From a Backup Technology to a Strategy-Outlining Approach

The Success Story of Cryopreservation

Gábor Vajta; Anikó Reichart; Filippo Ubaldi; Laura Rienzi

Disclosures

Expert Rev of Obstet Gynecol. 2013;8(2):181-190. 

In This Article

Perspectives

Pretreatment for Better Cryotolerance

During the past decades, several attempts have been made to apply treatments to improve the general condition and the specific tolerance of oocytes and embryos to ensure better survival and developmental competence after cryopreservation, including the application of diverse chemicals, such as cytoskeleton relaxants or antifreeze proteins. So far, only one treatment seems to have had a positive effect for all reproductive cells, all developmental stages and all species tested, the application of sublethal stress before the intervention. The stress may induce changes in the gene function, protein conformation, metabolism or in other structures and functions in the cells, and after a recovery period oocytes, spermatozoa or embryos become more tolerant to the subsequent injury (i.e., cryopreservation). Various stressors may be used including heat treatment, toxic chemicals, osmotic agents and high hydrostatic pressure. The latter intervention provides the most consistent injury, as the pressure can be perfectly regulated, and the effect starts and at each point of each sample at exactly the same time – unlike chemical and osmotic injuries.

As oocytes and embryos are surprisingly tolerant to high hydrostatic pressure, a special device is required to produce a sublethal (200–800 bar) effect for approximately 1 h, with both parameters depending on the species, sample and stage of development. After a subsequent recovery period for another hour, cryopreservation (alongside other manipulations, including fertilization, somatic cell nuclear transfer or just embryo transfer) shows significantly better outcome. Alongside numerous research papers, extensive reviews of the application of high hydrostatic pressure in reproductive biology have been published recently,[82–84] based almost exclusively on domestic or experimental animal results. As the procedure has been found harmless in many species – in some of them with a follow-up to many generations – human application seems to be a feasible possibility in the not too distant future.

Standardization

So far, the most distressing consequence of the confusion described earlier regarding various tools, solutions and parameters is the problem with transportation of vitrified samples from one laboratory to another – a common practice that has been applied for traditionally frozen embryos for decades. Embryologists of the recipient laboratory have to use tools and methods they are not familiar with, resulting in a considerable chance of decreased survival, compromised quality or even loss of the sample. Considering that warming is supposedly the most important step of the whole vitrification procedure, the responsibility of the recipient laboratory is disproportionally high. Each situation may require individual technical solution, ordering of special kits, using media of unknown composition and seeing features during warming that may be completely different of those seen in the technique the laboratory is experienced in.

There were some successful attempts to find a 'general warming procedure' after vitrification[85] but deviation from the standard procedure is risky and the responsibility, including legal responsibility, may be enormous. The authors do not expect a rapid and radical solution to resolve this problem, but the process of standardization should be started now. Apart from resolving the problem with transportation, it may help to find the best procedure, the best parameters and to achieve the best possible outcome. This process has happened rather rapidly in traditional slow-rate freezing decades ago, and it should happen even more rapidly now with vitrification.

Work Safety Issues

The authors do not want to focus the attention on authorities of this problem, but this rarely discussed aspect should also be part of a review dealing with vitrification. In contrast to the most disputed and never proven danger of disease transmission, where hundreds of thousands of transferred embryos have never resulted in a single infection – proving that open vitrification is a less dangerous intervention for the patient than a blood pressure measurement – present vitrification methods impose a continuous and real health hazard for embryologists.

Decades ago, pioneers of the field had to use homemade tools and equipment, including the iconic foam box and simple carriers. These scientists were fully aware of and acknowledged the danger and risked their health for the advancement of science and for the improvement of assisted reproduction. This situation is not entirely acceptable but is tolerated in many fields of research. However, today vitrification is a routine procedure used worldwide in many, probably most, human ART laboratories, and it is the duty of the staff to perform vitrification every day, in large series, by using exactly the same tools and foam boxes. The industrial design provided by some producers may be attractive and may help to increase the price, but with very rare exceptions not at all the safety. Almost all laboratories use some homemade or ad-hoc techniques to fill these boxes with liquid nitrogen, transport them to the laboratory bench, immerse the carrier tools, prepare and transport these tools for storage, and discard or collect the used liquid nitrogen. Each step of this procedure is a definite hazard, with strict work safety standards to be applied.

In an embryology laboratory, none of these standards are used; liquid nitrogen is usually handled like tap water. Protective heavy cryogloves are useless for delicate manipulations, and protective safety goggles hamper parallel microscopic observation. Heavy rubber boots, face masks and protective clothing that are suggested or required in other workplaces are not only regarded as unnecessary, but are mostly unknown by embryologists. Moreover, the danger of storing liquid nitrogen dewars in a small under-ventilated room is often underestimated or disregarded, although this neglection has already resulted in deaths in at least one embryo unit.

A sudden implementation of the highest safety standards would result in catastrophic consequences for reproductive cryobiology and in general to human ART. However, the absurdity of the situation should be realized and definitive steps should be made by researchers, industrial producers and authorities to ensure reasonable progress towards a safe solution, preserving the present efficiency of vitrification, but consistently and rapidly increasing and the safety standards in laboratories, before serious accidents force us to do so.

Automation

The final solution for standardization, consistency, high efficiency and also work safety should be automation of the entire vitrification procedure including equilibration, cooling, preparation for storage, as well as removal from the storage container, warming and dilution of cryoprotectants. The task is difficult and would require considerable intellectual and financial investment. However, science and technology of the 21st century has already resolved the more complicated issues. Automation of vitrification is not just a commercial possibility, but a kind of categorical imperative, a duty that should be accomplished as soon as possible, to provide an ultimate and relaxing stage for this complicated, controversial, but extremely promising issue.

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