The first live births following frozen-thawed embryo transfer were reported in 1984 and 1985 by groups in Australia, the Netherlands and the United Kingdom. Since that time, the original protocols have been modified and simplified such that cryopreservation with successful survival of sperm, oocytes and embryos is now an essential component of every routine IVF program. Pregnancy and live birth rates after frozen embryo transfer contribute significantly to cumulative conception rates after fresh transfer. In recent years, traditional methods of freezing and thawing have been increasingly replaced by protocols for vitrification/warming. For both slow freezing and vitrification, an understanding of the basic principles of cryobiology involved is essential to ensure that the methodology is correctly and successfully applied, in order to minimize cell damage during the processes of freezing/vitrification and thawing/warming.
The first live births following frozen-thawed embryo transfer were reported in 1984 and 1985 by groups in Australia, the Netherlands and the United Kingdom. Since that time, the original protocols have been modified and simplified such that cryopreservation with successful survival of sperm, oocytes and embryos is now an essential component of every routine IVF program. Pregnancy and live birth rates after frozen embryo transfer contribute significantly to cumulative conception rates after fresh transfer. In recent years, traditional methods of freezing and thawing have been increasingly replaced by protocols for vitrification/warming. For both slow freezing and vitrification, an understanding of the basic principles of cryobiology involved is essential to ensure that the methodology is correctly and successfully applied, in order to minimize cell damage during the processes of freezing/vitrification and thawing/warming. There are two major classes of physical stresses that cells are exposed to during cryopreservation:
1. Direct effects of reduced temperature
2. Physical changes associated with ice formation.
‘Chilling injury’ is the damage to cell structure and function caused by cooling; the type and extent of damage, associated with modifications in membrane permeability and changes in the cytoskeletal structure, differ between different types of mammalian cells (reviewed by Watson & Morris, 1987 and Fahy & Wowk, 2015). The effect is species-specific and well documented for spermatozoa (cattle, pig), oocytes and embryos (pig). Oocytes are particularly susceptible to damage, and sublethal injuries can also occur during cryopreservation of oocytes and ovarian and testicular tissue, particularly in relation to breakdown of the cellular spindle apparatus.
Physical Changes Associated With Ice Formation
Temperature changes observed during the freezing of an aqueous solution are illustrated in Figure 12.1. Water and aqueous solutions have a strong tendency to cool below their melting point before nucleation of ice occurs: this phenomenon is referred to as supercooling, or more correctly undercooling. For example, whilst 0°C is the melting point of ice, the temperature of water may be reduced significantly below 0°C before ice formation occurs, and in carefully controlled conditions water may be cooled to approximately –40°C before ice nucleation becomes inevitable. The homogeneous nucleation temperature (Th) is the lowest temperature to which small samples can be cooled without ice formation; Th decreases with increasing solute concentration.
Following ice nucleation and initial crystal growth, the temperature rises to its melting point and remains relatively constant at that temperature during the subsequent phase change to ice (‘latent heat plateau’), when the temperature then changes more rapidly to the environment temperature.
The tendency of a system to supercool is related to a number of factors including temperature, rate of cooling, volume, exclusion of atmospheric ice nuclei and purity of particulates. In cryopreservation of cells and tissues in IVF systems, there is thus a strong tendency for supercooling to occur. In order to avoid the damaging effects of supercooling on cells and in particular embryos (see below), slow-freezing protocols initiate ice formation in a controlled manner. This is commonly referred to as ‘seeding’ – although, strictly speaking, this term refers to the introduction of a crystal to an under-cooled solution. ‘Nucleation’ is the initiation of ice other than by seeding, and this is the process generally practiced in slow-freezing protocols for IVF.
Supercooling and Cell Survival
Controlled ice formation during freezing is recognized to be a key factor in determining the viability of embryos following freezing and thawing (see Whittingham, 1977). In a carefully controlled series of experiments, samples which were nucleated below –9°C had a low viability, whilst nucleation at higher subzero temperatures of –5°C to –7.5°C resulted in much higher viability (Figure 12.2).
An analysis of the spontaneous nucleation behavior of straws (Figure 12.3) clearly demonstrates that if nucleation is not controlled, a poor recovery of embryos would be expected.
Figure 12.3 The measured nucleation temperatures within 0.25-mL straws cooling at 0.3°C/min.
The physical basis of this injury is clear from examining thermal histories of supercooled straws (Figure 12.4). The differences between laboratories that achieve good results and those that are less successful can often be attributed to the practical step of ice nucleation, or ‘seeding.’ Straws can be frozen horizontally or vertically – this has no effect on viability or ease of ice nucleation. Embryos sink in the cryoprotective additive and will always be found at the wall of the straw when frozen horizontally, or at the bottom of the column of liquid when frozen vertically. Following thermal equilibration (‘holding’) at the nucleation temperature (–7°C), ice formation is initiated by touching the outside of the straw or ampule with a liquid nitrogen cooled spatula, forceps, cotton bud etc., at the level of the meniscus (seeding at both ends of a horizontal straw has also been advocated). This causes a local cold spot on the vessel wall, which leads to ice nucleation. Immediately following ice nucleation, the temperature will rise rapidly (cf. Figure 12.1), and the ice front will propagate through the sample. Following ice formation, the temperature returns at a rate of 2.5°C/min to –7°C. Cellular dehydration then occurs during subsequent slow cooling.
Figure 12.4 Measured temperatures within straws. During conventional cryopreservation, the straws are held at a temperature of –7°C and nucleated; the resultant rise in temperature following ice nucleation is small. In the absence of induced nucleation the straws may reach very low temperatures before spontaneous nucleation occurs. A large rise in temperature to the melting point of the suspending medium then occurs followed by a rapid reduction in temperature; this will inevitably result in intracellular ice formation within embryos or oocytes.
By contrast, in a straw supercooled to –15°C, spontaneous ice formation again results in a temperature rise followed by a rapid rate of cooling at 10°C/min to –5°C. The combination of a rapid rate of cooling and a large reduction in temperature does not allow the cell to dehydrate, and lethal intracellular ice formation is then inevitable (Table 12.1). This has been observed by direct cryomicroscopy.
|System||Type/cause of damage|
|All||Intracellular ice formation, extracellular ice formation, apoptosis, toxicity, calcium imbalance, free radicals, ATP levels, general metabolism, fertilization failure, cleavage failure, intracellular pH, parthenogenetic activation, cleavage|
|Membrane||Rupture, leakage, fusion, microvilli, phase transition|
|Chromosomes||Loss/gain, polyspermy, polygny (failure to extrude polar body), tetraploidy|
|DNA||Apoptosis, fusion, rearrangements|
|Cytoskeleton||Microtubules dissolve, actin|
|Proteins/enzymes||Dehydration, loss of function|
|Ultrastructure||Microvilli, mitochondria, vesicles, cortical granules, zona pellucida|
|Zona pellucida||Hardening, fracture|
Ice crystallization in an aqueous solution effectively removes some water from solution. The remaining aqueous phase becomes more concentrated, and a two-phase system of ice and concentrated solution coexists. As the temperature is reduced, more ice forms and the residual unfrozen phase becomes increasingly concentrated. For example, in glycerol and water a two-phase system occurs at all temperatures to –45°C. At –45°C the nonfrozen phase solidifies, with a glycerol concentration of 64% w/v; this is the eutectic temperature. In dilute aqueous solutions such as culture media, there is a dramatic increase in ionic composition following ice formation and by –10°C the salt concentration reaches c. 3 molal – not surprisingly, this is lethal to cells. Cryoprotective additives reduce cellular damage during freezing and thawing by simply increasing the volume of the residual unfrozen phase. This reduces the ionic composition of the solution at any subzero temperature. It must also be noted that all other physical parameters of the solution change following the formation of ice, including gas content, viscosity and pH.
The addition of cryoprotectant agents (CPAs) is thought to protect cells by stabilizing intracellular proteins, reducing or eliminating lethal intracellular ice formation, and by moderating the impact of concentrated intra- and extracellular electrolytes. The first successful use of a cryoprotectant was in 1949, when Polge et al. used glycerol to freeze semen; glycerol is still commonly used as a cryoprotectant today. All cryoprotectants are low molecular weight compounds, are completely miscible with water, easily permeate cell membranes and depress the freezing point of water to very low subzero temperatures, e.g., <−60º C. Some (but not all) are nontoxic when the cells are exposed to them at room temperature for limited times, even at high concentrations. All are hyperosmotic, and they can be divided into two groups:
1. Permeating: glycerol, ethylene glycol, 1,2-propanediol (PROH) and dimethylsulfoxide (DMSO) penetrate the cell membrane, although more slowly than water. Inside the cell, they stabilize intracellular proteins, reduce the temperature at which intracellular ice forms and minimize osmotic damage due to electrolyte concentration effects. Glycerol penetrates tissue less readily than DMSO, PROH and EG. These cryoprotectants have high water solubility, rapid tissue penetration and induce less osmotic damage at high concentrations.
2. Non-permeating: a variety of sugars, polymers and amphipathic compounds have been used as non-permeating cryoprotectants. Raffinose and lactose decrease the percentage of unfrozen water and/or decrease salt concentrations. Glycine, proline and trehalose are amphipathic compounds that interact with membrane lipids and proteins to alter phase transitions and hydration status.
Cryoprotectants should always be used in combination with nonpermeating osmolytes such as sucrose or mannitol. These partially dehydrate the cells and act as osmotic buffers to protect against cell swelling during the addition/removal of cryoprotectants. Egg yolk has been added to cryopreservation medium used to preserve animal sperm, since the low-density lipoprotein fraction of egg yolk is thought to protect against cold shock. However, this is not recommended for freezing human cells, as it introduces batch-to-batch variation and the possibility of bacterial contamination.
The pioneering studies on mammalian embryo cryopreservation used either glycerol or DMSO as cryoprotectants, but these were subsequently superseded by the protocol of Lasalle et al. (1985), which uses PROH. PROH is considered to have a higher permeability to human embryos than either glycerol or DMSO, and is less toxic than DMSO. The real toxicity of cryoprotectants in cells is largely unknown; Stanic et al. (2000) reported that most of the decrease in sperm motility during cryopreservation was due to exposure to cryoprotectants rather than to the freezing process.
Permeability to CPA
Studies investigating the permeability coefficients of oocytes and zygotes demonstrate that the oocyte is much less permeable to CPAs than the zygote, and the activation energy of oocyte permeability is higher than that of the zygote. This means that as temperature is lowered, a CPA will penetrate an oocyte far more slowly than it will penetrate a zygote, with resulting effects on the rates of water loss from the cells. The permeability of oocytes has also been found to vary between individuals, with differences ranging from 3- to 6-fold, i.e., cohorts of oocytes from different women may differ in their permeability to CPAs. This feature has also been demonstrated in mice and in cattle, suggesting that properties of cell membranes may have a genetic basis (see Leibo & Pool, 2011 for review).
The ratio of surface area to volume in a cell influences permeability characteristics, and this will determine the optimum cooling rate for that cell. In general, the larger the cell, the slower it must be cooled to survive freezing. For example, a human oocyte, the largest cell in the body, is a sphere measuring 120 µm in diameter with a large volume, whereas the flattened paddle-shaped sperm cell has a much higher surface area to volume ratio. Consequently, when exposed to CPAs, spermatozoa reach osmotic equilibrium much faster than oocytes, and optimum cooling rates for spermatozoa are much higher than those for oocytes and embryos. However, although oocytes and zygotes have nearly identical surface area/volume ratios, the oocyte is less permeable to CPAs than the zygote. Germinal vesicle stage oocytes differ from metaphase II oocytes in their activation energy of water permeability, so that decreasing temperature has a greater effect on the osmotic response of a GV oocyte than on a metaphase II oocyte, although both are equally susceptible to chilling injury.
At temperatures near 0°C, abrupt changes in volume can immediately damage cells and also make them more susceptible to stress during subsequent cooling or thawing procedures; therefore, extreme fluctuations in cell volume must be avoided during CPA equilibration. The duration of exposure to these potentially toxic chemicals should also be minimized: CPA toxicity can be reduced by lowering the temperature of exposure, but this would require a longer exposure time.
When large cells such as oocytes are frozen in molar concentrations of CPAs, their survival is strongly dependent on cooling rate, and the specific optimum cooling rate depends on both the type and the concentration of CPA. Cell survival is equally dependent on warming rate, and the optimum warming rate depends on both the CPA and its concentration, as well as on the cooling rate that preceded it. Cryobiology studies have shown that different types of cell have different optimal cooling rates, even when frozen in the same solution. This is especially relevant to the cryopreservation of tissues such as ovarian cortex that are made up of many different types of cell, each with its own characteristic size, shape and permeability properties. Cooling and warming conditions that are optimal for one cell type may be harmful to others; warming rate rather than cooling rate is more important in determining ultimate viability of the cell (Jin et al., 2014).
As mentioned above, when aqueous solutions are frozen, water is removed in the form of ice, causing the cells to become increasingly concentrated as the temperature falls. The reverse occurs during thawing. Cells in suspension are not punctured by ice crystals (see Figure 12.5), nor are they mechanically damaged by ice. Cells partition into the unfrozen fraction and are exposed to increasing hypertonic solutions. Varying amounts of water may be removed osmotically from the cell, dependent on the rate of cooling. At ‘slow’ rates of cooling, cells may remain essentially in equilibrium with the external solution. As the rate of cooling is increased, there is less time for water to move from the cell, which becomes increasingly supercooled until eventually intracellular ice is formed. An optimum rate of cooling results from the balance of these two phenomena. At rates of cooling slower than the optimum, cell death is due to long periods of exposure to hypertonic conditions. At rates of cooling faster than the optimum, cell death is associated with intracellular ice formation, which is inevitably lethal. The actual value of the optimum rate is determined by a number of biophysical factors, including cell volume and surface area, permeability to water, Arrhenius activation energy, and type and concentration of cryoprotective additives. As the cells are frozen, they must respond osmotically to large changes in extracellular fluid concentrations: the efflux of water during slow freezing (<2°C/min) causes oocytes to undergo osmotic dehydration, with resulting contraction.
Although cells in suspension can tolerate exposure to very high concentrations of CPAs, whether or not they survive the freeze–thaw process depends on how the CPA solution is removed. When frozen cells are warmed rapidly, the melting process is equivalent to rapid dilution of the CPA that was concentrated during the freezing process. As the extracellular milieu begins to melt, the rapid influx of water into the cells can cause osmotic shock at subzero temperatures. Sensitivity to osmotic shock is therefore a function of the cell’s permeability to water and solutes. This shock can be reduced by using a nonpermeating substance such as sucrose as an osmotic buffer. Tissues are even more sensitive to osmotic effects than are cell suspensions, because cells located in the interior of a piece of tissue can respond osmotically only when the neighboring cells have also responded.
In summary, cells that are cooled too slowly can be damaged by changes in solution composition and osmotic effects of shrinkage; cells that are cooled too quickly can be damaged by ice formation. The formation of ice during either cooling or warming depends upon how much time is available for ice nucleation and growth: optimal high cooling and warming rates can ‘outrun’ the kinetics of ice formation, minimizing the chance of injury. CPAs change the optimal cooling rate in a manner that is dependent on the cell type.
Two basic types of cryopreservation are used for cells and tissues: slow freezing and vitrification.
The procedures used for slow freezing of oocytes, embryos and ovarian cortex are generally quite similar. The cells or tissues are equilibrated in an aqueous solution containing an optimal concentration (1.0–1.5 M) of cryoprotectant and sucrose (0.1 M) and frozen in straws or ampules. Following CPA exposure, the temperature is slowly lowered, and ice crystal growth is initiated in the solution (‘seeding’). The ampules or straws are seeded at –6.5 to –7°C, cooled slowly at 0.3–0.5°C/min to approximately –40°C, then quickly cooled to –150°C before final transfer into liquid nitrogen for storage. Embryos and spermatozoa do not deteriorate when stored even for decades in liquid nitrogen.
Freezing: Water molecules are reorganized into ice crystals.
Thawing: Melting of ice.
Nucleation: A small number of ions, atoms or molecules arrange into a crystalline pattern, forming a site for crystal growth by addition of further particles.
Homogeneous: does not involve foreign particles or atoms, takes place away from surfaces. Homogeneous nucleation temperature (Th) is the lowest temperature to which small samples can be cooled without ice formation.
Heterogeneous: a different substance, such as dust particle or wall of the container, acts as the center for crystal formation; takes place at nucleation sites such as interfaces, surface of impurities, etc. Heterogeneous nucleators mimic the structure of ice, with a lower surface energy.
Cryoprotectant agent (CPA): An agent that reduces or prevents chilling/freezing injury.
Critical cooling rate (CCR): The rate above which ice formation is not observed.
Critical warming rate (CWR): Warming rate that suppresses ice formation during warming.
CCR and CWR depend on total solute content and the chemical nature of the solute.
Vitrification: Transition of a solution from liquid to glass phase.
Glass transition temperature (TG): The temperature at which vitrification takes place on cooling.
Devitrification: Formation of ice during warming after previous devitrification.
Rewarming: Warming of a previously cryopreserved system, whether frozen or vitrified.
Recrystallization: Transfer of water molecules from small ice crystals to larger crystals, which cause greater damage.
Vitrification combines the use of concentrated cryoprotectant solutions with ultra-rapid cooling in order to avoid the formation of ice. The high osmotic pressure of concentrated CPAs causes rapid dehydration, and the contracted samples are placed into a vitrification device that can be immediately plunged into liquid nitrogen. Samples reach very low temperatures (−196ºC) in a state that has the molecular structure of a viscous liquid without crystals, forming an amorphous glassy solid. The transition from liquid to glass phase is a kinetic phenomenon: increased viscosity delays thermodynamic intermolecular rearrangements and ‘locks in’ a non-equilibrium thermodynamic state. The goal of vitrification is to completely eliminate ice formation in the medium containing the sample throughout all stages of cooling, storage and warming.
Since rapid cooling is effected via direct contact with liquid nitrogen, programmable freezing machines are not needed. There is an inverse correlation between the cryoprotectant concentrations and cooling rates required, and successful vitrification is based on applying extremely high cooling rates in combination with very high concentrations of cryoprotectant or cryoprotectant mixtures. Cooling is too rapid for ice to form or grow appreciably, and the solute concentration remains constant during cooling.
A balance must be achieved between the lowest level of least hazardous CPA and maximal cooling rate: higher CPA concentrations allow lower cooling rates, and vice versa. Since highly concentrated CPAs may cause toxic and osmotic damage, a preferred strategy is to use the highest possible cooling and warming rates, and then use the lowest concentration of CPA that will prevent ice formation.
Adequate dehydration and permeation of cells is essential, and therefore exposure time is important.
In order to avoid the formation of both intracellular and extracellular ice, the initial cooling rate must exceed the critical cooling rate (CCR) of the solution.
Warming rates are critical to survival: total development of ice is much more rapid during warming, and warming rates required to avoid significant devitrification are far higher than cooling rates required to achieve vitrification.
The cooling rate is affected by several parameters, and different methods have been employed in order to find an effective and practical solution, by varying cryoprotectant solutions, combinations, exposure times and temperature. The addition of sugars (sucrose, trehalose, fructose, sorbitol saccharose, raffinose) reduces the concentration of CPA required; the permeability of mixtures is higher than that of individual components, and different combinations of CPA have also been tried. Vitrification solutions are developed with the lowest possible concentration of CPA compatible with achieving glass formation. Reducing volume to a minimum reduces potential toxicity and osmotic damage, and methods have been devised to reduce the volume of CPA down to between 0.1 µL and 2 µL. Reducing the drop size or increasing the number of embryos per drop risks diluting the CPA with medium carried over from the culture drop, and this could allow the sample to freeze, with lethal results.
Low concentrations of permeating + nonpermeating cryoprotectants (1.5/0.1–0.3), PROH or DMSO/sucrose.
Requires controlled rate freezing machine, 2°C/min, then 0.3°C/min.
Nucleation (seeding) and transfer to liquid nitrogen is critical.
Well-established protocols and techniques.
Avoids formation of ice that can damage membranes.
Rapid method, simple equipment.
No specialized controlled rate freezer required.
Application in clinical practice has been slow, due to concerns about toxicity of high CPA concentrations (up to 6 M).
Use of very low volumes reduces toxicity risk.
Requires critical process control: zero tolerance to any changes/fluctuations.
Samples must be handled and moved very rapidly.
Avoid accidental warming: stored samples are very fragile and could be susceptible to mini-devitrification cycles during routine dewar use in a busy IVF laboratory.
The thawing rate must also be rapid, in order to prevent devitrification and ice crystal formation during the transition state. The samples are kept in air (room temperature) for 1–3 seconds; for open systems, the sample is then immersed in dilution medium at 37°C, and for closed systems, the carrier is immersed in a 37°C water bath before transferring the sample to the dilution medium. The CPA is diluted in several steps, in order to counterbalance osmotic effects as the CPA leaves the cell.
Although the methodology and protocols that accompany vitrification systems for IVF appear deceptively simple, the principles that ensure viability after warming are a highly complex combination of thermodynamics and cell and molecular biology. Numerous kinetic variables surrounding the physics of aqueous solutions and biological survival can jeopardize success. (See Fahy & Wowk, 2015 for detailed review.) Different types of vitrification systems are available which involve different principles:
1. Unstable vitrification: solute concentrations that are too low to prevent homogeneous ice nucleation will nucleate at large numbers of points in the solution, i.e., >2500/µm3. Cooling requires thousands of degrees per minute or more, to prevent high ice nucleation and growth rates of homogeneous nucleation. Cells can survive if warming is sufficiently rapid.
2. Metastable vitrification: solute concentrations are high enough to avoid homogeneous nucleation, but low enough to thermodynamically favor ice formation. Cells can be supercooled to glass transition without necessarily nucleating ice, but ice can be formed in discrete locations where heterogeneous nucleators are present. Cooling rates on the order of 10ºC/minute are possible.
3. Stable/equilibrium vitrification: solute concentrations are so high that ice cannot exist in the solution; arbitrarily low cooling rates are possible, but complete stability requires high concentrations of solute.
4. Kinetic vitrification: ultrafast cooling (>10,000ºC/min) usually requires very small sample volumes but lower CPA concentrations.
The basic principles regarding CPA concentrations/cooling rates outlined above were brought into question by Jin & Mazur (2015), who demonstrated >90% survival of mouse oocytes and embryos when cooled at much slower rates, in solutions containing one-third of the ‘standard’ solute concentrations, provided that they are warmed ultra-rapidly (107ºC/min) using a laser pulse. They suggest that survival of a cell after vitrification is highly dependent on its dehydration, due to the fact that the rate of recrystallization of intracellular ice on warming is highly sensitive to its residual intracellular water content after vitrification: i.e., the osmotic withdrawal of a large fraction of intracellular water prior to cooling is the most important feature during vitrification, provided that warming is ultra-rapid.
Vitrification instead of slow freezing is now routinely used for human oocytes and embryos. However, questions remain about potential external contamination, as well as the long-term stability of the ‘glassy state’ of the vitrified cells, which are prone to fracture; this may be a hazard under normal working conditions in the IVF laboratory with routine access to storage tanks.
At least 30 different carrier tools have been described in published literature, and at least 15 versions are commercially available (see Vajta et al., 2015 for review).
1. ‘Fully open’ systems allow direct contact between the sample and liquid nitrogen, so that both cooling and warming rates can be extremely high. These types of open tools carry potential contamination risks. Examples include Open-pulled straw (OPS), Cryotop, Cryolock, Cryoleaf, Vitri-Anga and Cryoloop.
2. Open cooling and closed storage: after cooling, the carrier tool is inserted into a precooled sterile container that is resistant to extreme changes in temperature and then sealed (Cryotop SC).
3. Semiclosed: vitrification takes place on the surface of a metal block (Cryohook) or a container straw (Rapid I) that is partially submerged in liquid nitrogen. Samples are exposed to nitrogen vapor, with the risk of vapor-mediated contamination.
4. Closed thin-walled narrow capillaries: the device is heat sealed before cooling, and opened only after warming (Cryotip, Cryopette). The devices are warmed in a water bath, then cut to allow contents to be expelled into the medium. The surface of the straw may be contaminated either from the liquid nitrogen or from the water bath. Cooling and warming rates are slower than those obtained with open devices, and CPA dilution after warming is delayed.
5. Carrier tools are sealed into a container that separates them from liquid nitrogen during cooling, storage and warming (OPS high security, Vitrisafe); these offer the highest protection, but cooling rates may be seriously compromised.
A novel device (KrioBlastTM) has recently been introduced that provides a platform for hyperfast cooling (kinetic vitrification) based on hyperfast spray cooling. The system provides cooling rates of 100,000–600,000ºC/min and can be used for sample volumes up to 4000 µl using 15% glycerol as CPA, thus eliminating the need for more toxic agents. Initial trials with human pluripotent stem cells and spermatozoa show promising results (Katkov et al., 2018).
From Elder et al. (2015)
1. Pre-equilibrate all media to the correct temperature prior to dispensing, and invert the vials immediately before use to make sure that the solutions are fully mixed.
2. Label each vitrification device fully prior to use. Remember that timing is crucial to effective vitrification and warming, and make sure that everything is ‘ready to go’ prior to starting the procedure.
3. Prepare a liquid nitrogen (LN2) bath and place it next to the flowhood, ready for plunging the loaded vitrification devices. The vessel should be a properly functioning container dedicated for the purpose of holding LN2. Portable insulated containers (‘eskies’) used for media transport/deliveries are not designed for use as LN2 baths, and using them for this purpose is highly inadvisable. Any leak of LN2 could have disastrous consequences. Place the LN2 bath on a flat secure surface; do not use a chair or stool!
4. Carry out all vitrification/warming procedures in an area that will remain free of distraction throughout.
5. Adhere to the correct timings strictly, using a timer. Using two separate timers can prevent the loss of vital seconds in resetting a single timer. It is also helpful to have a second embryologist to assist with the timings, etc.
6. If a straw sealer is used, make sure that it is switched on and ready to use before starting the vitrification process.
7. Some media companies recommend using 1- to 2-mL solution volumes for each vitrification/warming event, but 100- to 200-µl droplets are equally effective, provided that the oocyte/embryo is fully equilibrated in each solution. Larger volumes for warming have the advantage that the vitrification device can be easily submerged into a large volume, making removal of the oocyte/embryo easier. Using larger volumes of the initial equilibration solutions also means that they can be warmed for up to 30 minutes without major pH or osmolality disturbance.
8. Ensure that there are no air bubbles on the surface of the initial warming solutions, as embryos tend to adhere to bubbles; this will hinder full submersion into the solution and also affect the precise timing of the warming process.
9. The speed of warming (at least 20,000°C/min) is more important in avoiding lysis than is the cooling speed. If warming is too slow, the intracellular CPA concentration is too low to prevent ice from re-crystallizing, and the supercooled liquid forms lethal ice crystals.
10. Embryo survival may not be immediately obvious after warming, and survival/morphology is routinely assessed after a minimum period of 2 hours in culture. This is particularly important in the case of blastocysts, which may appear collapsed immediately after warming but will re-expand within 2 hours when cultured under optimal conditions.
During the early 1990s the transmission of hepatitis B virus between frozen bone marrow samples in a liquid nitrogen storage tank was demonstrated. This incident raised the possibility of pathogen transmission between samples in ART laboratories and led to further consideration of potential sources of contamination and strategies to avoid the transmission of infection. Although no disease transmission caused by liquid nitrogen or other source related to cryopreservation has been reported in mammalian and human assisted reproduction, theoretical sources of contamination include:
1. Within the freezing apparatus. Vapor phase-controlled rate freezers spray nonsterile liquid nitrogen directly onto the samples. This may be further compounded by liquid condensation that may accumulate within ducting between freezing runs. Ideally, a freezing apparatus should have the capability of being sterilized between freezing runs, but this is not a practical option.
2. During storage. Straws may be contaminated on the outside, or seals and plugs may leak. Particulates may then transfer via the liquid nitrogen within the storage vessel.
3. From liquid nitrogen. Generally, liquid nitrogen has a very low microbial count when it is manufactured. However, contamination may occur during storage and distribution. Any part of the distribution chain that periodically warms up, in particular transfer dewars or dry shippers, may become heavily contaminated. The microbial quality of the liquid nitrogen when delivered from the manufacturer varies widely with geographical region, and more extreme reports of microbial contamination may reflect local industrial practices. Although this raised concern about the safety of ‘open’ devices used for vitrification, no infection attributable to the procedures has yet been reported (Vajta et al., 2015).
The HFEA in the United Kingdom prepared a consultation document with guidelines for safe storage of human gametes in liquid nitrogen (Human Fertilisation and Embryology Authority, 1998); basic recommendations include patient screening for hepatitis B, hepatitis C and HIV, careful hygiene throughout, double containment of storage straws and the use of sealed ampules. The risks of cross-contamination during the quarantine period need to be assessed and procedures put in place to minimize these risks. However, the literature, as well as experience in both animal models and human IVF, suggests that in practice, the risk of cross-contamination in IVF working conditions is negligible (Pomeroy et al., 2009; Vajta et al., 2015).
Following fresh embryo transfer in a stimulated IVF cycle, supernumerary embryos are available for cryopreservation in a large number of cycles. In a routine IVF practice, more than half of stimulated IVF cycles may yield surplus embryos suitable for cryopreservation (although this is now subject to legislative control in particular countries of the world). In addition to enhancing the clinical benefits and cumulative conception rate possible for a couple following a single cycle of ovarian stimulation and IVF, a successful cryopreservation program offers other benefits including the possibility of avoiding fresh embryo transfer in stimulated cycles with a potential for ovarian hyperstimulation syndrome, or in which factors that may jeopardize implantation are apparent (e.g., bleeding, unfavorable endometrium, polyps or an extremely difficult embryo transfer).
A unit that offers embryo cryopreservation must also be aware of logistic, legal, moral and ethical problems that can arise, and ensure that all patients are fully informed and counseled. Both partners must sign comprehensive consent forms indicating how long the embryos are to be stored, and define legal ownership in case of divorce or separation, death of one of the partners, or loss of contact between the Unit and the couple. Cryopreserved samples cannot in practice be maintained in storage indefinitely, and there must be a clear clinic policy to ensure that records are correctly maintained, with regular audits of the storage banks. Clinic administration may mandate that all couples with cryopreserved embryos in storage must be contacted annually and asked to return a signed form indicating whether they wish to continue storage. In the United Kingdom, options for couples include:
1. Continue storage.
2. Return for frozen embryo transfer.
3. Donate their embryos for research projects approved by appropriate ethics committees/Internal Review Boards and the HFEA.
4. Donate their frozen embryos for transfer to another infertile couple.
5. Have the embryos thawed and disposed of.
Using PROH as cryoprotectant, embryos can be frozen at either the pronucleate or early cleavage stages. Careful selection of viable embryos will optimize their potential for surviving freeze-thawing.
The cell should have an intact zona pellucida and healthy cytoplasm with two distinct pronuclei clearly visible. Accurate timing of zygote freezing is essential to avoid periods of the cell cycle that are highly sensitive to cooling. For example, during the period when pronuclei start to migrate before syngamy, with DNA synthesis and formation of the mitotic spindle, the microtubular system is highly vulnerable to temperature fluctuation, leading to possible scattering of the chromosomes. Zygotes processed for freezing at this stage will no longer survive cryopreservation. The timing of pronucleate freezing is crucial, and the process must be initiated while the pronuclei are still distinctly apparent, no later than 20–22 hours after insemination.
Two- to eight-cell embryos should be of good quality, Grade 1 or 2, with less than 20% cytoplasmic fragments. Uneven blastomeres and a high degree of fragmentation jeopardize survival potential; embryos with damage after thawing may still be viable and result in pregnancies, but their prognosis for implantation is reduced.
Details of each patient and the associated embryos must be carefully recorded on appropriate data sheets. Meticulous and complete record keeping is crucial, and must include the patient’s date of birth, medical number, date of oocyte retrieval (OCR), date of cryopreservation, number and type of embryos frozen, and number of straws or ampules used, together with clear and accurate identification of storage vessel and location within the storage vessel. The data sheets should also confirm that both partners have signed consent forms. Ampules and straws have been successfully used for embryo storage, and each has advantages and disadvantages. The choice between them is a matter of individual preference, as well as availability of storage space and laboratory time to prepare and sterilize ampules. When straws are used, they must be handled with care to avoid external contamination, and to avoid inadvertent temperature fluctuations during seeding or transfer to the storage dewar. The measured temperature excursions within straws can be very dramatic (see Figure 12.6). It is likely that in straws frozen horizontally the embryos will be adjacent to the wall, where they will be exposed to the highest thermal gradient; great care must be taken in handling cryopreserved material. Plastic cryovials are not recommended for embryo freezing.
Figure 12.6 Measured temperatures within straws following removal from a controlled rate freezer or from a liquid nitrogen vessel at various points during the freezing cycle. Prior to nucleation the temperature rise within 5 seconds is sufficient to prevent ice nucleation. At –30°C the sample temperature may rise very quickly and if transfer to liquid nitrogen is carried out at this point of the freezing program, care must be taken to ensure that the increase in temperature is minimized. Following liquid nitrogen immersion, the temperature of straws may rise by 130°C within 20 seconds.
Ready-to-use media for freezing and thawing embryos are available from the majority of companies who supply culture media. Individual methods and protocols vary slightly with the different preparations, and manufacturers’ instructions should be followed for each.
Ensure that no air bubbles are trapped within the freezing medium after the sample has been loaded, into either ampules or straws. Air bubbles can sometimes be seen in both vessels on thawing, and these present a hazard to the fragile dehydrated embryo. Warming solutions to 37°C before starting the procedure may effectively act as a ‘degassing’ mechanism.
It is common practice to cool human embryos within the controlled rate freezing apparatus down to below –100°C after the slow cooling to –30°C, before transfer to liquid nitrogen. In veterinary IVF cryopreservation, straws are often transferred to liquid nitrogen directly from –30°C. This procedure would give equally good results for human embryos and is indeed used by some laboratories with no reduction in viability. However, it is essential that the transfer is carried out rapidly (within 5 seconds) because the temperature of the straws may rise very quickly when they are removed from the controlled rate device (see Figure 12.6). Cooling to temperatures below –100°C within the freezing machine carries less risk, but does consume considerably more liquid nitrogen.
1. Because straws have a large surface area, small diameter and a thin wall, very rapid warming occurs when they are removed from a cold environment. Measured temperature excursions that occur at different points of the cryopreservation procedure are illustrated in Figure 12.6. If straws are removed from the controlled rate freezing apparatus for excessive lengths of time during the nucleation procedure, they can warm to a temperature that is too high for ice nucleation to occur. Ice nucleation may occur because of the local cooling induced by the nucleating tool, but it is possible that the bulk temperature of the fluid may not allow ice crystal growth to propagate through the sample. In some laboratories, it is common practice to check that ice propagation has occurred throughout the sample, usually 1 minute after the seeding procedure. If straws are removed from the controlled rate cooling equipment, this in itself may cause melting of the nucleated ice.
2. Thermal control of the freezing apparatus may not be sufficiently accurate or stable at the nucleation temperature. The temperature achieved may allow nucleation to occur because of the thermal mass of the nucleating tool, but may not be sufficiently low to allow subsequent ice propagation. Any thermal fluctuations within the freezing apparatus may also lead to ice melting.
3. Within straws, nucleation of ice at temperatures very close to the melting point results in a very slow propagation of ice through the sample. In some cases, ice propagation can actually become blocked, and embryos are then effectively supercooled. In this case the embryos would not be expected to survive further cooling.
Sample Protocol for Embryo Slow Freezing
1. Equilibrate selected and washed embryos in 1,2-propanediol (1.5 M) at room temperature, to allow uptake of the CP into the cells. This is usually done in two steps, the second step incorporating 0.1 M sucrose.
2. Load equilibrated embryos into straws or ampules.
3. Cool the samples at a rate of 2°C/min to –7°C, and ‘hold’ at this temperature to allow thermal equilibration before ice nucleation (seeding).
4. Following seeding, with initiation and growth of ice crystals, cool the samples at a slow rate, –0.3°C/min, down to –30°C.
5. Cool the samples rapidly to LN2 temperatures, then plunge and store in LN2.
Sample Protocol for Embryo Thawing after Slow Freezing
1. Samples are thawed in two stages: hold straws in air for 40 seconds, and then transfer to a 30°C water bath for a further minute.
2. Remove cryoprotectant by dilution through solutions containing 0.2 M sucrose, and then wash three times in culture medium.
The thawing protocol is carried out at room temperature, and the embryos placed in equilibrated culture medium at room temperature before being allowed to warm gradually to 37°C in the incubator. Pronucleate embryos may be cultured overnight to confirm continued development, and cleavage stage embryos are incubated for a minimum of 1 hour before transfer.
The first reports of successful human blastocyst cryopreservation were published in 1985 (Cohen et al., 1985; Fehilly et al., 1985), but blastocyst freezing became routine in IVF only after media for effective extended culture became available during the 1990s. Using Vero cell co-culture to enhance extended culture, Ménézo et al. (1992) explored the use of a combination of glycerol and sucrose as cryoprotectants to freeze surplus expanded blastocysts, and the protocols were later modified to obtain satisfactory freeze–thaw rates. Inconsistent success rates were reported initially, but this may have been partly due to lack of experience with selection criteria for freezing, and also a need to understand the subtleties of cryopreservation and the impact that even the slightest variation might have on consistency. Extended culture to blastocyst stage is now routine in many IVF laboratories, and slow-freezing protocols using glycerol as cryoprotectant have largely been replaced by the more successful technique of blastocyst vitrification.
Using strict criteria to select potentially viable blastocysts is crucial to success:
Growth rate: expanded blastocyst stage on Day 5/Day 6.
Overall cell number >60 cells (depending on day of development).
Relative cell allocation to trophectoderm/inner cell mass.
Original quality of early stage embryo: pronucleus formation and orientation, blastomere regularity, mono-nucleation, fragmentation, appropriate cleavage stage for time of development.
Tissue culture washed borosilicate glass ampules with a fine-drawn neck can be used for embryo cryostorage.
Fill the ampule with approximately 0.4 mL of the sucrose/PROH solution using a needle and syringe.
Carefully transfer the embryos using a fine-drawn Pasteur pipette.
Using a high-intensity flame, carefully heat seal the neck of the ampule. It is important to ensure (under the microscope) that the seal is complete, without leaks: leakage of LN2 into the ampule during freezing will cause it to explode immediately upon thawing. It is often impossible to detect whether the glass neck is completely sealed, and the possibility of explosion can be avoided by opening the ampule under LN2 before thawing.
Blastocyst vitrification protocols now yield very favorable survival, implantation and clinical pregnancy rates. Commercial kits for blastocyst vitrification are available – as always, the ultimate success of the protocol will be related to the operator’s experience and careful attention to detail. In common with all aspects of human ART, careful research into the consequences of such new therapies continues to be essential. In large expanded blastocysts, collapsing the blastocoelic cavity with an ICSI needle immediately before processing increases survival rates after both slow freezing and vitrification (Kader et al., 2009). Poor morphology and delayed expansion (to Day 7) have a negative impact on survival post-vitrification.
Freeze-thawing is known to cause hardening of the zona pellucida, and the application of assisted hatching, particularly at the blastocyst stage, has been suggested as beneficial to implantation after freeze-thawing (Tucker, 1991). In some cases, zona pellucida fracture can be a routine result of some cryopreservation protocols (Van den Abbeel et al., 2000). Embryos with existing holes in the zona pellucida following PGD procedures can successfully survive and implant (Magli et al., 2006). A recent systematic review confirmed that assisted hatching is consistently of benefit after thawing frozen/vitrified blastocysts (Alteri et al., 2018).
Parmegiani et al. (2014) postulated that the same warming procedure could be used for both slow-frozen and vitrified oocytes, and carried out a prospective study to investigate this proposal. Using slow-frozen sibling oocytes randomized for either conventional thawing or rapid vitrification warming, their results showed better survival with rapid warming (90%) than with conventional thawing (75%). Chromosomal configuration and the meiotic spindle examined by confocal microscopy showed no differences using either procedure. The authors suggest that a single warming protocol/solution may be used for both slow-frozen and vitrified oocytes; slow-frozen oocytes thawed with this protocol show increased survival rates that are comparable to those obtained after vitrification. This substitution can also potentially be applied for slow-frozen zygotes and embryos (Kojima et al., 2012).
Freeze-thawed embryos must be transferred to a uterus that is optimally receptive for implantation, in a postovulatory secretory phase. Patients with regular ovulatory cycles and an adequate luteal phase may have their embryos transferred in a natural cycle, monitored by ultrasound and blood or urine luteinizing hormone (LH) levels in order to pinpoint ovulation. Older patients or those with irregular cycles may have their embryos transferred in an artificial cycle: hormone replacement therapy with exogenous steroids is administered after creating an artificial menopause by downregulation with a gonadotropin-releasing hormone (GnRH) agonist.
1. Patient selection: oligomenorrhea/irregular cycles, or age >38 years.
3. Administer estradiol valerate:
4. Progesterone from Day 15 to 16, choice between:
Double the dose from Day 17 onwards (100 mg Gestone, 400 mg twice daily Cyclogest, 200 mg three times daily Utrogestan, 8% Crinone gel PV, twice daily).
5. Embryo transfer:
(a) Pronucleate: thaw on Day 16 of the artificial cycle, culture overnight before transfer on Day 17 or 18.
(b) Cleavage stage embryos: thaw and replace on Day 17 or 18.
(c) Blastocysts: thaw and replace on Day 19 or 20.
6. If pregnancy is established, continue hormone replacement therapy (HRT) with 8 mg estradiol valerate and the higher dose of progesterone supplement daily until Day 77 after embryo transfer. Gradually withdraw the drugs with monitoring of blood P4 (progesterone) levels. This protocol is also successfully used for the treatment of agonadal women who require ovum or embryo donation. In combination with prior GnRH pituitary suppression, the artificial cycle can be timed to a prescheduled program according to the patient’s (or clinic’s) convenience.
1. Patient selection: regular cycles, 28 ± 3 days, previously assay luteal phase progesterone to confirm ovulation. A commercially available ovulation ‘kit’ can also be used in a previous cycle to confirm that the patient has regular ovulatory cycles.
2. Cycle monitoring from Day 10 until ovulation is confirmed by ultrasound scan and plasma LH. Ultrasound scan should also confirm appropriate endometrial development; the cycle should be canceled if the endometrial thickness is <8 mm at the time of the LH surge.
3. Timing of the embryo transfer:
a. Pronucleate embryos: thaw on Day 1 after ovulation (3 days after the LH surge: LH + 3), culture overnight
b. Cleavage stage embryos: thaw and transfer on Day 2 or 3 after ovulation (LH + 4/5)
c. Blastocysts: thaw and transfer on Day 4 or 5 after ovulation (LH + 6/7).
Patients with irregular cycles may be induced to ovulate using clomifene citrate or gonadotropins, and embryo transfer timed in relation to the endogenous LH surge or following administration of human chorionic gonadotropin (hCG). Although it is possible to estimate embryo transfer time using an ovulation ‘kit’ to detect the LH surge, this may be less accurate, and does present a risk of inappropriate timing.
Prior to 1997, the options for preserving a young woman’s fertility after treatment for malignant disease were very limited: a full IVF treatment cycle with cryopreservation of embryos prior to the initiation of chemotherapy, or oocyte or embryo donation following recovery from the malignant disease. The first option is available only to women with partners to provide a semen sample for fertilization of the harvested oocytes. However, the success of frozen embryo cryopreservation in a competent IVF program is such that these patients maintain a very good chance of achieving a pregnancy after transfer of frozen-thawed embryos following recovery from their disease. On the other hand, this strategy also raises the risk of creating embryos with a higher than average chance of being orphaned. Many of the legal and ethical problems created by the cryopreservation and storage of embryos can be overcome by preserving oocytes, especially for young women about to undergo treatment for malignant disease that will result in loss of ovarian function. Oocyte cryopreservation is also indicated in patients with a known family history of premature ovarian failure, and can be advantageous in various clinical scenarios, such as in ovarian hyperstimulation syndrome, unexpected lack of sperm following oocyte retrieval, egg donation programs and in order to extend the duration of natural fertility.
Human oocytes are particularly susceptible to freeze–thaw damage due to their size and complexity. They must not only survive thawing, but also preserve their potential for fertilization and development. The first pregnancies with human oocyte freezing were reported in the 1980s (Chen, 1986; Al-Hasani et al., 1987), but the procedure was abandoned for approximately 10 years due to low survival and fertilization rates, thought to be due primarily to hardening of the zona pellucida and to spindle damage causing aneuploidy. Since 1997, focus intensified on modifying protocols to increase survival rates, in particular to avoid activation/premature release of cortical granules, zona pellucida hardening and the detection/avoidance of spindle damage and aneuploidy. In 2009, Noyes et al. reported the birth of more than 900 babies after oocyte cryopreservation, with no apparent increased incidence of congenital anomalies (Noyes et al., 2009). Attempts to monitor alterations in the permeability of the plasma membrane, assess warming and rehydration protocols, and use ICSI to improve fertilization rates have resulted in significant clinical progress.
Several intrinsic difficulties are associated with human oocyte freezing, due mainly to their high volume:surface area ratio and low membrane permeability. Intracellular ice formation causes critical damage to the cytoskeleton, which is also sensitive to osmotic stress. Disruption of the meiotic spindle can cause chromosome defects and aneuploidy. Lowering the temperature, or the cryoprotectant agents themselves, may cause an increase in intracellular Ca2+ leading to changes in the intracellular signaling mechanisms and oocyte activation. Finally, since the zona pellucida hardens after freezing, it is necessary to employ ICSI for fertilization of the thawed oocyte.
Freezing can result in parthenogenetic activation, leading to premature release of cortical granules (CGs). It is also important to consider the cytoplasmic maturity of the oocyte at freezing and the potentially toxic effects of cryoprotectants.
Modifications found to improve the effectiveness of oocyte freezing include:
1. Complete removal of the cumulus and coronal mass, which increases survival rates.
2. Alteration of sucrose concentrations from 0.1 to 0.2, 0.3 or 0.5 mol/L, which increases oocyte dehydration and survival.
3. Choline has been used as a substitute for sodium (Boldt et al., 2006; Stachecki et al., 2006) on the basis that cryodamage to the Na+/K+ pump might lead to high intracellular concentrations of Na+ with a resulting efflux of protons. Choline does not cross the plasma membrane, is less toxic than high sucrose and does not affect osmotic pressure of the cell.
Not surprisingly, damage caused by oocyte freezing appears to be protocol-dependent (Rienzi et al., 2004). Using a Polscope to observe the meiotic spindle following freeze–thaw procedures, these authors observed that the spindle disintegrates during freeze-thawing, and oocytes must reconstruct their spindles after thawing. Other authors using confocal or electron microscopy have shown that elevated sucrose concentrations may prevent spindle damage (Coticchio et al., 2006; Nottola et al., 2008). The timing of freezing after oocyte retrieval also seems to be important, with lower pregnancy rates reported from oocytes that were frozen more than 2 hours after OCR (Parmegiani et al., 2009). Germinal vesicle stage oocytes show better survival (Sereni et al., 2000), and retrieval of immature oocytes has now become another option for fertility preservation. The protocols require minimal hormonal stimulation, and oocytes can be collected within a short time following the diagnosis of cancer
The thawing process is equally fraught with difficulties. Osmotic stress caused by rehydration must be minimized in order to prevent degeneration, and reassembly of the spindle post-thaw takes at least 3–4 hours.
Vitrification of human oocytes has proved to be superior to slow freezing, and this is now the routine method used for oocyte cryopreservation. The first live birth was reported by Kuleshova et al. in 1999, and numerous studies with favorable results were published between 2005 and 2009. The main concerns with vitrification are the toxicity of high concentrations of cryoprotectant and extreme osmotic changes. Huang et al. (2007) showed less damage to the spindle and chromosomes after vitrification compared to slow freezing. Cobo et al. (2008) compared sibling fresh oocytes with vitrified donor oocytes using the Cryotop method, reporting very high survival rates after warming (97%), and fertilization, blastocyst development and pregnancy rates for recipients that were equivalent to those obtained with the use of fresh donor oocytes. Differences in participant characteristics and study design, as well as ethical and legal issues related to oocyte cryopreservation in different countries, mean that heterogeneous results can be observed in different studies. Nevertheless, a systematic review and meta-analysis carried out for results published between 1980 and 2013 confirmed that vitrified oocytes have better survival, fertilization and cleavage rates compared with slow-freezing protocols (see Potdar et al., 2014 for review).