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Middle East Fertility Society Journal, Vol. 10, No. 3, 2005, pp. 209-211
DEBATE Vitrification versus conventional cryopreservation technique - Comment by: Khaled Elnomrosy, MRCOG, MD., Bassam Elhelw, MRCOG & Mostafa El Sadek, M.D. Khaled Elnomrosy, MRCOG, M.D., Bassam Elhelw, MRCOG, MFFP, Mostafa El Sadek, M.D. Middle East Fertility Center, Cairo, Egypt Code Number: mf05036 Related Articles: Cryopreservation of different types of living cells including oocytes, embryos even stem cells has become mandatory in infertility treatment and play important role in improving the results. There are different techniques that have developed in that field. All aim to be cell friendly; that is to choose low temperature technology that is likely to minimize cell damage and enhance survival rate of the living material that is stored for infertility treatment. This debate is to compare slow cooling with vitrification process to evaluate, which is likely to be more suitable for assisted reproductive technology as currently practiced i.e. which of them cause the least damage to the gametes during cryostorage. Vitrification is a process that produces glass like solidification of living cells that completely avoids ice crystal formation during cooling, and more importantly during thawing, which is fundamental issue in cryopreservation as ice should never be allowed to appear and grow inside the cells or tissue as this leads to damage and death of the living system. This is the same goal of the slow cooling process, as ice crystal formation is very detrimental to the living cells. Vitrification simply avoid ice crystal formation by cooling the living cells so quickly that ice wouldn't have time to form, that included the use of very high concentration of cryoprotectants to support the cytoplasm. The moment the cytoplasm is sufficiently concentrated the cooling process starts rapidly (1). To achieve this aim vitrification has passed through several steps. Initially the cryoprotectant used were very toxic to the cells, several attempts were made to formulate a non toxic and efficient vitrification solution, as high concentrations of cryoprotectant can be very detrimental to cells. However, Ethylene glycol has recently been widely accepted as the cryoprotectant of choice, which has resulted in successful pregnancies and deliveries by using it in vitrification of human embryo (2), because it penetrates embryos more rapidly than either propandiol or glycerol and is well tolerated by embryos. Furthermore, the survival rate of embryos frozen in ethylene glycol was much higher than propylene glycerol, which was used initially in vitrification, suggesting that ethylene glycol has a better effect on the embryonic cells. But still has to be used in high concentrations to allow the vitrification process to be done successfully. This has been proved to be one of the main factors that deteriorates the survival of embryos and oocytes (3). This is on of the large conflicts about vitrification process. The way around this problem is to shorten the contact between the embryos/oocytes and the cryoprotectant to a very short period of time to avoid its toxic effects. As the shorter the time of exposure the better survival of embryos and oocytes (4). However there are limitations in reducing the time of exposure as there is time required to load the embryos into straw/vials and seal them properly before final immersion in liquid nitrogen, which leads to the other conflicting issue about vitrification process. As in order to reduce this time, the embryos/oocytes loading should be very fast and the sealing process of the straws/vials should be minimum. Therefore different techniques have evolved, such as using open pulled straw technique (OPS), Cryoloop technique. All these techniques depend on no sealing of the straws, which leads to direct contact between the gametes and the liquid nitrogen. This increases the risk of contamination between gametes and liquid nitrogen, as infectious agents may be transmitted to cryopreserved embryos/oocytes and vice versa (5). Although no diseases transmitted by liquid nitrogen has been reported in human or domestic animals embryology, theoretical risk cannot be excluded. A technique of using fixed caps on the open end of the straw and other end was plugged with cotton to avoid direct contact with liquid nitrogen and shorten the contact time has been suggested, but this also was not airtight i.e. there was some leakage of liquid nitrogen to the gametes. Others have suggested straw vitrification of human embryos, where standard 0.25ml straw containing vitrification medium was located inside another 0.5ml straw which was closed before plugging into liquid nitrogen but this doesn't allow for rapid warming and simultaneous removal of cryoprotectant during this process (6). In spite of the previous controversies about vitrification, there are several advantages over the slow cooling process. Mainly the interaction between the vitrification solution with oocytes or embryos is smoother, which leads to the least damage especially in oocytes, this is due to the good control of solute penetration and the dehydration rate of the cells. Also, the vitrification process decreases the chilling injuries during the cooling process due to the ultra rapid cooling process which will decrease the mechanical damage i.e. fracture of the zona pellucida, and osmotic stress to the cells and this improves the survival of cell (7). Therefore, there is some progress in freezing embryos by vitrification, as there are some pregnancies from transferring vitrified and thawed embryos (8). But the main success was in vitrification of human oocytes especially germinal vesicle type (GV), as it has no spindle formed yet and the chromatid is well spread. Therefore it has better fertilization and better quality of embryos than the slow cooling method (9). CONCLUSION The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the development stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. REFERENCES
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