Vitrification: Will It Replace the Conventional Gamete Cryopreservation Techniques?
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Vol. 10, No. 3, 2005 Middle East Fertility Society Journal © Copyright Middle East Fertility Society REVIEW Vitrification: will it replace the conventional gamete cryopreservation techniques? Yasser Orief, M.D.* Konstantinos Dafopoulos, M.D.† Askan Schultze-Mosgau, M.D. † Safaa Al-Hasani, M.D.† Department of Obstetrics and Gynecology, Shatby maternity hospital, Faculty of Medicine, Alexandria University, Egypt, and Department of Obstetrics and Gynecology, Medical University of Lubick, Germany ABSTRACT The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human- assisted reproduction. This may be due to the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all pre-implantation stages is encouraging for further research and clinical implementation. Keywords: vitrification, ,cryopreservation, cryoprotectants. INTRODUCTION In 1985, ice-free cryopreservation of mouse embryos at -196°C by vitrification was reported as In January 1977, Robert Edward stated: “The an alternative approach to traditional slow- storage of human pre-implantation embryos at cooling/rapid-thaw protocols, vitrification lower temperature could be valuable in clinical techniques have entered more and more the practice for the cure of infertility and possibly to mainstream of animal reproduction. In addition, avert inherited defects in children”. The same the last few years have seen a significant revival of author suggested that it might be beneficial to interest in the potential benefits of vitrification cryopreserve human oocytes. Since the birth of protocols and techniques in human-assisted Louise Brown in 1978, more than one million reproductive technologies. children have been born as a result of assisted The cryopreservation of human oocytes, reproductive techniques. Although difficult to zygotes, early cleavage-stage embryos, and document specific numbers, it has been estimated blastocysts has become an integral part of almost that tens of thousands of children have developed every human in vitro fertilization (IVF) program. from embryos that had been cryopreserved. Since the first report of human pregnancy following cryopreservation, thawing, and transfer *Department of Obstetrics and Gynecology, Shatby maternity hospital, Faculty of Medicine, Alexandria University, Egypt. of an 8-cell embryo (1), IVF centers have been †Department of Obstetrics and Gynecology, Medical using traditional slow-rate or equilibrium freezing University of Lubick, Germany. protocols fairly successfully. The time taken to Vol. 10, No. 3, 2005 Orief et al. Vitrification versus conventional cryopreservation techniques 171 complete these freezing procedures for human pulled straws in 1997, the successful vitrification of embryos ranges from 90 minutes to 5 hours. early stage bovine in vitro-produced embryos was Freezing includes the precipitation of water as ice, reported (5). with the resulting separation of the water from the In the field of assisted reproductive technologies dissolved substances. Both intracellular ice crystal (ART) in 1999 and 2000, successful pregnancies and formation and the high concentration of dissolved deliveries following vitrification techniques and substances pose problems. Therefore, a slow rate protocols for human oocytes have been reported of cooling attempts to maintain a very delicate (6,7). Since this time, the number of scientific balance between those factors that may result in publications concerning vitrification has clearly risen. damage, mostly by ice crystallization but also by osmotic and chilling injury, zona and blastomere fracture, and alterations of the cytoskeleton. Vitrification in nature Many studies have been undertaken to reduce the time of the freezing procedure and to try to Although most living organisms are composed eliminate the cost of expensive, programmable of large amounts of water, it is not inevitable that freezing equipment. One way to avoid ice freezing these organisms results in ice-formation. crystallization damage is through the use of Among amphibians and insects that can tolerate vitrification protocols. These cryopreservation freezing, there is wide variation in the amount of methods present an alternative to conventional freezing they can tolerate (8). Woolly bear caterpillars "may spend 10 months of the year freezing with equilibration. o The strategy behind vitrification is more radical: a frozen solid at temperatures that descend to –50 C. total elimination of ice formation, then an attempt to Species of frogs can spend days or weeks with as reduce toxic and osmotic changes. The physical much as 65 percent of their total body water as ice. process of vitrification can be defined as the glass Some amphibians achieve their protection due to like solidification of solutions at low temperate, the glycerol manufactured by their livers. Glycerol without ice crystal formation. This phenomenon is "antifreeze", it reduces ice formation and lowers could be achieved by increasing the concentration of freezing point. Such substances are called the cryoprotectant, and/or increasing cooling and "cryoprotectants". The sugar glucose is also a warming rates. Other factors that may facilitate cryoprotectant and arctic frogs have a special form vitrification include decreasing the volume of the of insulin that accelerates glucose release and solutions and increasing the hydrostatic pressure, absorption into cells as temperatures approach although the latter has a very few practical freezing. A cryoprotectant can make water harden consequences in embryology. like glass, with no crystal formation. That Vitrification techniques are mainly used for the phenomenon is called vitrification. cryopreservation of oocytes or blastocysts. The late The glycerol, sugars, and other cryoprotectants interest of the technique could be explained by the which are produced naturally in these organisms, apprehension of many researchers to expose are not found in levels that adequately explain embryos to high concentrations of cryoprotectants (with current knowledge of cryobiology) the (30% to 50%) necessary to obtain a vitrified state. remarkable freezing-tolerance. Experiments with As early as 1985, ice crystal-free cryopreservation cryoprotectants in mammals in the laboratory still of mouse embryos at -196°C by vitrification was produce results far inferior to those observed in initially reported (2) in an attempted alternative nature in frogs and insects. approach to cryostorage. Approximately 8 years later, the successful vitrification of mouse embryos was Approaching Vitrification demonstrated (3). Time travel is a solved problem. Einstein In 1996, Martino et al. (4) showed that by using showed that if you travel in a spaceship for months high cooling rates, bovine oocytes after at speeds close to the speed of light, you can return vitrification are still able to develop to the to earth centuries in the future. Unfortunately for blastocyst stage. With the introduction of open- would-be time travelers, such spacecraft will not 172 Orief et al. Vitrification versus conventional cryopreservation techniques MEFSJ be available until centuries in the future. pockets lock into place, and cells remain preserved Rather than Einstein, nature relies on Arrhenius inside the glassy water-cryoprotectant mixture to achieve time travel. The Arrhenius equation of between ice crystals. chemistry describes how chemical reactions slow This approach for preserving individual cells by down as temperature is reduced. Since life is freezing was first demonstrated half a century ago chemistry, life itself slows down at cooler (14). It is now used routinely for many different temperatures. Hibernating animals use this cell types, including human embryos. Preserving principle to time travel from summer to summer, organized tissue by freezing has proven to be more skipping winters when food is scarce. difficult. While isolated cells can accommodate as Medicine already uses this kind of biological much as 80% of the water around them turning time travel. When transplantable organs such as into ice, organs are much less forgiving because hearts or kidneys are removed from donors, the there is no room between cells for ice to grow (15). organs begin dying as soon as their blood supply In 1984 cryobiologist Greg Fahy proposed a stops. Removed organs have only minutes to live. new approach to the problem of complex tissue However with special preservation solutions and preservation at low temperature (16). Instead of cooling in ice, organs can be moved across hours freezing, Fahy proposed loading tissue with so of time and thousands of miles to waiting much cryoprotectant that ice formation would be recipients. Cold slows chemical processes that completely prevented at all temperatures. Below would otherwise be quickly fatal. the glass transition temperature, entire organs Some surgeries