The following review was written by Dr. Gábor Vajta at the request of the Practice Committee of the American College of Embryology (EMBCOL). Based on this review, EMBCOL has issued this opinion.
The issue
Application of vitrification for cryopreservation of oocytes and embryos is one of the greatest achievements of human assisted reproduction in the past decade. The new approach improved considerably the in vitro and in vivo development of cryopreserved blastocysts and oocytes. It opened new perspectives for extended embryo culture, blastocyst biopsy and alternative ways for fertility preservation or oocyte donation.
However, the currently applied vitrification techniques differ from each other in many technical details including solutions, equilibration and dilution parameters, carrier tools, cooling, storage and warming methods. One of the fundamental differences between various groups is the open versus closed vitrification. The closed systems keep the samples physically separated from liquid nitrogen or vapor phase nitrogen during the entire cooling, storage and warming procedure. The open systems - to improve the overall efficiency of vitrification by increased cooling rate - allow definite or possible direct contact to liquid/vapor phase nitrogen.
As liquid or vapor phase nitrogen may contain infective agents, therefore, the direct contact theoretically may mean a possibility for infection and disease transfer, even though, such infection has never been reported after an estimated 600,000 to 1,000,000 transfers worldwide, involving vitrified embryos or embryos derived from vitrified oocytes using open systems.
Open vs. closed systems - classification
For most embryologists, the two categories are easily distinguishable. Open systems allow and closed systems prevent direct contact between the medium containing oocytes/embryos and liquid/vapor phase nitrogen. However, in some cases it is difficult to qualify a particular system as clearly open or close.
The goal of vitrification is to completely eliminate ice formation in the medium that contains the sample during all phases (cooling, storage and warming) of the procedure (1). It can be achieved either by increased cooling and warming rates, or increased concentration of cryoprotectants. In practical situations both approaches are applied. The higher the cryoprotectant concentration, the lower cooling rate is required and vice versa. As highly concentrated cryoprotectants may cause toxic and osmotic injury, the preferred strategy is to use the lowest concentration of cryoprotectants and apply the highest possible cooling and warming rates to ensure safe ice-free solidification (2-5). The high cooling and warming rates may also be beneficial to avoid chilling injury (6).
The easiest way to achieve high cooling and warming rates is to use the smallest solution volume and the highest temperature conductivity between the medium-containing sample and the cooling or warming agent, preferably liquid nitrogen (7).
Decreasing the thickness of the wall of the sample-holding container, for example straw, may be helpful. Obviously, a total elimination of the thermo-insulating layer is the best solution. However, the seemingly easiest approach - small droplets freely plunged into the liquid nitrogen (8) - is suboptimal. To form a drop an excessive amount (>3 µl) of solution is required, and the nitrogen vapor coat that surrounds the warm medium will keep the drop for a relatively long period (8-10) over the surface of liquid nitrogen decreasing considerably the cooling rate. Accordingly, carrier tools were introduced to hold small amount, and ensure rapid submersion and fast elimination of the vapor coat (9, 10). The small (>1, >0.5 µl) amount of solution is also beneficial to minimize the danger of heterogenous ice formation (11).
Most carriers are based on homemade, simple tools, later modified for industrial production, although these modifications did not always increase the practical value and safety. At least 30 different carrier tools were published; at least 15 versions are commercially available. Most of them are slightly modified versions of the initially introduced carrier tools (12,13,14). Most of the "closed" systems represent modifications of these open systems.
A thorough structural and functional investigation of the existing vitrification systems reveals various levels of openness. Although they are important, these differences are commonly disregarded in the current laboratory practice.
1. Fully open systems
In these systems, samples contact directly with liquid nitrogen during cooling, and are not protected from further contact with nitrogen. Therefore, potential of cross-contamination during storage exists. On the other hand, both cooling and warming rates can be extremely high.
2. Open cooling and closed storage systems
As clear from the definition, the systems belonging to this group are open systems described above, but after cooling, the carrier tool is inserted into a pre-cooled sterile container that is heat-sealed and is capable to resist the extreme temperature changes (15). The cooling and warming rates are as high as in the previous group; there are no indications that a properly performed wrapping procedure decreases survival or in vitro/in vivo developmental rates.
3. Semi-closed cooling connected to either open or closed storage systems
In these systems the vitrification is performed on the surface of a metal block that is half-submerged into liquid nitrogen or inside of a container straw half-submerged into liquid nitrogen. In these systems, the presence of nitrogen vapors is unavoidable and therefore the possibility of nitrogen vapor-mediated contamination (16) cannot be excluded. Accordingly, these systems –have to be considered semi-closed. Cooling rates may be high on metal surfaces and somewhat compromised in vapor cooling; warming rates may be identical with those achieved in the previous groups.
4. Closed thin walled, narrow capillaries
In these systems the device is heat sealed before cooling, and opened only after warming. At warming, these devices are immersed in a water bath, and then the straws are cut and the content is expelled into the appropriate medium. Although in such systems the specimen itself is not coming into direct contact with nitrogen during cooling/warming it is not clear whether they are contamination proof. This is because the surface of the container may remain contaminated from the liquid nitrogen, and from the water bath. Furthermore, when straws are opened with cutting devices, the infection agent may theoretically enter the space shared with the embryos or eggs (17, 18). It should also be noted that currently available methods, i.e. wiping the surface of the capillary with ethanol or similar agents, even though they may decrease the chance of contamination they are not 100% safe either - see discussed later. Therefore, the safety of these closed systems may be compromised at the final phase. Also, compared to the previous groups, a decrease in both cooling and warming rates may occur, and the dilution of cryoprotectants after warming is delayed significantly.
5. Carrier tools sealed into a container that separates them safely during cooling, storage and warming
These devices may offer the highest protection against cross-contamination during the entire vitrification process ,and from this point of view, may even be safer than sealed straws used for traditional freezing. However, they may seriously compromise the cooling rates.
In summary, four out of the five groups of currently available vitrification tools do not completely prevent liquid nitrogen or nitrogen vapor mediated contamination. Only devices belonging to the group 5, which are relatively rarely used for vitrification, can be regarded safe from the point of liquid nitrogen mediated contamination, but their design does not allow to deliver the cooling rate deemed necessary to take a full advantage of vitrification.
The theoretical and practical risk of disease transmission via liquid/vapor phase nitrogen mediated infection
The subject has been summarized in detail in the review by Bielanski and Vajta (19). Since then no new relevant data was published, therefore, the most important points from this review are given below.
Exposure to low temperatures with or without the use of cryoprotectants and sophisticated cryopreservation strategies may or may not decrease the viability of infective agents. The survival depends on the species, complexity, size and the way these agents are exposed to low temperatures. Unfortunately methods developed for cryopreservation of mammalian oocytes and embryos may also support cryosurvival of different kinds of microorganisms.
Liquid nitrogen, and also vapor phase nitrogen (16) may act as a vector between two contaminated samples. The phenomenon has been observed in the food industry, in dermatology, and also under experimental conditions during storage of semen pellets and artificially infected samples stored in an open vitrification device. However, the only published human infection clearly attributable to liquid nitrogen mediated cross contamination has happened between large amounts of blood samples stored in leaky plastic containers. In reproductive biology including mammalian and human assisted reproduction, no disease transmission caused by liquid nitrogen mediated cross-contamination, or other cryopreservation-related source has been reported, in spite of the following:
- Collection of semen is not a sterile procedure
- Oocytes are contaminated with blood during collection
- Many containers are inappropriately sealed or closed by non-hermetical methods
- The outer surface of straws and vials are always contaminated
- Storage tools (canisters, holders) are not sterilized
- Openings of dewars, mix air with LN2 vapor and may cause infection
- Factory derived sterile LN2 is usually not transported under aseptic conditions, therefore, cannot be regarded as sterile
- Contaminated samples (sperm, oocytes) cannot be completely decontaminated
- In the majority of IVF labs, Dewars are not decontaminated regularly.
It has to be noted that LN2 tanks and LN2 in tanks should always be presumed contaminated.
The sources of potential cross contamination
The only completely safe method for hermetical closing of containers is heat sealing. Cotton plugs, beads commonly used for sperm, seemingly waterproof mechanical closures including screwed caps of cryovials or protective caps of several vitrification devices cannot be considered truly sealed.
Heat sealing, including commercially available, is a demanding procedure, and the outcome depends on many factors including the material, wall thickness and diameter of the straw, temperature, power of pressure and length of heating. Both insufficient and extensive melting may result in leaks that are hard to detect, or damage of the wall that is prone to leak or explosion during the extreme temperature changes the material is exposed to.
The procedure of warming and expelling samples from hermetically closed containers after cryostorage is a problem that has not been resolved yet since the introduction of the very first cryopreservation procedure. Vessels used for water baths are not sterilized, and not filled with sterile water. The wiping of vessels with 70% ethanol-soaked tissue is banned by many laboratories due to concerns with the potential toxicity of the alcohol. There is no generally acceptable procedure for using other disinfectants as none can be considered completely non-embryo toxic. Scissors or blades used to cut the straws are usually not sterilized between straws and patients, and in many clinics, the cut end of the straw is immersed into the medium during expelling. During all these manipulations, there is a risk of contamination from the liquid nitrogen or from the cutting device, although probably not very significant. Considering all the possibilities of infection of the cryo-stored sample, the fact that no infection attributable to the cryopreservation procedures has been reported is reassuring. Furtheremore, no infection transmission has been reported as a result of any ART procedures in both humans and domestic animals. There is no evidences that reproductive samples, including sperm, eggs, or embryos can retain any blood born pathogens by the time they are cryopererved. Semen wash and IVF are standard techniques for procreation of couples discordant for a blood born infection. No single infection has been reported to date following ART with processed reproductive samples (20, 21)
The most feasible explanation is that the threshold level required to cause a clinical infection is high in the female reproductive track (probably as a natural protection against infective agents relatively abundant in the semen), and washing-dilution procedures during routine ART interventions usually dilute the infective agents to the levels where they can longer be considered pathogenic.
However, even if the probability is extremely low ( the estimated >500.000 transfers after the use of fully open - group 1 - vitrification systems did not result in a single detected infection, ie. the probability seems to be less than 0.0002%), it would be wrong to become complacent. It is a role of embryology practitioners to continually improve patients safety while carefully considering the appropriate balance between the risks and benefits of all procedures.
The effectiveness of open vs. closed systems in human embryology
As traditional freezing was successfully applied since decades for pre-compaction stage human embryos, the introduction of vitrification has resulted in breakthrough in two main areas: cryopreservation of blastocysts and MII phase oocytes. These initial successes have been achieved with fully open systems belonging to group 1 (22, 23), which worldwide remain the most commonly used for vitrification of reproductive samples(24).
In 2013, two papers from the same group (17;18) have published two prospective randomized studies comparing open and closed vitrification systems for cryopreservation of human blastocysts and oocytes, respectively. There is no measurable difference in any outcome between the two systems reported for blastocysts. On the other hand, after oocyte vitrification, the survival rate for closed system was found to be significantly lower.
An observational study in an oocyte donation program performed vitrification in high-security closed vitrification system and found 90% survival; 78% fertilization, and 62% good quality embryos on D3. Only good quality embryos were transferred, with 45% ongoing pregnancy rate. Although authors evaluated their results as excellent the validity of their methodology was questioned (26).
Earlier publications were summarized in a recent review (27) . Fourteen publications reported on the results for blastocyst vitrification with open systems and 3 with closed systems. The overall tendency was a slightly compromised outcome with closed systems, but the diverse approaches did not allow statistical analysis. For human oocytes, however, the 18 papers dealing with open systems reported cumulatively higher overall success than that of reported in only 2 publications using closed systems.
It should also be considered that
- Worldwide, the majority of human IVF laboratories continue to use open vitrification systems for embryos, and the overwhelming majority performing oocyte vitrification uses open systems (groups 1 or 2).
- In the absence of reports of cross-infection, there is no strong incentive to switch away from something that works well to something new with no proven advantages in effectiveness.
It is a promising fact is that both papers reporting clinically acceptable results after the use of a closed system for oocyte vitrification, used systems that were completely closed (group 5). It should be noted that any results of vitrification obtained in species other then human have to be interpreted with caution and mere morphological survival cannot be considered a valid end-point (27, 28-30).
In summary, compared to the overwhelming majority of published and unpublished excellent results with open systems used for oocyte vitrification, with tens of thousands of healthy babies born worldwide during the past 8 years, evidences supporting the suitability of closed systems are scarce, published by isolated research groups, resulted in only 13 births and 20 ongoing pregnancies in total. Right now, these attempts should be regarded as experimental, and extensive work including multicenter prospective randomized trials are still required to explain why are these procedures are successful when applied in one lab and inefficient in others, and to prove that the suggested methods are really competitive alternatives of open systems at every outcome check-point.
It should be noted that the recent Guidelines of the American Society for Reproductive Medicine regarding human MII phase oocyte cryopreservation (31) stating that the technique should no longer be considered experimental is based exclusively on the success of open systems.
Concluding notes
The future research will determine whether the closed systems can deliver the effectiveness of oocytes vitrification equal to open system, while providing complete protection from any possibility of infection.
Meantime certain additional steps can be taken by those who continue to use open systems to increase patients safety.
For example, if factory-derived liquid nitrogen is separately stored and exclusively used for cooling, the risk of infection with the most feared HIV and HBV is negligible, as these are not airborne viruses (19). Liquid nitrogen can be sterile filtered with disposable filters (15) large-capacity industrial devices. It can also be decontaminated by exposition with UV light (32). Any tool/vessel that contacts with liquid nitrogen (but not with the samples or sample-containing solutions) can be decontaminated easily by chemical agents, then by repeated flush with sterile distilled water (19)
Many leading laboratories have implemented one or two of these measures to prove their devotion towards measures to avoid liquid nitrogen or nitrogen vapor mediated cross contamination. Although in the lack of any documented disease transfer it is difficult to evaluate the need and efficiency of these measures, they may help to maintain the awareness and high laboratory standards.
Reference List
(1) Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 2-14-1985;313:573-5.
(2) Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004;9:164-70.
(3) Fuller B, Paynter S. Fundamentals of cryobiology in reproductive medicine. Reprod Biomed Online 2004;9:680-91.
(4) Stachecki JJ, Cohen J. An overview of oocyte cryopreservation. Reprod Biomed Online 2004;9:152-63.
(5) Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006;12:779-96.
(6) Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Hum Reprod 2005;20:3385-9.
(7) Arav A. Vitrification of oocytes and embryos. In: A Lauria & F Gandolfi, eds. New Trends in Embryo Transfer, Cambridge:Portland Press., 1992:255-64 .
(8) Landa V, Tepla O. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biol (Praha) 1990;36:153-8.
(9) Steponkus PL, Myers SP, Lynch DV, Gardner L, Bronshteyn V, Leibo SP, et al. Cryopreservation of Drosophila melanogaster embryos. Nature 5-10-1990;345:170-2.
(10) Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996;54:1059-69.
(11) Rall WF, Wood MJ, Kirby C, Whittingham DG. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987;80:499-504.
(12) Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53-8.
(13) Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999;72:1073-8.
(14) Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999;51:165.
(15) Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H, . Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryo Letters 1998;19:389-92.
(16) Grout BW, Morris GJ. Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer. Theriogenology 4-15-2009;71:1079-82.
(17) Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Prapas N, Zikopoulos K, Georgiou I, et al. Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study. Reprod Biomed Online 2013;26:595-602.
(18) Panagiotidis Y, Vanderzwalmen P, Prapas Y, Kasapi E, Goudakou M, Papatheodorou A, et al. Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reprod Biomed Online 2013;26:470-6.
(19) Bielanski A, Vajta G. Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units. Hum Reprod 2009;24:2457-67.
(20) Vitorino RL, Grinsztejn BG, de Andrade CA, Hokerberg YH, de Souza CT, Friedman RK, et al. Systematic review of the effectiveness and safety of assisted reproduction techniques in couples serodiscordant for human immunodeficiency virus where the man is positive. Fertil Steril 2011;95:1684-90.
(21) Savasi V, Oneta M, Parrilla B, Cetin I. Should HCV discordant couples with a seropositive male partner be treated with assisted reproduction techniques (ART)? Eur J Obstet Gynecol Reprod Biol 2013;167:181-4.
(22) Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300-8.
(23) Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001;76:618-20.
(24) Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11:608-14.
(25) Stoop D, De MN, Jansen E, Platteau P, Van den Abbeel E, Verheyen G, et al. Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online, 2012; 24, 180– 185
(26) Vanderzwalmen, P. Adjustment of cryoprotectant exposure time to counteract reduced cooling rates. Reprod BioMed Online (2012) 24, 684.
(27) Vajta, G., Reichart, A., Ubaldi, F., Rienzi, L. From a backup technology to a strategy-outlining approach: the success story of cryopreservation. Expert Rev. Obstet. Gynecol. March 2013, Vol. 8, No. 2, 181-190 , DOI 10.1586/eog.12.80
(28) Seki S, Mazur P. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice. Biol Reprod 2008;79:727-37.
(29) Seki S, Mazur P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology 2009;59:75-82.
(30) Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to -196 degrees C at 95 degrees to 70,000 degrees C/min and warmed at 610 degrees to 118,000 degrees C/min: A new paradigm for cryopreservation by vitrification. Cryobiology 2011;62:1-7.
(31) Mature oocyte cryopreservation: a guideline. Fertil Steril 2013;99:37-43.
(32) Parmegiani L, Cognigni GE, Filicori M. Ultra-violet sterilization of liquid nitrogen prior to vitrification. Hum Reprod 2009;24:2969.
Conflict of interest disclosure: Prof. Dr Vajta declares that he is an inventor and small scale producer of the Open Pulled Straw.