In the past decades, slow cooling procedures was used for cryopreservation, but over the last few years, it has been suggested that the vitrification method may be a valuable alternative to slow cooling procedures. In comparing the principles, procedures and results of slow cooling and vitrification protocols, it was stated that the both methods resulted in the successful of cryopreservation of human oocytes and embryos. Vitrification is an ultra rapid cooling technique, offers a new prospective in attempts to develop an optimal cryopreservation, by producing a glass like solidification of cell, completely without intracellular ice crystallization during the cooling and warming process.
Methods of fertility preservation in women
Oocyte cryopreservation is useful for prepubertal girls and young women wishing to avoid using a sperm donor, or who wish to retain a choice of male partner and it avoids the custody battles if the relationship between the couple breaks up in the future (in spite of the presence of many legal and moral restriction in the different cultures and religions). There are several unique biological characters of the human oocytes that might be susceptible to damage during the cyopreservation procedure such as meiotic spindle, the cytoskeletal elements and cortical granules well as the extra- and intracellular ice formation are critical factors affecting the viability of the oocyte
The technical difficulties of the method were highlighted in a series of reports published after the first successful attempts and were conducted as early as the mid-1980s; they include a low pregnancy rate and an increased percentage of aneuploidy after gamete exposure to cryoprotectants and the freezing- thawing process. On the other hand, recent studies showed no increase in the number of abnormal or stray chromosomes in previously cryopreserved oocytes
Vitrification procedures circumvent two of the major limiting factors for achieving optimal cryopreservation: chilling injury and ice formation. Chilling injury can be defined as irreversible damage after the exposure of the cells to low temperatures, from +15 c to -5 c which affect mainly the cytoskeleton and cell membranes. These two major problems and those mentioned before are subsided by the application of vitrification by cryotop method. Once oocytes are loaded onto the cryotop, almost the entire loading solution is removed by aspiration before direct immersion into the liquid nitrogen.
This extremely low volume is also useful to achieve higher warming rates (42.000c/minute), avoiding ice crystal formation during warming. In addition, this method reduce the concentration of the permeable cryoprotectant to30%, minimizing any potential toxic effects
Cryopreservation of embryos is an established method offered by many fertility centers world wide. However, it is not feasible for pubertal girls, requiring spermatozoa and requiring an expensive programmable freezing system
There are many factors may limit the cryopreservation success. Ice crystals formation intra- and extra-cellular during the freezing and thawing processes has a harmful effect on the cell membrane leading to fracture of zona pellucida and/or cytoplasm and decreases the cell survival). Another harmful factor is the toxic effect of the permeable cryoprotectants. They replace some of the bound water molecules in and around proteins, DNA and other intracellular components the non-penetrating additives stay outside and aid in dehydration before and during preservation; they may also help stabilize the membrane by stabilizing the phospholipids head groups. All these permeable cryoprotectants are toxic. Particularly at high concentration, they may cause osmotic shock to the cells
The embryo developmental stage at which the process of cryopreservation will be performed is an important factor of cryopreservation success. Two-pronuclei (2PN)-stage embryo shows no signs of their developmental competence. While cleavage-stage embryo after thawing, damaged blastomeres often coexist with intact ones). Blastocyst-stage embryos are preimplantation embryos that have successfully passed the critical step of genomic activation and so have a high developmental potential. As blastocysts contain many cells, loss of some cells during freezing will cause lower harmful effect for the embryo development. However, development of embryos with reduced viability will arrest after extended culture.
Blastocysts freezing has three major rationales: (1) the superiority of blastocyst-stage freezing over earlier stage freezing in terms of implantation per thawed embryos transferred improves overall exceptions for the cryopreservation programme; (2) maximization of the cumulative pregnancy rates per oocyte retrieval; and (3) extended in-vitro culture of human embryos is becoming more common, encouraging the routine use of blastocysts transfer in IVF programmes and reducing multiple pregnancies.
More attention has been focused on vitrification technology as an alternative to the slow freezing technique, especially after reporting of the first embryo vitrification pregnancy. With slow freezing technique, it is difficult to eliminate the ice crystals formation and its hazards. Before the storage of the embryos in liquid nitrogen, controlled-rate freezing equipment and a long period of time for routine calibration and maintenance are obligations for the cryopreservation success. Selection of the most appropriate cryoprotectants, the time, the adequate temperature and the number of addition and removal steps are important to improve the success of slow freezing cryopreservation.
Vitrification technology had been developed as a method of cryopreservation to get around the difficulties of slow freezing method. It has been claimed to be a more safe procedure due to the total prevention of intra-cellular ice crystals formation. However, vitrification requires high cooling rate along with use of higher concentration of cryoprotectants, which may cause toxic and osmotic effect on cells. Thus, an increased probability of all other forms of cell injury except from the formation of ice crystals has been hypothesed. However, vitrification resulted in higher clinical and laboratory results with mounting data those introduced by different centres than those resulted by slow freezing technique in the subsequent years.
In comparison, vitrification is simpler than slow freezing technique. Vitrification requires no expensive programmable freezing equipment and it needs a very small volume of vitrification medium which must be cooled at extreme rates not obtainable in regular enclosed cryostraws and cryovials. Vitrification can be observed and analyzed while slow freezing cannot. Unlike slow freezing method, vitrification offers the ability to control the solute penetration, control the dehydration rate, and the maintenance of physiological temperature during equilibrium procedure.
Preservation of women fertility for those under threats may affects their ovarian reserve, represents a very important issue. Woman fertility could be affected by many factors. It is not only a mater of onclogical diseases, chronic diseases (e.g. autoimmune diseases, hematological, etc…), or even due to genetic predisposition, it may be due to her wish to postpone pregnancy. So the application of the best method of cryopreservation, become a right should be delivered to our patients. Now we can say that vitrification is the best method should be applied for having better results, even in comparison with slow freezing protocols in all the means of preservation of fertility. We can find that nitrification shows better results and easier protocol. It is cheaper, and can be done by only one embryologist.
Safaa Al-Hasani (1), Benkhalifa Moncef (2), Paul Cohen Bacrie (2), Anne Marie Junca (2), Martine Dumont (2), Stéphanie Belloc (2), Martine Cohen Bacrie (2), Alain dalleac (2), Yves Menezo (2)
1) Frauenklink der Univesritat Schleswig-Holstein. Campus Lubeck. Ratzuburger allee 160. 23538 Lubeck Germany 2) Département de Biologie et Génétique de la Reproduction. Laboratoire Eylau. 55-57 Rue Saint Didier. 75116 Paris