Oocyte cryopreservation, also egg freezing, is an assisted reproduction technique used to preserve these cells at very low temperatures, exactly -196 °C in liquid nitrogen, without affecting their viability and for an indefinite time.
Its main indication is the preservation of fertility, that is to say, to freeze the eggs of a woman when she is still young in order to be able to be a mother in the future.
Mainly, there are two methods to freeze eggs, which we will detail and compare throughout this post:
- Slow freezing
The different sections of this article have been assembled into the following table of contents.
Factors influencing freezing
The egg is the largest cell in the human body. It has a size of 0.14 mm and a high water content. Moreover, before fertilization, the egg is in a very complex stage known as metaphase II, in which the division of its genetic material is arrested.
Because of this, it is extremely difficult to freeze eggs without damaging them. Low temperatures can lead to depolymerization of microtubules, which leads to disorganization and dispersion of chromosomes.
Also, the high water content increases the likelihood of ice crystals forming, which would cause damage to the egg's internal structures and cell membrane.
Finally, excessive dehydration of the egg during freezing can also lead to the so-called solute effect (increase in the concentration of intracellular salts) and the theory of minimum volume, which can lead to irreversible collapse and cell death.
Therefore, in order to prevent all of these problems from arising during egg freezing and to increase survival rates, it is very important to control the following factors:
- The cooling rate or speed
- Dehydration and the fate of intracellular water
- The surface/volume ratio of the egg
- The use of cryoprotective agents
What are cryoprotectors?
Cryoprotectors are substances used in all egg freezing protocols, as they protect eggs from all the harmful effects mentioned in the previous section.
Depending on their ability to penetrate inside the cell, cryoprotectors are classified into:
- replace the aqueous liquid inside the egg, prevent the formation of ice crystals and help counteract the effect of high concentrations of solutes. The most commonly used are glycerol, dimethyl sulfoxide (DMSO), propanediol (PROH) and ethylene glycol (EG).
- promote controlled cell dehydration, which also prevents the formation of ice crystals and cell collapse. The most commonly used are polyvinylpyrrolidone (PVP), dextran, albumin and sugars such as sucrose and glucose.
Normally, permeable and non-permeable cryoprotectors are used together for greater efficiency.
However, the survival rates of this slow method after thawing were very low and, as a result, today it has been practically replaced by the ultra-fast technique known as vitrification.
In the next section, we are going to explain what each of the two methods for freezing eggs consist of, as well as what their advantages and disadvantages are.
Slow freezing of eggs
The technique of slow egg freezing consists of making the temperature fall little by little, at the same time that the cell is dehydrated with the use of cryoprotectors.
This minimizes the formation of ice crystals, although there may be a toxic effect due to prolonged exposure time to cryoprotectors.
To carry out the process, a programmable freezer is needed in which the temperature will gradually drop to a temperature between -40 and -70 °C. The eggs are then immersed in liquid nitrogen, where they will quickly reach -196 °C.
For the thawing of the eggs, the straws in which they are contained are extracted from the liquid nitrogen, kept at room temperature for 30 seconds and immersed in a bath at 31 °C, where the eggs are released from the inside of the straw.
The eggs are sequentially rehydrated in different culture media, the remains of the cryoprotector are removed and finally regain their normal structure.
Vitrification of eggs
Vitrification is an ultra-fast freezing technique with a cooling speed of up to 23,000 °C/min. Thanks to this, the water inside the cell does not have time to crystallize, it goes from a liquid state to a vitreous state, an amorphous solid similar to a hard gelatin.
Vitrification requires a high concentration of cryoprotectors in order to be successful. However, in order to avoid toxicity, two strategies have been carried out:
- Reducing exposure time as much as possible
- Using the minimum volume of medium to vitrify
These strategies make it possible to increase the cooling speed and avoid possible damage to the eggs.
During the vitrification process, the eggs are passed through balancing media with increasing concentrations of cryoprotectors. The eggs are then dehydrated and placed on a vitrification support with as little volume of medium as possible.
As a last step, the eggs are introduced directly into liquid nitrogen at -196 °C, so freezing is immediate.
The devitrification technique also follows the same procedure of passing the eggs through balancing media to rehydrate the cell and eliminate cryoprotectors.
Advantages and disadvantages
In general, vitrification has multiple advantages over the slow freezing of eggs:
- The egg survival rate is much higher with vitrification, around 90%.
- The implantation rate of embryos from vitrified eggs is higher than that of embryos from slow-frozen eggs.
- By using vitrification, the risk of crystallization and cryofracture of the eggs is lower.
As a possible disadvantage of vitrification, we can say that it is a very laborious practice and that it requires a lot of experience on the part of the embryologist who carries it out.
For all that has been said throughout this post, vitrification is the technique of choice in egg cryopreservation today.
All assisted reproduction laboratories use it regularly, which has allowed the creation of donor egg banks and the preservation of fertility in women with cancer or for those who want to delay their maternity.
If you are considering freezing your oocytes to have a baby in the future, we recommend that you get your Fertility Report now. In 3 simple steps, it will show you a list of clinics that fit your preferences and meet our strict quality criteria. Moreover, you will receive a report via email with useful tips to visit a fertility clinic for the first time.
FAQs from users
Is slow oocyte freezing still in use?
The slow freezing of oocytes was the first oocyte conservation technique to develop. Although it offers good results in sperm conservation, it does not show good survival results in oocytes. The crystals formed as a consequence of slow freezing damage the oocyte, decreasing the success rates in assisted reproduction treatments.
Since the last decade, oocyte vitrification has been the main technique of choice for the conservation of oocytes. Cryopreservation allows us an ultra-fast freezing, which prevents the formation of ice from the water contained in the cell in question (the oocyte), thus avoiding any deterioration. During the vitrification process, we introduce the oocyte into a liquid nitrogen solution and reach a temperature of -196 °C in less than one second. The rapid speed means that the water contained in the oocyte does not freeze and vitrify, becoming a state similar to that of a consistent gelatine.
Until what age can eggs be frozen?
There is no age limit for freezing eggs, but you have to bear in mind that their quality decreases as women get older. For this reason, it would be advisable to preserve fertility before the age of 30-35.
Is special treatment required for egg vitrification?
Yes, in order to vitrify the eggs, it is necessary to extract them through follicular puncture. In addition, in order to obtain a greater number of eggs in this surgical intervention, the patient will follow a controlled ovarian stimulation treatment.
Therefore, the woman who is going to vitrify her eggs, either to preserve fertility, to undergo an IVF process or for egg donation, will have to go through these two processes: medical stimulation of ovulation and ovarian puncture.
Suggested for you
If you want to know more about the process of egg vitrification, we recommend you read on in the following post: How does egg vitrification work and what are its advantages?
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Levi-Setti PE, Patrizio P, Scaravelli G. Evolution of human oocyte cryopreservation: slow freezing versus vitrification. Curr Opin Endocrinol Diabetes Obes. 2016;23(6):445-450.
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