1
When fresh embryos were exposed to EFS40 at 25°C for 2, 5 or 10 min and recovered without vitrification, the survival rates were 94, 22 and 0% respectively (Table II).
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Vitrified embryos
As shown in Table II, when embryos were vitrified after exposure to EFS40 for 2 or 5 min at 25°C, the survival rates were 40 and 0% respectively.
The appearance of embryos vitrified after 5 min of exposure is shown in Figure 2D. Just like embryos that were injured by the toxicity without vitrification (Figure 2C
), embryos looked normal in 0.5 mol/l sucrose/PB1 and PB1 medium, but showed decompaction after 1 h of culture and then degenerated after 24 h.
Cooled embryos
Table II shows the survival of embryos held in EFS40 at 20°C for various periods. After 1 h of holding, 91% of the embryos survived. However, after 3 and 6 h of holding, survival rates decreased to 39 and 0% respectively. The decrease was attributed to the toxicity of the cryoprotectant, because no ice formed in EFS40.
The appearance of the embryos held for 3 h at 20°C is shown in Figure 2E. All the embryos looked completely normal in 0.5 mol/l sucrose/PB1 and PB1 medium. After 1h of culture, however, one embryo shrank and its blastomeres showed decompaction. This embryo, presumably, was dead after 24 h of culture, whereas a less decompacted embryo formed a small blastocoele. The rest of the embryos re-expanded.
Fresh embryos 2
During the toxicity experiment, we happened to find that some embryos that had been given a higher dose of the cryoprotectant looked different from embryos that showed a decompaction of the blastomeres (here called the `fixed' type). To produce this injury, embryos were exposed to EFS40 at 25°C for up to 60 min. After 30 min of exposure, 60% of the embryos had the `fixed' type appearance, and after 60 min of exposure, all the embryos had this type of appearance (Table III).
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Osmotic swelling
Fresh embryos
As shown in Figure 2G, when fresh embryos treated in a hypotonic solution were suspended in 0.5 mol/l sucrose/PB1 medium, they shrank. If treated embryos were directly recovered in PB1 medium, some shrank with a clear cytoplasm, but others had collapsed blastomeres and the contents of the cells were dispersed within the zona pellucida. After 1 h of culture, the former embryos had a clear outline of the cytoplasm, but the latter ones did not. After 24 h of culture, the former embryos were found to have survived, whereas injured embryos shrank further.
Vitrified embryos
To induce osmotic injury during dilution of the cryoprotectant after cryopreservation, embryos vitrified in EFS40 were diluted with 0.5 mol/l sucrose/PB1 or with isotonic PB1 medium at 0°C (Table IV). Compared with the survival rate of embryos treated by the control method (96%), the survival rate decreased significantly (63%) when the dilution temperature was lowered to 0°C. However, no further decrease was observed by prolonging the exposure time in 0.5 mol/l sucrose/PB1 from 5 min to 30 min (61%). When the sample was diluted with PB1 medium, the survival rates decreased only slightly (61 versus 63% and 50 versus 61%).
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Osmotic shrinkage
Fresh embryos
As shown in Figure 2I, when embryos were suspended in a hypertonic sucrose solution at 25°C without cryopreservation, all the embryos were shrunken with a clear outline of the cytoplasm. However, on being transferred to PB1 medium, blastomeres of the embryos swelled and the outline of the cytoplasm became vague. After 1 h of culture, most of the blastomeres had collapsed and the embryos remained shrunken. They did not re-expand after 24h of culture.
Vitrified embryos
To induce injury from osmotic shrinkage in cryopreserved embryos, vitrified embryos were first diluted with 0.5 mol/l sucrose/PB1 at 25°C and then suspended in 1.0 mol/l sucrose/PB1 for 30 min. When embryos were suspended in 1.0 mol/l sucrose/PB1 at 25°C, the survival rate was 50% (Table V). So, this decrease must be caused by the treatment in this hypertonic solution. On the other hand, when embryos were suspended in 1.0 mol/l sucrose/PB1 at 0°C, almost all survived (98%). This shows that the injury from osmotic shrinkage is dependent on the temperature.
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Fracture damage
As shown in Figure 2K, in embryos with fracture damage, the zona pellucida, and in many cases the cytoplasm, were found to be dissected physically. Unaffected blastomeres formed a blastocoele after 24 h of culture.
Extracellular ice
When embryos were frozen in isotonic PB1 medium without cryoprotectant to various subzero temperatures, 100% of embryos survived at 5°C, but the survival rate decreased to 53% at 10°C and 0% at 15°C (Table VI).
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Discussion |
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When embryos were vitrified after exposure to EFS40 for only 10 s without pretreatment, none survived (Table I), indicating that they were injured by insufficient permeation of ethylene glycol and insufficient concentration of the cytoplasm, and thus by the formation of intracellular ice. The normal appearance of the embryos in 0.5 mol/l sucrose/PB1 (Figure 2B1
) shows that the cell membrane can react to the osmosis normally. Therefore, a normal shrunken appearance in the sucrose solution does not mean that the embryo survives. On transfer to isotonic PB1 medium, however, the embryos swelled and were damaged. Although the reason for the swelling is not known, the osmolality of the cytoplasm might be higher than isotonic. However, since the exposure time of embryos was only 10 s, the amount of permeated ethylene glycol must be small. Therefore, in cells injured by intracellular ice, permeated cryoprotectant might be difficult to diffuse out.
The toxicity of the solution is a major obstacle in vitrification. The vitrification solution, EFS40, contained ethylene glycol as the only permeating component, sucrose as a non-permeating sugar which promotes dehydration by its osmolality, and Ficoll as a macromolecule to promote vitrification of the solution (Kasai et al., 1990). One study (Zhu et al., 1993
) showed that ethylene glycol is responsible for the toxicity of the vitrification solution, although ethylene glycol is the least toxic permeating cryoprotectant for mouse embryos (Kasai, 1996
; Mukaida et al., 1998
). The toxicity is dependent not only on the concentration of the permeating component but also on the time and temperature of exposure (Kasai et al., 1992b
). In the present study, the toxic injury was induced by increasing the exposure time. By increasing the exposure time, permeation of ethylene glycol would increase, and the probability of osmotic swelling would also increase. However, osmotic injury is less likely in this case, because the observation that mouse blastocysts shrink only slightly in 10% ethylene glycol/PB1 (Zhu et al., 1993
) shows that they are quite permeable to ethylene glycol. Actually, the present osmotic experiment showed that 61% of embryos survived even when the sample was diluted with PB1 medium at 0°C (Table IV
). In addition, sucrose in EFS40 makes embryos shrink, which would restrict the permeation of ethylene glycol.
Usually, the toxicity of the cryoprotectant was detectable after 1 h of culture by decompaction of blastomeres of embryos, regardless of whether the embryos were treated at 25°C or at 20°C, and whether embryos were vitrified or not. In PB1 medium, the embryos looked normal, especially when the toxicity was not very high, as in the case of embryos kept in EFS40 at 20°C for 3 h (Figure 2E). Therefore, to detect the toxic injury, a short period of culture is necessary. In mouse morulae, it has also been shown that the toxicity of EFS40 is related to decompaction of the blastomeres (Kasai et al., 1992b
). However, in embryos at early cleavage stages before compaction of the blastomeres (e.g. 28-cell stages), this toxic injury may not be detected until an inability of cleavage of the blastomeres is observed. Usually, this type of injury would not occur in slow freezing, but it might occur when embryos are suspended in cryoprotectant solution at high temperature for a long time, or when samples under slow freezing are kept in a freezer at relatively high subzero temperature for a long period.
When embryos were exposed to extremely high toxicity, they did not collapse and looked as if they were fixed (Figure 2F). So, the cell membrane of the embryos must have become unresponsive to the osmosis. This type of appearance was first found when embryos were suspended in a small amount of EFS40 that had been left on the bench for
30 min. Therefore, evaporation of the vitrification solution must elevate the toxicity. In addition, an elevation of the temperature must also increase the toxicity. Although the mechanism of the `fixed' appearance is not known, the results of the toxicity experiment show that toxic injury from the cryoprotectant (at least from ethylene glycol) is not related to mechanical destruction of the cytoplasm.
When osmotic over-swelling was induced in embryos in a hypotonic solution without cooling, the cell membrane of the embryos physically collapsed (Figure 2G). However, it is known that embryos just after recovery from cryopreservation are much more sensitive to osmotic swelling (Pedro et al., 1997b
; Edashige et al., 1999
). So, we tried to observe this type of injury in the process of dilution of vitrified embryos. Since the permeability of embryos correlates to the temperature (Jackowski et al., 1980
), vitrified and warmed embryos were suspended in 0.5 mol/l sucrose/PB1 at 0°C where diffusion of ethylene glycol would be slow. Although 63% of the embryos survived this treatment (Table IV
), probably because the permeability of mouse blastocysts to ethylene glycol is quite high (as mentioned above), the significant decrease in survival would be attributable to osmotic swelling during dilution. However, this injury was not clearly detectable in mouse blastocysts until an inability to develop was observed after 24 h of culture. To observe this injury more closely, the use of embryos having a lower permeability to cryoprotectant (e.g. 2-cell embryos) would be preferable.
To prevent injury from osmotic swelling, sucrose solution is widely used for diluting the cryoprotectant solution as a hypertonic solution with a non-permeating solute. However, after diffusion of the permeated cryoprotectant out of the cell, osmotic shrinkage can cause injury (Pedro et al., 1997a). The results of the present study show that the effect of hypertonic treatment on mouse blastocysts is largely dependent on the temperature; at 0°C, the survival rate did not decrease (Table V
). It is known that sucrose and other sugars even have a protective action on the survival of refrigerated morulae in the mouse and the rat (Kasai et al., 1983
; Kasai, 1986
). Thus, the injury observed in the present study is not attributable to shrinkage per se. At higher temperature, the permeability of the cell membrane increases. And since the injury of embryos by osmotic shrinkage was characterized by swelling of the cells in PB1 medium (Figure 2J
), it is suggested that a small amount of sucrose permeates at room temperature and causes osmotic swelling in PB1 medium. An observation (Yang et al., 1990
) that the volume of rabbit embryos suspended in a hypertonic solution containing 1.5 mol/l sucrose at room temperature increased gradually supports this interpretation.
Fracture damage is a well-known injury (Rall and Meyer, 1989; Kasai et al., 1996
). This injury can be identified easily by physical dissection of the zona pellucida, and sometimes of the cytoplasm of embryos. However, since this injury is physical, embryos which were not dissected by the fracture plane could survive (Figure 2K
). It is possible to prevent fracture damage completely in vitrification, if the straw sample is cooled and warmed in the gas phase when the sample passes through the glass transition temperature (Kasai et al., 1996
).
To induce physical injury caused by extracellular ice, embryos were frozen in isotonic PB1 medium without cryoprotectant. When embryos were frozen to 5°C, the zona pellucida was not elongated (figures not shown), probably because the channel where embryos were located was large, as was shown for erythrocytes (Rapatz et al., 1966). However, the elongation of the zona pellucida of embryos frozen to 10°C (Figure 2L
) strongly suggests that the embryos were injured mechanically by the extracellular ice. In this condition, salts would be concentrated, which could influence the survival, but this is improbable since the exposure time was only 2 min and the temperature was low. Actually, this physical injury would not occur in conventional cryopreservation since the inclusion of a certain amount of cryoprotectant will reserve the channel space. In other words, the fact that this type of appearance is not observed would indicate that embryos were not injured by the extracellular ice.
In addition to the injuries examined in the present study, some types of embryos are injured just by chilling to <20°C. This injury was not examined because mouse blastocysts are not sensitive to chilling. The appearance of embryos injured by chilling, including chilling-sensitive types such as pig embryos (Polge, 1977), is of interest.
In summary, the normal shrinkage of embryos in sucrose solution does not indicate that the embryos are viable. The swelling in PB1 medium shows that the embryos might be injured by intracellular ice; a similar swelling can result from osmotic shrinkage but this is less likely in ordinary protocols. Fracture damage and extracellular ice are easily detectable by examining the morphology of the zona pellucida, i.e. physical dissection and elongation. Shrinkage of embryos with intact blastomeres after culture may be related to either cryoprotectant toxicity or osmotic swelling. If decompaction of the blastomere is distinct, the injury is most likely to be attributable to the toxicity. In many cases, different types of cryoinjury can give similar morphological appearances in the end phase. Thus it is the entire sequence of events until 24 h of culture that has to be followed.
The present study shows that it is possible to deduce many types of injuries by observing the appearance of embryos under a microscope. The morphological characteristics shown in the present study will be useful for those who handle embryos under the microscope to adjust and optimize the cryopreservation protocol. For instance, to prevent intracellular ice from forming, the loading of more cryoprotectant into the embryo is necessary, whereas to prevent toxic injury, the effect of the cryoprotectant should be reduced; this can be achieved by adjusting the exposure time and step, exposure temperature and/or the concentration of cryoprotectant. To prevent osmotic injuries, adjustment of the concentration of sucrose and dilution temperature is important. The morphological characteristics observed in vitrified mouse blastocysts will be applicable to blastocysts in other species including human, since vitrification using ethylene glycol-based solutions has proven an acceptable strategy for cryopreservation of human embryos at the blastocyst stage (Mukaida et al., 2001; Yokota et al., 2001
). In addition, the results could also be applicable to embryos at other stages of development.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 29, 2001; accepted on March 8, 2002.