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
Provided below is an index with the 6 points we are going to expand on in this article.
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.
Can thawed eggs be re-vitrified?
It is possible, but not recommended. Vitrification of eggs that have been previously thawed will only be performed in cases of force majeure, for example, when it has not been possible to obtain sperm for insemination. Normally, when eggs are thawed it is for use in an assisted reproduction treatment. These eggs will be inseminated and embryos will be created. These embryos can be re-vitrified without any problem.
Re-vitrifying an egg that has just been devitrified would increase the osmotic stress, and although it is possible to do so, it is also possible that an egg could be damaged in the process. It must be taken into account that the ovum is the largest cell in the human body, and therefore, the most sensitive in the vitrification and devitrification process.
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.
Which is better? Egg freezing or egg vitrification?
Without a doubt, vitrification. This technique is the one that has really made it possible to offer the technique of female preservation with all the guarantees.
First of all it would be necessary to distinguish the two techniques and their history, as well as why the conventional freezing technique was relatively valid for embryos but not for oocytes.
With the conventional freezing technique, cryoprotectants are used in low concentration for a relatively long time and a slow progressive cooling. This sometimes results in the formation of crystals that are lethal to the cells. The larger the cell, the greater the possibility of crystals forming, and if we take into account that the egg is the largest cell in the human body, this explains why this technique has produced very poor results with eggs.
The vitrification technique consists of exposing embryos and oocytes to high concentrations of cryoprotectant in a relatively short time and ultra-fast cooling (in the order of 20,000 ºC/min). As a result of this process a vitreous mass is formed (hence the name vitrification) avoiding the formation of crystals that would damage the cell.
Therefore, vitrification of eggs is undoubtedly better than freezing. It is this technique that has really made it possible to offer the technique of female preservation in a standardized way with all the guarantees.
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.
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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Cao YX, Xing Q, Li L, Cong L, Zhang ZG, Wei ZL, Zhou P. Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification. Fertility and Sterility 2009; 92 (4): 1306-1311
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.
Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, Vanderpoel S, Racowsky C. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23(2):139-155.
FAQs from users: 'Is slow oocyte freezing still in use?', 'Can thawed eggs be re-vitrified?', 'Until what age can eggs be frozen?', 'Which is better? Egg freezing or egg vitrification?' and 'Is special treatment required for egg vitrification?'.
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