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The starting point of human embryo cryopreservation can be found in the early

seventies in a unique collaboration between three scientists. These scientists were

David Whittingham from the UK, and Stanley Leibo and Peter Mazur from the USA.

David Whittingham had already had some experience in the cryopreservation of

mouse embryos beforehand and published the results of his research in 1969

together with Wales and in 1971. Stanley Leibo and Peter Mazur based their

investigations on emerging understanding of the freezing of mammalian embryos

and published their findings in a number of articles between 1963 and 1972. The

collaboration between these three scientists in 1972 led to a live birth of mice after

thawing and transferring mouse embryos previously frozen at -196°C and -269°C.

Independently from this work, Wilmut also succeeded in freezing mouse embryos in

1972.

The work carried out by Wilmut, Whittingham, Leibo, and Mazur soon became the

model for cryopreservation of cow embryos as reported by Wilmut et al. in 1975, rat

embryos as demonstrated by Whittingham in 1975, sheep embryos as reported by

Willadsen et al. in 1976, and rabbit embryos as demonstrated by Whittingham et al.

in 1976.

For human embryo cryopreservation, the breakthrough came in January 1977,

when CIBA Foundation symposium on “The Freezing of Mammalian Embryos” was

held in London. During this symposium, Bob Edwards, a human embryologist,

lectured on “The Relevance of the Frozen Storage of Human Embryos”, discussing

for the first time the optimum stage of human preimplantation development for

storage at low temperatures, and on cryobiological aspects of cleaving human

Embryo cryopreservation has numerous applications in reproductive medicine.

These applications include the following:

• First of all, any supernumerary embryo can be cryopreserved for a certain time

period, that depends on various medical and legal (regulatory factors), allowing

a second attempt in case the first implantation has failed (for example because

of a suboptimal endometrium). As a consequence, embryos could be

transferred in non-stimulated cycles and in several subsequent cycles. Also, the

number of stimulated treatment cycles per patient could be reduced.

• Second, cryopreservation is particularly valuable in facilitating synchronization

between an oocyte donor’s cycle and the cycle of a female recipient, who

cannot produce a normal oocyte herself. If embryo preservation was not

available, it would be purely a matter of chance that a potential donor might be

synchronized with a recipient, as stated by Devroey et al. in 1988.

• Third, in cases when the risk of ovarian hyperstimulation syndrome (OHSS)

becomes apparent after oocyte retrieval, freezing all the embryos is certainly an

option, as demonstrated by Wada et al. in 1992.

• Fourth, if for any other specific medical or technical reason embryos cannot be

transferred during the oocyte retrieval cycle, they can be cryopreserved and

transferred during a subsequent cycle.

• Finally, cryopreservation is a useful option for patients who are at risk of losing

their ovarian function because of radio- or chemotherapy.

Several authors have reported the importance of embryo cryopreservation in daily

ART practice. Cryopreservation has been highlighted either as a method to reduce

multiple births in ART, as demonstrated by Tiitinen et al. in 2003, Schnorr et al. in

2001, Gerris et al. in 2003 and Kolibianakis et al. in 2004; or as a possibility to

Cryopreservation of reproductive cells or embryos is performed to stop all biological

processes in the cells. An efficient way to do this is to expose the cells to very low

temperatures, such as the temperature of liquid nitrogen which is -196°C. At this

temperature no metabolic process in the cell can occur.

However, cooling the cells down to very low temperatures is not risk-free.

Intracellular space of human oocytes and embryos consists of about 80 % water, so

exposing the cells to low temperatures will cause the formation of lethal ice crystals

and will therefore inevitably damage the cells. Two different methods exist on how to

deal with the intracellular water – slow freezing and vitrification – both successfully

used for oocyte and embryo cryopreservation.

For reproductive cells, there are basically two different slow freezing techniques:

1) nonequilibrium slow freezing and

2) equilibrium slow freezing;

and there are two different vitrification techniques:

1) nonequilibrium vitrification and

2) equilibrium vitrification.

These techniques are schematically presented in this slide and will be discussed in

more detail in the following slides.

During non-equilibrium slow freezing, the cell is first cooled from 37°C to 22°C

(room temperature), at which point a cryoprotectant is added to the cell. After

loading the cell, submerged in a basic salt solution, with a cryoprotectant (CPA), the

cell is cooled to -7°C at which point extracellular ice crystal formation is induced.

Since these ice crystals typically consist of pure water, and since the cell is

submerged in a physiological salt solution, the salt concentration in the extracellular

environment will rise and the osmolality will increase. As a response to this increase

in osmolality, the cell will start to lose water and will shrink.

What happens further will depend on the cooling rate and the size of the cell. When

controlled-rate cooling is slow enough (for oocytes and embryos typically

0.3°C/min), the temperature drops, ice crystals are formed outside the cells and the

cell will respond by gradually losing water, thus resulting in cell shrinkage. When the

cell reaches the temperature of let us say -30°C, the cell has lost most of its water

(is sufficiently dehydrated) so that the intracellular environment solidifies without the

formation of lethal intracellular ice crystals. However, when the cell is plunged into

LN2 at -30°C, it will contain microscopic intracellular ice crystals, which are

innocuous, provided the cell is thawed very rapidly (> 300°C/min) in order to prevent

the ice crystals from growing to detrimental sizes. Upon return to 22°C the CPA will

be diluted out, before the cell is returned to 37°C.

The first steps in the process of the equilibrium slow freezing are identical to those

of non-equilibrium slow freezing. The cell is first cooled from 37°C to 22°C (room

temperature) at which temperature a cryoprotectant is added to the cell. After

loading the cell, submerged in a basic salt solution, with a cryoprotectant (CPA), the

cell is cooled to -7°C at which temperature extracellular ice crystal formation is

induced. Since these ice crystals typically consist of pure water and since the cell is

suspended in a physiological salt solution, the salt concentration in the extracellular

environment will rise and the osmolality will increase. As a response to this increase

in osmolality, the cell will start to lose water and will shrink.

What happens further will depend on the cooling rate and the size of the cell. When

controlled-rate cooling is slow enough (for oocytes and embryos typically

0.3°C/min), the temperature drops, ice crystals are formed outside the cell and the

cell will respond by gradually losing water. When the cell reaches the temperature of

-80°C, it has lost all of the freezable water and can be plunged into LN2 without any

risk of intracellular ice crystals formation. When plunging occurs at -80°C, the cells

must be thawed slowly (< 25°C/min) to prevent osmotic shock. Upon returning to

22°C the CPA will then be diluted out before the cell is returned to 37°C.

Most cryobiologists date the beginning of the modern cryobiology era of

reproductive cells to the accidental discovery of the cryoprotective properties of

glycerol by Polge et al. in 1949. Since this highly significant discovery,

cryoprotective agents have been used to secure the survival of living cells at low

temperatures.

Although the mechanisms responsible for protection during cryopreservation are not fully understood, all cryoprotectants share a number of common properties. They are mixable with water, they reduce the freezing point of aqueous solutions,

increase the viscosity of aqueous solutions, and lower the ice nucleation

temperatures of cells or solutions, as stated by Rall et al. in 1983.

Despite some degree of success, however, only few cryopreservation techniques

result in 100 % survival of the cells after freezing and thawing. In other words,

cryoprotectants are no panacea, even when optimized cooling and warming rates

are used. At least part of the problem is due to the cryoprotectants themselves. First

of all, toxicity of the cryoprotectants limits the concentrations of additives that can be

used before freezing and therefore restrains the cryoprotective efficacy of these

agents. Additionally, there is some evidence that beside their benefits,

cryoprotective agents may also play a direct role in producing cryoinjury, as reported

by Fahy in 1986.

Polge C, Smith A, Parkes A (1949) Revival of spermatozoa after virtrication and

dehydration at low temperatures. Nature, 164, 666.

Fahy G (1986) The relevance of cryoprotectant toxicity to cryobiology. Cryobiology

23, 1-13.

Cryoprotectants can be divided into two groups: penetrating and non-penetrating

cryoprotectants, both successfully used for slow freezing and vitrification

The following substances belong to the group of penetrating cryoprotectants:

dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG)

and methanol. In 1954 Lovelock demonstrated that these cryoprotectants are

capable of passing oocyte and embryo membranes and that they provide protection

both within and around the cells.

Non-penetrating cryoprotectants, as reported by Ashwood-Smith in 1986, include

monosaccharides, like galactose, disaccharides, like sucrose and trehalose,

polysaccharides, like dextrans and hydroxyethyl starch, and polymers, like

polyethylene glycol, polyvinylpyrrolidone or polyvinylalcolhol. Cryoprotective

qualities of this group of cryoprotectants were studied by a number of scientists.

Franks et al. and Crowe et al. published the results of their research in 1977 and

1990 respectively, reporting that non-penetrating cryoprotectants protect the cells

through dehydration, stabilization of lipid bilayers and proteins, or by changing the

water properties in the vicinity of the membranes. When oocytes or embryos are

exposed to mono- or disaccharides, cells respond osmotically by loosing water.

Since these cryoprotectants do not pass membranes, cells remain contracted when

equilibrium is reached.

Several variables determine the tolerance with regard to chemical toxicity and (or)

osmotic stress of oocytes and embryos exposed to penetrating or non-penetrating

cryoprotectants. In 1987 Rall reported that these variables include the permeability

characteristics of the cell membranes to water and cryoprotectants, the

Several devices and media can be used for freezing of oocytes and embryos.

Devices include the use of glass ampoules, plastic vials, or plastic straws. At

present, plastic straws are most commonly used. They prevent possible crosscontamination

problems, for example a possible transmission of viruses to

cryopreserved embryos via contaminated liquid nitrogen.

For equilibrium slow freezing, mostly dimethyl sulfoxide is used as the

cryoprotectant.

For non-equilibrium slow freezing glycerol, propylene glycol, and dimethyl sulfoxide

are used mostly in combination with sucrose. Sucrose is a non-penetrating

cryoprotectant; it dehydrates the cells before the actual freezing begins.

This slide summarizes the technical challenges of slow freezing:

• When cells are cooled slowly, their survival depends on the cooling and/or

warming rates.

• Extracellular and intracellular ice crystal formations have to be avoided.

• Cells may be killed by cooling to ~0°C.

• Cells may survive freezing, but be then killed by osmotic shock during thawing.

• Various chemicals, the so-called cryoprotectants, can be used to protect the

cells from cryopreservation damage.

• Expensive equipment is required for both slow freezing and vitrification, for

example biological freezers

• Cryopreservation can be a time-consuming procedure (3 to 5 hours depending

on the slow freezing end-point).

• Robust cryopreservation procedures are required to provide repeatable results.

This slide describes schematically the basic differences between equilibrium and

non-equilibrium vitrification techniques. For both vitrification procedures, the basic

principle is plunging CPA loaded cells directly into liquid nitrogen.

Vitrification refers to the phenomenon that solutions can be converted directly into a

glass phase without the formation of ice crystals. The basic aspects of vitrification

as well as the factors influencing vitrification have been described in detail by Fahy

et al. in 1984 and Rall in 1987.

Briefly, vitrification refers to the physical process by which concentrated solutions of

cryoprotectant agents solidify during cooling without crystallization. The solid state

retains the normal molecular and ionic distributions of the liquid state and can be

considered as an extremely viscous solid glass. Vitreous transformation can be

assessed by techniques such as differential scanning calorimetry, X-ray diffraction,

and direct visual observation.

References:

Fahy G et al. (1984) Vitrification as an approach to cryopreservation. Cryobiology

21, 407-426.

Rall W (1987) Factors affecting the survival of mouse embryos cryopreserved by

vitrification. Cryobiology 24, 387-402.

There are two important variables in vitrification: cooling and warming rates, and

cryoprotective agents. For vitrification to occur at moderate or low cooling rates

(equilibrium vitrification), extremely high concentrations of cryoprotective agents are

required, typically in the range of 6 to 7 M . At these concentrations, cryoprotective

agents are considered to be chemically and/or osmotically toxic. This osmotic

toxicity can be reduced by adding cryoprotective agents to the cells stepwise and by

using osmotic buffers for diluting cryoprotective agents out of the cells. The

chemical toxicity of cryoprotective agents can be reduced by:

adding specific toxicity neutralizers;

using cryoprotective agents with a high glass forming tendency;

using mixtures of cryoprotective agents;

including non-penetrating cryoprotective agents such as sugars or polymers to

increase the viscosity of the solution and to aid in a pre-freezing dehydration;

increasing the hydrostatic pressure on the solution or

lowering the temperature of the exposure.

The latter has, however, profound effects on cryoprotective agent’s permeability

characteristics, which makes working at lower temperatures less attractive, as

reported by Fahy et al. in 1984 and Rall in 1987. Therefore, most attempts to vitrify

embryos have focused on increasing the cooling and warming rates, and on

keeping the concentration of cryoprotective agents within “tolerable” limits. This nonequilibrium

vitrification is usually accomplished by:

reducing the sample size and/or using specific carrier systems;

using the least toxic cryoprotective agent mixture that will produce vitreous

transformation with achievable cooling and warming rates, and

Successful vitrification depends on “sufficient” penetration of permeating

cryoprotective agents and “sufficient” dehydration by non-permeating cryoprotective

agents during vitrification. Sufficient permeation and sufficient dehydration are

directly related to the specific permeability characteristics of oocytes and embryos

to water and cryoprotective agents and therefore these characteristics have to be

considered in a vitrification procedure.

First of all, permeability characteristics of oocytes and embryos are known to be

very temperature dependent. The lower the temperature, the lower the permeability

of the cell membrane to water and cryoprotective agent. Therefore, temperature in

the vitrification lab should be strictly controlled and maintained throughout the year

in order to avoid fluctuations in the outcome of vitrification. Preferentially

temperature should range between 22 and 27 °C.

Second, permeability characteristics vary among different oocytes or embryos from

one patient and also between patients. Consequently, the optimal time period,

required for permeation of the cryoprotective agent during equilibration step, will

vary amongst oocytes and embryos.

Third, the membrane permeability characteristics of oocytes and embryos are

known to be dependent on the type of cryoprotective agent used. The membrane is

less permeable to glycerol than to ethylene glycol or dimethyl sulfoxide or propylene

glycol, as demonstrated by Van den Abbeel et al, in 2007. Mixtures of cryoprotective

agents with different permeability characteristics are therefore often used for

vitrification.

Finally, oocytes are known to be less permeable to water and cryoprotective agents

Several commercially available media formulations can be used for the vitrification

of reproductive cells. These include dimethyl sulfoxide, ethylene glycol and sucrose

formulations and propylene glycol, ethylene glycol and sucrose formulations. Also

glycerol, ethylene glycol and sucrose formulations can be used, especially for

blastocysts. Ethylene glycol in combination with only sucrose can be used as well.

Trehalose can be used as a substitute for sucrose.

There are also several commercially available devices, which are suitable for the

vitrification in very low volumes. Some of them are open devices, meaning that

during the vitrification step and during storage the sample is in direct contact with

liquid nitrogen. Other devices are closed. In these devises, no contact with liquid

nitrogen is possible.

In comparison to slow freezing, vitrification has several advantages.

First, there is no risk of extracellular or intracellular ice crystals formation, which can

damage the cell.

Secondly, during vitrification the cells lose water or are dehydrated before cooling.

During slow freezing, on the other hand, cell dehydration is initiated by extracellular

ice induction (seeding) as a consequence of the increasing salt concentration in the

extracellular milieu. This increase in salt concentration is called the solution effect

and is known to be detrimental to cells. During vitrification this extracellular increase

in salt concentration does not occur, consequently there is no risk of solution

effects.

Thirdly, during slow freezing cells are cooled slowly to -7°C. Since meiotic and also

mitotic spindles are temperature sensitive, particularly cooling sensitive, freezing

these structures might cause their irreversible damage, possibly leading to

aneuploidies. During vitrification the cooling phase is extremely rapid and

consequently a possible cooling injury is suppressed.

The final advantage of vitrification is its flexibility. Vitrification of the oocytes or

embryos could be organized at any time during the day when the oocytes and

embryos are in the best condition.

A slow freezing procedure for reproductive cells typically begins with adding 1.5

mol/l propylene glycol and 0.1 mol/l sucrose to the cell, for 10 to 15 min, at 22°C.

After loading the sample into a device (straw, ampoule or vial) the cells are cooled

at a rate of 2°C/min to -7°C in a biological freezer. At -7°C extracellular ice crystal

formation (seeding) is induced by touching the device (with the sample inside) with

liquid nitrogen cold forceps. After seeding, the sample is further cooled at a rate of

0.3°C/min to -30°C, at which temperature the sample is plunged into liquid nitrogen.

After that, the samples are stored in containers filled with liquid nitrogen.

For thawing, the sample is taken from liquid nitrogen and put on the bench of a

laminar air flow at 22°C for 40 seconds. After this, the sample is warmed in a 37°C

warm water bath until all ice has disappeared (~5 sec).

The cryoprotectants are then diluted out stepwise at 22°C. The first 10 min in a

solution containing 1 mol/l propylene glycol and 0.2 mol/l sucrose, then 10 min in

0.5 mol/l propylene glycol and 0.2 mol/l sucrose, then 5 min in 0.2 mol/l sucrose, 5

min in 0.1 mol/l sucrose and finally 5 min in a solution without sucrose. The cells are

then put in culture until transfer to the patient.

In a typical vitrification procedure, cells are loaded in an equilibration step with

cryoprotectants. Cells are first exposed to a solution containing 7.5 % ethylene

glycol and 7.5 % dimethyl sulfoxide for 10 to 15 min. Following this equilibration

step, samples are loaded in a device with less than 1 microliter of vitrification

solution containing 15 % ethylene glycol, 15 % dimethyl sulfoxide, and 0.5 mol/l

sucrose and then vitrified for 90 seconds by plunging the device into liquid nitrogen.

The devices are then stored in containers filled with liquid nitrogen.

For thawing, the device with the sample is put directly from liquid nitrogen into a

37°C warm solution containing 1 mol/l sucrose and then kept for 5 min at 22°C.

After this, the sample is transferred to a solution containing 0.5 mol/l sucrose for 5

min at 22°C. After further 5 min at 22°C in a solution containing 0.2 mol/l sucrose,

the cells are put for 5 min at 22°C in a solution without sucrose. After this, the cells

are cultured in vitro until transfer.

https://www.globalfertilityacademy.org/static/resources-en/5/5-08.pdf

Hoping this will be helpful,

Rafik

dear rafik You're very generous thank you very much.Life for Palestine.

Brother Kamal from Algeria.

good questions dear gaci