Centre for Early Human Development, Monash Institute of Reproduction and Development and Monash University, Clayton, Victoria, Australia
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Abstract |
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Key words: contamination risk/cryobiology/embryo/polymer/vitrification
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Introduction |
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In the food industry, experiments have shown that bacteria could be transmitted between carcasses which were rapidly chilled by immersion in liquid nitrogen (Berry et al., 1998). Although there are studies which have analysed the liquid nitrogen from sperm storage tanks (Piasecka-Serafin, 1972
), and others which have calculated the risk of transmission of diseases in association with international movement of livestock embryos (Sutmoller and Wrathall, 1997a
,b
), it is unclear how likely disease transmission by liquid nitrogen is in IVF.
As some, but not all, contaminating pathogens can be removed from embryos by rigorous washing steps (Bielanski and Jordan, 1996; Bielanski et al., 1997
; Stringfellow et al., 1997
; Kim et al., 1998
; Trachte et al., 1998
), it is best to adopt strategies which minimize the likelihood of either the straws or their contents from becoming contaminated. Several strategies aimed at minimizing the likelihood of disease transmission during storage have been suggested. Contamination could be reduced by storing spermatozoa, eggs and embryos in nitrogen vapour, but the risk of accidental thawing or the effects of greater fluctuations in storage temperature may counter the benefits of using this storage procedure (Wood, 1999
). Russell et al. (1997) and Wood (1999) showed that properly sealed insemination straws do not leak dye, Escherichia coli or Newcastle Disease virus, and should therefore provide good protection. However, some sealing strategies were better than others (Russell et al., 1997
). Therefore extra protection, e.g. wrapping the straws in a plastic film, may be needed to further reduce the risk of leakage/contamination (Wood, 1999
).
Developing protocols to prevent contamination of very rapidly cooled or vitrified materials may prove more difficult. One strategy for achieving very rapid cooling rates has been to minimize the size of the sample, e.g. by placing the specimen on electron microscope grids (50 000180 000°C/min, Martino et al., 1996; Park et al., 1999; Hong et al., 1999), loops (Lane et al., 1999a,b
) or aluminium foil (Choo et al., 1999
; Dinnyes et al., 2000
), or inside heat-softened and pulled straws (~18 000°C/min, Vajta et al., 1998). The disadvantage of each of these approaches is that the vitrification solution comes in direct contact with liquid nitrogen during cooling or storage. Storage of such rapidly cooled specimens in nitrogen vapour would reduce the likelihood of cross-contamination, but the storage temperature is higher and more variable than in liquid nitrogen. Small specimens warm significantly faster than large specimens, with the consequence that current practices, such as pulling canes partially out of a tank to check the information on a specimen before removing it, could inadvertently thaw the specimens. Alternative strategies for reducing contamination should therefore be sought. The risk of contamination by larger pathogens could be reduced by filtering the liquid nitrogen through a 0.2 µm filter (Vajta et al., 1998
), UV irradiation of the liquid nitrogen, or by inserting the cryopreserved specimen into an additional outer protective container before they are moved to a storage tank (Vajta et al., 1998
). The latter approach has disadvantages, as it is difficult to manipulate small specimens and to seal containers at low temperatures.
This study was therefore aimed at developing a simpler protocol, which would allow rapid cooling of embryos with glass-like solidification (vitrification), while simultaneously reducing the risk of contamination from the liquid nitrogen. Although blastocyst transfer has become more popular in human in-vitro fertilization, we deemed day 2 and day 3 mouse embryos to be an appropriate animal model. To minimize the likelihood of contamination, we aimed to develop protein/serum-free cryoprotectants, which would allow embryos to be immersed directly into liquid nitrogen while packaged inside a double straw.
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Materials and methods |
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Embryo transfer procedures
Mature C57BLxCBA F1 females were placed with vasectomized C57BLxCBA F1 males to induce pseudopregnancy. Females with a mating plug were anaesthetized with 0.4 mg Rompun (Troy Laboratories, NSW, Australia) and 2 mg ketamine (Parnell Laboratories, Alexandria, NSW, Australia) per mouse and 56 embryos transferred to each oviduct. Females were killed on day 15 of gestation and the number of fetuses and implantation sites assessed.
Cryopreservation procedures
Preparation of solutions
The solutions for pre-equilibration and cryopreservation were prepared as follows. Pre-equilibration solution: 2.5 ml ethylene glycol 62.07 mol. wt (EG, Sigma-Aldrich, NSW, Australia), and 7.5 ml handling medium. Two different final cryopreservation solutions were made: (i) 2.5 ml EG, 3.5 g Ficoll 70 000 mol. wt (Sigma), 4 ml protein free PBS. This solution is referred to as EG25:F35; (ii) 2.5 ml EG, 3.5 g dextran 60 00090 000 mol. wt (clinical grade, Sigma), 4 ml protein-free PBS. This solution is referred to as EG25:D35.
Cryopreservation protocol
Two-cell mouse embryos were pre-equilibrated in 25% volume ethylene glycol in PBS + 4 mg/ml BSA in 35 mm tissue culture dishes (Falcon; Becton Dickinson, Melbourne, Australia) at room temperature for 23 min and then inserted in plastic 0.25 ml straws containing 30 µl of a final solution (EG25:F35 or EG25:D35) at the same temperature. After a 30 s equilibration period with the final solution, the straws were either sealed and immersed directly into liquid nitrogen (experiment 1), or sealed and placed inside a 0.5 ml straw which in turn was sealed (`straw-in-straw') before being immersed into liquid nitrogen (experiments 2 and 3). In experiment 1, the straws were warmed by holding them in air for 5 s before immersing them into a 31°C waterbath. In experiment 2, the larger (outer) straw was removed before the thawing step. In experiment 3, the outer straw was kept in place until after they were warmed by immersion in a waterbath at 38°C. The larger (outer) straw was removed before the dilution procedure. A two-step dilution protocol was used for all experiments, followed by in-vitro culture in M16 or MTF. All treatments were carried out with 310 replicates in each experimental group (>28 embryos/group). Control embryos were placed in culture without treatment.
Protocol for sealing the straws
The 0.25 ml straws for the embryos were shortened before the start of the experiment so that they could fit inside the outer straw. In order to save time in the straw-in-straw protocol, one end of the outer 500 µl straw was sealed before the start of the experiment. Two sealing strategies were examined, heat sealing and sealing with polyvinylalcohol (PVA) sealing powder. The former, which flattened and widened the straw, was very effective for the outer straw, but was less suitable for the inner straw as it made it difficult to insert into the outer straw. It also caused stretching of the outer straw, which may compromise its strength and integrity. With practice, effective seals could be created with either method.
Statistical analysis
Differences between individual replicates in vitro were determined using 2x2 contingency analysis for proportions. Development rates of non-vitrified and vitrified warmed embryos were compared to the untreated control embryos using simple factor analysis of variance (ANOVA) for the replicates of embryos developing into blastocysts.
In-vivo data are expressed as the proportion of live fetuses from the total number of embryos transferred with between 410 replicates in each experimental group. Data were analysed by one-tailed Fisher exact test to determine if the number of live fetuses from vitrified embryos was significantly less than the number of fetuses in control experiments. All statistical analyses were computed using Microsoft Excel and P < 0.05 was taken as a significant result in all cases.
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Results |
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The survival rates of day 3 mouse embryos (8-cell stage), pre-equilibrated in 25% by volume EG at room temperature and then vitrified by three different protocols in the above mentioned Ficoll- and dextran-based solutions are shown in Table III. There was no difference in the proportion of day 3 (8-cell) embryos which developed to the expanded blastocyst stage in-vitro following cryopreservation in the single or straw-in-straw configuration, irrespective of the warming protocol. The results were not influenced by whether the larger (outer) straw was left in place or removed before thawing (Table III
). All day 3 embryos that were vitrified-warmed, using the three different protocols, were recovered with an intact zona pellucida and developed into expanded blastocyst stage in culture.
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Discussion |
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The cryoprotectant used in this study which contains a high concentration of high molecular weight polymers was developed for mouse embryos. The concentration of penetrating cryoprotectant in this solution (25%) is more than double that used for conventional slow cooling (~11%) but is only half that which is currently used in most vitrification solutions (>40%). This EG concentration was chosen in an attempt to minimize the toxicity associated with the penetrating cryoprotectant, while maintaining a high total solute concentration (Shaw et al., 1997; Kuleshova et al., 1999a
, b
). While we have not ascertained whether other vitrification solutions can be used with the straw-in-straw configuration described here, the high polymer content solutions developed in this study have several advantages. Most vitrification solutions depend on high concentrations of penetrating cryoprotectants in order to vitrify, with the result that embryos must be processed rapidly in order to reduce toxic effects. By contrast 2-cell mouse embryos can be kept in the solutions used in this study for up to 20 min without loss of viability (unpublished data). It is possible that the viscous solutions used in this study slow the movement of water and solutes, thereby reducing both the rate of cryoprotectant penetration and the toxicity of the penetrating cryoprotectants to the embryo. Vitrification solutions which have a high total solute concentration, but substitute a large amount of the potentially toxic penetrating agents for less toxic sugars or polymers, have recently been effectively employed for human oocyte and embryo cryopreservation (Mukaida et al., 1998
; Hong et al., 1999
; Kuleshova et al., 1999c
; Chung et al., 2000
).
The solutions used in this study vitrify on cooling when used in either a single 0.25 ml straw or when used in the straw-in-straw configuration; however, they devitrify in both configurations during warming. It is not known whether human material would be damaged by the ice crystals that form as a solution devitrifies during warming. Although it is thought that solutions that remain vitreous throughout the cooling and warming procedure should be better for embryo cryopreservation as they avoid ice formation, previous studies have found no chromosomal or developmental anomalies associated with solutions which devitrify only on warming (Shaw et al., 1991). Solutions which vitrified on cooling but devitrified on warming also were found to be applicable for rapid freezing of bovine oocytes (Martino et al., 1996
; Vajta et al., 1998
). By contrast, conditions that allow ice crystal formation during cooling have the potential to cause chromosomal damage (Shaw et al., 1991
).
The protocol developed here provides a simple strategy for preventing cross-contamination and has the advantage that the specimen can be warmed while still inside the double straw. While we have used it in combination with vitrifying solutions, it can also be used in combination with slow cooling. In both cases further studies are needed, in particular if this approach is to be applied to human cells, to ascertain the optimal temperature for the waterbath, to avoid overheating of cells during warming. Since the solutions used in this study do not contain components of human or animal origin, there is no risk that it will introduce unwanted infectious contaminants, and should therefore be useful for embryos which are to be shipped internationally. The finding that both dextran and Ficoll were suitable was not unexpected. Both polymers have similar effects on the physical vitrification properties of ethylene glycolsaline based solutions (Shaw et al., 1997). Although cryoprotectant solutions containing PVP and EG have been used before (Leibo and Oda, 1993
; Murakami et al., 1998
), we have found that dextran and Ficoll are both significantly less toxic to embryos than either dialysed or non-dialysed PVP (Kuleshova et al., 1999b
). It is not clear how these polymers influence the solution, but it appears to make them less likely to fracture (Shaw et al., 1997
). The incidence of fracture damage in mouse embryos vitrified in 0.25 ml straws in Kasai's vitrification solution (Kasai et al., 1990) is lower with moderate cooling (vapour cooling, ~120°C/min) and warming (in air, 300°C/min) rates, than in straws cooled by direct immersion into liquid nitrogen (~2900°C/min) and/or warmed by direct immersion into a waterbath (1900°C/min) (Kasai et al., 1996
). These rates are calculated for the temperature range 0 to 150°C. A 0.25 ml straw within a 0.5 ml straw which is plunged directly into liquid nitrogen cools at around 400°C/min and warms at 120°C/min (in air) or 650°C/min (in water). This indicates that when a double straw is plunged directly into liquid nitrogen or directly into a waterbath the inner straw will have a cooling and warming rate which is comparable with the `moderate' rates used successfully for mouse (Kasai et al., 1996
) and human (Mukaida et al., 1998
) embryos. Vapour cooling has also been shown to be effective for human blastocysts (Yokota et al., 2000
). While it was not specifically tested in this study, it is likely that some but not all existing vitrification solutions could also be used in combination with this straw-in-straw procedure.
Rapid cryopreservation protocols are starting to be used for human oocytes and embryos at different stages of development including the blastocyst stage (Mukaida et al., 1998; Choi et al., 1999
; Hong et al., 1999
; Kuleshova et al., 1999c
; Lane et al., 1999b
; Chung et al., 2000
). Recently, it has been suggested that one of the techniques which promotes the viability of cryopreserved mammalian oocytes may also be appropriate for human oocytes. As a result of these efforts, for the first time in June 1999 the birth of a normal infant was reported (Kuleshova et al., 1999c
) following a transfer of an embryo derived from a vitrified mature oocyte. The birth of healthy twin babies following the vitrification of day 2 and 3 human embryos at a significantly lower cooling rate (2500°C/ min) in 0.25 ml straws has been reported (Mukaida et al., 1998
). Using the same approach, a further 10 pregnancies had been achieved by October 1999 (Dr M.Kasai, personal communication). It remains to be ascertained whether the cryoprotectant solutions and the straw-in-straw configuration developed here can be adapted to human oocytes and embryos.
We conclude that embryo cryopreservation in the proposed glass-like vitrifying solutions allows effective cooling in protein/serum-free solutions which minimizes the risk of exposure to biological contaminants. This, in combination with the inner straw placed inside an outer sealed protective container to prevent the inner straw from ever coming into contact with liquid nitrogen, provides a simple, rapid and effective strategy for reducing or eliminating the risk of contamination during cryopreservation.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 30, 2000; accepted on September 12, 2000.