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Theriogenology 81 (2014) 96–102

Contents lists ava

Theriogenology

journal homepage: www.theriojournal .com

40th Anniversary Special Issue

Cryopreservation of oocytes and embryos

A. Arav*

FertileSafe, Shlomzion Hamalca, Tel Aviv, Israel 62266

a r t i c l e i n f o

Article history:Received 15 July 2013Received in revised form 11 September 2013Accepted 11 September 2013

Keywords:FreezingVitrificationDryingOocytesEmbryosCryopreservation

* Corresponding author. Tel.: þ972 523 638022.E-mail address: arav@agri.huji.ac.il.

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.09.0

a b s t r a c t

Two hundred years have passed since the first description of supercooled water by Gey-Lussac to the recently high survival rates of embryo and oocytes after vitrification. Thisreview discusses important milestones that have made vitrification the method of choicefor oocytes and embryos cryopreservation. We will go through the first cells ever to survivelow temperature exposure in the beginning of the last century, the finding of glycerol inthe late 1940s and the first mouse and bovine embryos freezing in the 1970s. Duringthe 1980s, embryo vitrification began and the time since is a tribute to the development ofoocytes vitrification. Standardization and an automatic vitrification procedure arecurrently under development. The next evolutionary step in oocyte and embryo cryo-preservation will be preserving them in the dry state at room temperature, allowing homestorage for future use a reality.

� 2014 Elsevier Inc. All rights reserved.

1. Two hundred years of vitrification

Going back to the book “Life and Death at Low Tem-peratures” by Basile J. Luyet and Marie Pierre Gehenio,published at 1940 [1], I found out that the small volumevitrification (the minimum drop size technique) that isgenerally attributed to my work was already thought at thebeginning of the 19th century. It was done by the greatFrench chemist and physicist Joseph Louis Gay-Lussac. He isknown mostly for his two laws of gases and for his work onalcohol–water mixtures. Gay Lussac found that water canbe cooled to �12 �C without freezing, finding with thisdiscovery the basis of vitrification [2].

In 1804, Gay-Lussac was ascended in a hot air balloon(Fig. 1) and noticed that the drops in the clouds are notfrozen despite the subzero temperatures. He publishedlater the discovery of the effect of small volume of waterdroplets on supercooling. The size of water drops in cloudsis around 8 to 10 mm,whichmaintains them at a liquid stateat a subfreezing temperature of �5 �C.

Luyet described it in his book: “Some of the oldestinvestigations on subcooling were made by Gay-Lussac

. All rights reserved.11

(1836) who observed that water can be subcooled to�12 �C when it is enclosed in small tubes” [1,3].

Already in 1858, Johann Rudolf Albert Mousson sprayeddroplets of water less than 0.5 mm in diameter on a drysurface and observed that the smaller the drops the longerthey stayed subcooled [4]. Not only was volume importantto achieve supercooling, among other factors that mighthave an influence in inducing crystallization, as mentionedby Luyet, are cooling velocity and concentration of thesupercooled or supersaturated solutions. Luyet wrote, “Toavoid freezing, the temperature should drop at a rate ofsome hundred degrees per second, within the objectsthemselves,” and “The only method of vitrifying a sub-stance is to take it in the liquid or gas state and cool itrapidly so as to skip over the zone of crystallization tem-peratures in less time than is necessary for the material tofreeze.” Luyet further wrote, “It is evident that when crys-tals grow faster one must traverse the crystallization zonemore rapidly if one wants to avoid crystallization” [1].

2. Basic principles

The velocity of cooling depends on the thermal mass ofthe sample and on its surface area. To achieve rapid cooling,

Fig. 1. .Joseph Louis Gay Lussac (1778–1850) and portrait and an illustrationof from 1804 of Gay Lussac and Jean Baptiste Biot in the hot air balloon at4000 m.

A. Arav / Theriogenology 81 (2014) 96–102 97

we should use material with the lowest heat mass andmaximum surface to volume ratio.

Indeed, GregoryM. Fahy andWilliam F. Rall published in2007 the critical cooling rates needed to vitrify aqueoussolutions that contain different concentrations of cryopro-tectants (CPs) [5]. It was extrapolated that for pure waterover 100 � 106 �C/min are needed to form a glass statewithout crystallization. Also, it is interesting to note that for15% (v/v) of most CPs almost 1 � 106 �C/min is needed,which is difficult to achieve. We have shown that 15% (v/v)of CPs can vitrify at relative slow cooling rates when thevolume of the drop is 0.07 mL [6]. Therefore, it is muchmorefeasible to achieve vitrification by lowering the volumethan by increasing the cooling rate.

James H. Walton and Roy C. Judd measured the velocityof ice crystal growth and found that it is in the range of 65mm/s [7]. This means that if we want to avoid crystalli-zation in a drop placed on a cold metal surface and thathas a diameter of 0.1 mm, we need a velocity of 1/6500mm/s, which is 0.0001 second. If we cool from roomtemperature to �180 �C, this means we need to reduce200 �C at a rate of 0.1 ms or at 78 � 106 �C/min. This isactually the cooling rate that was estimated by Balds andby Bruggeller [1,5]. However, because this cooling rate isimpossible to achieve, the ability to reach vitrificationof pure water in a small drop can be achieved in relativelyslow cooling rates, which indicate that the smallvolume has an independent effect on the probability ofvitrification.

Later attempts at vitrifying pure water have been madeby a few investigators; Hawkes [8] published an experi-ment in which a drop of solid amorphous water was ob-tained, by chance, during rapid cooling. Burton and Oliver[9] obtained from steam some solid water in which x-rayanalysis did not reveal any crystalline structure. As we cansee, these achievements weremainly owing to the samples’small volume and not the velocity of cooling. A review onsupercooling of water can be found also in “Cryobiology” byMeryman published in1966 [10].

3. The first cells survival after vitrification

The most important year for cryobiology was 1938;Basile J. Luyet and Eugene L. Hodapp [11] published the firstsuccessful vitrification of sperm. Luyet began his researchwith colloids (gelatin, milk, or agar) and found that theirwater content determines the possibility or impossibility ofvitrification. In general, with 50% gelatin solutions, theyhad vitrified layers of 0.3 mm thick (by the method of im-mersion in liquid nitrogen [LN]), whereas with solutionscontaining 90% water, they could vitrify only smears of fewmicrons thick [3]. They were the first to demonstrate suc-cessful cryopreservation of frog sperm by vitrification using2 mol/L sucrose and small drops.

4. The beginning of slow freezing

In 1949, Christopher Polge, Audrey Smith, and AlanParkes [12], when trying to duplicate Luyet’s results, dis-covered by mistake the cryoprotective property of glyceroland so opened the field of slow freezing. Currently, thereare two methods for gametes cryopreservation: slowfreezing and vitrification. Slow freezing has the advantageof using low concentrations of CPs, which are associatedwith chemical toxicity and osmotic shock. Vitrification is arapid method, which reduces chilling sensitivity and crys-tallization damage caused to cells. Sherman and Lin [13]showed that mouse oocytes need 8 to 10 minutes forequilibration in a freezing solution containing 5% glycerolat 37 �C. In addition, he demonstrated that mouse oocyteswill survive supercooling to�20 �C after slow cooling at 0.6�C/min, however, oocytes that were cooled faster or tolower temperatures were damaged owing to intracellularcrystallization. In the early 1970s, two groups werecompeting on achieving the first success of slow freezing ofembryos. One group included Whittingham, Leibo, andMazur, and the other Wilmut and Polge. Whittingham hadpartially succeeded in freezing embryos to �79 �C for 30minutes using polyvinylpyrrolidone; however, theseexperiment could not be duplicated [14–16]. Both groupspublished in 1972 the first survival of mouse embryos afterslow freezing [15,16] and live offspring [15]. The techniqueincluded cooling at a slow rate in the presence of 1 mol/LDMSO, which most likely was the ingredient that enabledthis. In 1976, sheep embryos were slow frozen byWilladsenusing 1.5 mol/L DMSO and a cooling rate of 0.3 �C/min. [17].

However, the first farm animal to be born after trans-plantation of frozen and thawed embryos was a calf. Thiswas published by Wilmut and Rowson at 1973 [18]. Since

A. Arav / Theriogenology 81 (2014) 96–10298

then, dozens of species have been successfully cry-opreserved by slow freezing (for a review, see [19]).

5. The comeback of vitrification

For many years, slow freezing and not vitrification wasthe method of choice for embryo cryopreservation. Thiswas because vitrification was not achieved easily owing tothe need of high CPs concentrations and relatively highvolume samples. In 1985, the first successful vitrificationof mouse embryos using a relatively large volume samplewas done [20]. Rall and Fahy vitrifiedmouse embryos withmixture of DMSO, acetamide, and polyethylene glycol andin a relatively large volume inside a 0.25-mL strawplunged into LN. At that time, I was a veterinary student atthe University of Bologna, Italy, and I met Bill Rall, whotoldme about the exciting work he did onmouse embryos.Two years later, I started to work on cryomicoscopy ofoocytes and embryos. As in our laboratory, we used toprepare oocytes for histology evaluation by fixing themwith a small drop over a microscopic slide. I had the ideaof using the same technique for vitrification in a smalldrop, which I later named as the “minimum drop size”[6,21–24].

As noted, the probability of vitrification increases asthe volume of the sample decreases. Pure water is vitrifiedonly in very small droplets obtain from aerosols. Vitrifi-cation of thin layers (<1 mm) of viruses in water suspen-sion was achieved in rapid cooling for the purposes ofelectron microscopy [25]. The method we developed in1989 was based on this concept and called the “minimumdrop size” [21]. The volume we used for the vitrificationwas in the range of 0.07 mL (70 nL) and the concentrationof the vitrification solution (VS) was about 50% lower thanof the VS used for large-volume vitrification [22]. It wascalled “minimum drop size” because this was the minimalsize that maintained oocytes or embryos without damageowing to desiccation.

Vitrification of embryos, on the other hand, althoughinitially attempted in the late 1980s, has not been appliedclinically until recently. Vitrification is currently producingvery satisfactory outcomes bymeans of methodologies thatuse a minimal volume [26,27]. Three important factorsshould be considered.

1. Cooling rate and warming rate: A high cooling rate isachieved with LN or LN slush and a warm water bath forwarming. When using LN, the sample is plunged into LN,resulting in cooling rates of hundreds to tens of thousandsdegrees Celsius per minute, depending on the container,volume, thermal conductivity, solution composition, andso on [28]. To achieve LN slush, the LN needs to be cooledclose to its freezing point (�210 �C), for example, byapplying a negative pressure above LN [29]. When LNslush is formed, the cooling rate is dramatically increased.The cooling rate is especially enhanced in the first stage ofcooling when cooling down from room temperature to0 �C. The cooling rate is enhanced two to six times morethanplunging to LN (�196 �C)with 0.25-mL straws or anyother device, such as open-pulled straws or electron

microscope grids [23]. It was shown for oocytes andembryos that increasing the cooling rate improves sur-vival rates by up to 37% [19]. Recently, it was found thatwarming rate is an important variable for successfulvitrification of mouse oocytes [30].

2. Viscosity of the medium in which the embryos are sus-pended or the glass transition coeffieciencyof the solutionat low temperatures is defined by the concentration andbehavior of various CPs and other additives during vitri-fication. The higher the concentration of CPs, the higherthe glass transition temperature (Tg), thus lowering thechance of ice nucleation and crystallization. Different CPsand other additives have different toxicity, penetrationrate, and Tg. Viscosity of the medium in which the em-bryos are suspended is defined by the concentration andbehavior of various CPs and other additives during vitri-fication. The combination of different CPs is often used toincrease viscosity, increase Tg, and reduce the level oftoxicity. In the cattle industry, to avoid handling of thepost warmed embryos and allow direct transfer, ethyleneglycol (which was found by Voelkel and Hu [31]) is oftenused as the permeating CP owing to its high penetrationrate [32].

3. Volume: The smaller the volume, the higher the proba-bility of vitrification [13,21,23,24]. Smaller volumesallow better heat transfer, thus facilitating greatercooling rates. Furthermore, small volume has an inde-pendent effect on the probability of nucleation as wasdiscovered 200 years ago by Guy-Lassac. Many tech-niques have been developed to reduce sample volumewith an explosion of methods appearing in the litera-ture during the last decade.

These techniques can generally be divided into twocategories: surface techniques and tubing techniques [19].The surface techniques include electron microscope grid[33], minimum drop size technique [21], Cryotop [34,35],Cryoloop [36,37], Hemi-straw [38], solid surface [39],nylon mesh [40], Cryoleaf [41], direct cover vitrification[42], fiber plug [43], vitrification spatula [44], Cryo-E [45],plastic blade [46], and Vitri-Inga [47]. To the tubing tech-niques belongs the plastic straw [20], open-pulled straw[48,49], closed pulled straw [50], flexipet-denudingpipette [51], superfine open-pulled straw [52], CryoTip[53], pipette tip [54], high-security vitrification device[55], sealed pulled straw [56], Cryopette [57], Rapid-i [58],and JY Straw (RC Chian, personal communication). Each ofthese two groups has its specific advantages. In the surfacemethods, the size of the drop (0.1 mL) can be controlled, ahigh cooling rate is achieved because these systems areopen, and high warming rates are achieved by directexposure to the warming solution. The tubing systemshave the advantage of achieving high cooling rates inclosed systems, thus making them safer and easier tohandle. Decreasing the vitrified volume and increasing thecooling rate allow amoderate decrease in CP concentrationso as to minimize its toxic and osmotic hazardous ef-fects [22,56]. Combining these three factors can result inthe following general equation for the probability ofvitrification:

Probability of vitrification ¼ cooling and warming rate � viscocityvolume

A. Arav / Theriogenology 81 (2014) 96–102 99

6. Oocyte vitrification

Oocytes are very different from sperm or embryos withrespect to cryopreservation. The volume of the mammalianoocyte is in the range of three to four orders of magnitudelarger than that of the spermatozoa, thus substantiallydecreasing the surface-to-volume ratio. However, this isnot the reason why the oocytes are sensitive to low tem-peratures and slow freezing. Mature oocytes are very sen-sitive to slow freezing; however, after fertilization thevolume of the oocytes remains the same but their sensi-tivity is reduced to minimum and in fact this is the beststage for freezing human oocytes [59]. The reason for manyoocytes susceptibility to low temperatures is owing to theirchilling sensitivity which occurs at different cellular levels:the zona pellucida, plasma membrane, meiotic spindles,cytoskeleton, and so on.

The plasmamembrane of oocytes at themetaphase (M) IIstage has a low permeability coefficient, thus making themovement of CPs and water slower [60,61]. In addition, thefreeze–thaw process causes premature cortical granuleexocytosis, leading to zona pellucida hardening and makingsperm penetration and fertilization impossible [62,63], aprocess that can be overcome by the use of intracytoplasmicsperm injection or subzonal sperm insertion. Oocytes alsohave high cytoplasmic lipid content, which increases chillingsensitivity [60]. They have less submembranous actin mi-crotubules [64], making their membrane less robust. Cryo-preservation can cause cytoskeleton disorganization, andchromosome and DNA abnormalities [65]. The meioticspindle, which has been formed by the MII stage, is verysensitive to chilling andmay be compromised as well [66]. Itdoes, however, tend to recover to some extent after thawingor warming and IVC, a recovery that is faster after vitrifica-tion than after slow freezing [66]. Oocytes are also moresusceptible to damaging effects of reactive oxygen species[67]. Many of these parameters change after fertilization,making embryos less sensitive to chilling and easier tocryopreserve [64,67,68].

Fig. 2. Dry oocytes on a Cryotop carrier after vitrification and drying and on the rig

Vitrification requires the presence of high concentra-tions of CPs. It is therefore important to minimize thedamage caused to cells by the osmotic stress or chemicaltoxicity. No ideal CP that meets the requirements of alldifferent species and developmental embryonic stages hasbeen found; vitrification studies should therefore be pre-ceded by osmotic and cytotoxic studies. The presence of CPin the VS decreases the probability of intracellular crystal-lization which is considered to cause most damage whenvery rapid cooling takes place, but the high concentrationof the CP required is toxic and causes osmotic injury to theoocytes even without cooling.

Different methods have been used to reduce this ‘so-lution effect’: (i) short time of exposure to CPs [67–69], (ii)use of low toxicity CPs [31,70] or mixtures of them [71],(iii) addition of nonpermeating CPs [72], (iv) reduction theCP concentration [70], and (v) exposure at low tempera-tures [70]. Of these methods, the use of nonpermeatingCPs is very useful either because the shrinkage of theoocyte and consequently the amount of water inside thecell that may crystallize during rapid cooling and warmingis lower [70] or because of the reduction of the amount ofthe CP that penetrates the cell thus reducing the possibletoxic effect [73]. In addition, the carbohydrates used as anonpermeating CPs have a stabilizing effect on mem-branes [74]. In the study reported by us [75], trehalose wasless harmful than sucrose. Determination of the BoyleVan’t Hoff relationship for both sucrose and trehaloseproduced the same regression line, so it is possible thatthis beneficial effect could be a consequence of its inter-action with the membrane polar lipid groups [74]. Only 10minutes of exposure is required for equilibration in pro-pylene glycol and DMSO solutions or mixtures of them.The membrane is very permeable to both of them. Theresults of the vitrification provide evidence that propyleneglycol can be used successfully. Indeed, the IVF rate of theoocytes vitrified in a solution containing 40% (w/v) pro-pylene glycol was 37% and is not different from the resultsobtained using a slow freezing protocol [76]. The viability

ht after rehydration and staining with live/dead fluorescent stains (SYTO/PI).

A. Arav / Theriogenology 81 (2014) 96–102100

of vitrified mouse embryos was successfully increased byreducing the concentration of the CP [56]. However,concentrated solutions of permeating CP are required forsuccessful cryopreservation of oocytes when rapid coolingand warming rates are used. In earlier reports on imma-ture pig oocytes, we showed that when lower concentra-tions of CPs are used, despite apparent vitrification,membrane destruction was unavoidable [77]. In 1990,Kasai. et al. [78] were the first to describe the use ofethylene glycol for mouse embryo vitrification. Today, themost common solutions are based on a mixture of DMSOand ethylene glycol [53].

7. Small volume is the solution for most problemsoccur during vitrification

There are three major problems associated with vitri-fication: (1) crystallization (during cooling), (2) devitrifi-cation (crystallization during storage or during warming),and (3) fractures of the glassy solution, which can causedevitrification owing to release of energy by the fracture.Surprisingly, at 1 mL, fractures appeared only when theconcentration of the VS is high (100% VS ¼ 38% ethyleneglycol, 0.5 mol/L Trehalose, and 4% bovine serum albuminin TCM medium), but not at lower volumes [24]. Thismeans that the probability of fractures increases with theincreasing of Tg or the viscosity of VS. At the low concen-tration of VS (50% VS), fractures were observed only atvery high cooling rate. We suggest here a simple expla-nation of this phenomenon, based on the followingequations:

Probability of fracturing ¼ CR and WR � m � V

Because the probability of vitrification is ¼ CR and WR � m � 1=V

1. Increasing the cooling rate (CR) increases the probabilityof vitrification; however, it also increases the probabilityof fractures.

2. Increasing the viscosity (m) increases the probability ofvitrification because Tg increases [28]; therefore, it in-creases the probability of fractions.

3. The only parameter that increases the probability ofvitrification and at the same time decrease the proba-bility of fractures is reducing the volume (V) to the valueof the minimum drop size.

The reason for the increasing probability of fractures inhigh concentrations of VS is thought to be related to theglass transition temperature (Tg). We know that fracturescan form only at temperatures below that at which theliquid turns into glass (Tg) and above the LN temperature(�196 �C). We also know that a solution with a higher CPsconcentration have a higher Tg. Therefore, if the tempera-ture gradient increases, as in the case of higher Tg, then the

probability of fracturing also increases. Finally, the resultsof vitrification of bovine oocytes at the MII or germinalvesicle stage, with a concentration of 75% VS, have beenreported [79]. We achieved 72% and 38% cleavage andblastocyst rates formation, respectively, for the vitrified MIIoocytes and 27% and 14% cleavage and blastocyst ratesformation, respectively, for oocytes vitrified at the germinalvesicle stage. We conclude that the new vitrification pro-cedure, which features small volumes, direct contact withsupercooled LN, and low concentrations of VS, reduceschilling injury and provides a high probability of vitrifica-tion in the absence of glass fractures.

8. Drying bovine oocytes after vitrification: Atechnological breakthrough

Storage of cryopreserved oocytes in LN is very de-manding in terms of maintenance, storage space, equip-ment, and costs. We, therefore, sought an alternativemethod for gamete preservation: vitrification followed bydrying. Storage is done in a dry state. We perform acomparative study between freeze-drying and vitrificationdrying.

Three experiments in parallel compared various coolingmethods on the recovery and survival after freeze orvitrification drying of in vitro-matured MII bovine oocytes(n ¼ 68). Ten oocytes were cryopreserved with slowfreezing (using the MTG-1314 device, IMT Israel) at acooling rate of 4 �C/min (group A); 24 oocytes with rapidfreezing (using MTG 516 device IMT, Israel), at a coolingrate of 150 �C/min (group B); and 34with vitrification usingminimum drop size in IMT-4 solution (trehalose basedsolution) at a cooling rate of greater than 20,000 �C/min.

The lyophilization process was carried out with the VirTiswizard for 24 hours (shelf temperature was set to �55 �Cand vacuum was 10 mTorr). The rehydration process tookplace at room temperature using equilibrated TCM199supplemented with 0.5 mol/L trehalose. Oocyte survivalwas assessed with live/dead fluorescent stains (SYTO/PI).

For group A, 70% of the oocytes were recovered afterrehydration but only one in seven was stained as viable(14%). For group B, 71% were recovered and 10 of 17 werestained as viable (59%). For group C, 88% were recoveredand 23 of the 30 stained as viable (77%; P < .05; Fig. 2).

In conclusion, lyophilization of oocytes is a ground-breaking innovation for gamete bio-banking; vitrification isconfirmed as an essential method not only for preservationin LN, but also in the dry state.

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