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Middle East Fertility Society Journal Vol. 10, No. 3, 2005 © Copyright Middle East Fertility Society

DEBATE

Vitrification versus conventional technique

Comment by: Imam El-Danasouri, D.V.M., Ph.D. following factors:

Ulm, Germany 1- Increasing the concentration of : Helmy Selman, Ph.D. The cryoprotectant is usually used in much higher Perugia, Italy concentration than in the slow method. To avoid the toxic effect of the cryoprotectant, cells are equilibrated in a low concentration During in vitro fertilization treatment (IVF), a solution before immersion in the high surplus of oocytes is produced which results in a concentration solution (vitrification solution). The surplus of embryos which must be preserved. Due equilibration solution contains between 10 to 15% to the recent tendency in ART treatment to transfer of the cryoprotectant while the vitrification fewer embryos in order to avoid multiple solution contains between 35 and 40% (around 5.5 pregnancies, partly a result of new laws and Molar) of the cryoprotectant. Selecting the optimal guidelines aimed at controlling the number of cryoprotectant is critical. It should be of high embryos to be transferred and the number of permeability (low molecular weight) and as non- oocytes to be fertilized, the need for simpler, more toxic to the cells as possible. Ethylene Glycol effective methods for the cryopreservation of (EG) has these characteristics and, to lesser extent, embryos and oocytes is increasing. Currently, also DMSO. zygotes and embryos are typically cryopreserved 2- Dehydration of the cells: This is achieved by by means of the traditional slow-rate freezing using a high concentration of nonpermeating protocol, while oocyte freezing is still in the cryoprotectant of a large molecular weight such as experimental . disaccharide : or galactose at a However, during the slow freezing method, concentration up to 1 Molar. The end crystals are formed or produced, which has a concentration of the nonpermeating cryoprotectant deleterious effect on the cells. Intracellular ice is added to the vitrification solution. We cannot crystals can damage the cell wall and structure, overemphasize the importance of minimizing the while the extracellular precipitation of as ice cells' exposure time in the highly concentrated crystals increases the salt concentration to levels vitrification solution; successful vitrification that cause damage to the cell. A delicate balance procedures must limit such exposure to a few between these potentially harmful factors must be seconds. maintained throughout the slow freezing process to 3- Very fast freezing rate: The vitrification device ensure survival of the cells. containing the cells is directly plunged into In 1985, the ultra-fast freezing method through nitrogen which can achieve a cooling rate of more vitrification was introduced based on inducing than 20,000°C/ minute. The rate depends on the solidification of the cells and the surrounding vitrification device used and on the freezing vitrification solution (-like or vitreous) volume. No special equipment is needed to without formation of any ice . This achieve such a freezing rate. When the can be achieved through the combination of the vitrification device enters the , a

Vol. 10, No. 3, 2005 Debate Vitrification versus conventional cryopreservation technique 205 coat of liquid nitrogen is generated around it, dilutions, loading them into the vitrification device, creating an insulation layer that can lower the and plunging them into liquid nitrogen. cooling rate. Moving the device continuously 4- One of the advantages of vitrification is that it upon its entry into the liquid nitrogen until allows the operator to observe the cells during the complete solidification occurs easily eliminates vitrification process. None viable zygotes and this effect. blastomeres can be recognized since they do not 4- Increasing the viscosity of the vitrification contract upon placement into the first or the second solution: The viscosity of the vitrification solution vitrification solution. The presence of contracted increases during the cooling process until the cells during the warming process is a useful marker solution solidifies. The increased viscosity of the for cell survival with the exception of oocytes vitrification solution is automatically achieved which might survive the vitrification and warming through the rapid cooling process. The viscosity processes, but show degenerative signs can also be increased through the use of high approximately 10 to 15 minutes after the warming concentrations of both the permeating and is completed. nonpermeating and the addition of 5- The fact that no special equipment or macromolecules such as PVP or Ficoll. investments are needed for implementation of the 5- Minimizing the vitrification volume: Several vitrification procedure makes it extremely cost- devices have been developed and used successfully effective, and offers a tremendous economic for the vitrification of oocytes and embryos. Using advantage over traditional methods. a "plastic loop" reduces the vitrification volume to Vitrification is a very simple effective process a liquid film within the loop and the "open pulled and the skill needed to perform it can be acquired straws" allow for a very small vitrification volume in a short time through training on materials to be which does not exceed 2µl. In this context, the discarded. Several IVF programs have adopted the loading of the cells in the vitrification device and vitrification method as the sole procedure for day-3 the handling of the device are critical since this is human embryos and for human blastocysts, with done during the cells' exposure to the highly excellent survival and pregnancy rates (2, 3). The concentrated vitrification solution. The choice of challenge now is to find a protocol to successfully the device is a personal decision depending on the vitrify human oocytes, for which the slow freezing skill and preference of the individual operator. method has yet to produce acceptable survival and However, speed and dexterity are crucial as the development rates. goal is to minimize the cells' exposure to the high concentration solution prior to solidification. 6- Rapid warming rate: The vitrified cells should REFERENCE be warmed rapidly to avoid crystallization as in the 1. Rall WF, Fahy GM. Ice-free cryopreservation of mouse fast cooling process. The vitrified material is embryos at 196°C by vitrification. Nature 1985;313:573-5. immersed directly in the warmed thawing solution 2. El-Danasouri, I. and Selman, H. Successful pregnancies and the rehydration of the cells is usually and deliveries after a simple vitrification protocol for performed in two or three steps. day-three human embryos. Fertil Steril. 76: 400-2, 2001 3. Takahashi K, Mukaida T, Goto T, Oka C: Perinatal outcome of blastocyst transfer with vitrification using Advantages of vitrification cryoloop: a 4-year follow-up study. Fertil Steril. 2005 Jul;84 (1):88-92. 1- The vitrification process is both simple and fast; it can be completed in less than 10 minutes. 2- The prevention of ice crystal formation during the vitrification process successfully eliminates the Imam El-Danasouri, D.V.M., Ph.D. major factors that can cause cell damage during the Helmy Selman, Ph.D. slow freezing method. Endokrinilogikum, 3- The process is easy to master: Vitrification Ulm, Germany, involves simply moving the cells between two University of Perugia, Perugia, Italy

206 Debate Vitrification versus conventional cryopreservation technique MEFSJ