What’s the Difference between the Freezing and Vitrification of Eggs?

By BSc, MSc (embryologist), MD, PhD, MSc (gynecologist), BSc, MSc (embryologist), BSc, MSc (embryologist) and (invitra staff).
Last Update: 10/23/2019

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
  • Vitrification

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:

Permeable
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).
Non-permeable
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.

Cryopreservation methods

Slow freezing is the traditional technique used to freeze eggs and embryos in all in vitro fertilization (IVF) laboratories.

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?

By Dr. Paloma de la Fuente Vaquero MD, PhD, MSc (gynecologist).

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?

By Zaira Salvador BSc, MSc (embryologist).

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?

By Andrea Rodrigo BSc, MSc (embryologist).

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?

If you are also interested in knowing the indications for preserving fertility in both men and women, click here: Fertility preservation.

Our editors have made great efforts to create this content for you. By sharing this post, you are helping us to keep ourselves motivated to work even harder.

References

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?', 'Until what age can eggs be frozen?' and 'Is special treatment required for egg vitrification?'.

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Authors and contributors

 Andrea Rodrigo
Andrea Rodrigo
BSc, MSc
Embryologist
Bachelor's Degree in Biotechnology from the Polytechnic University of Valencia. Master's Degree in Biotechnology of Human Assisted Reproduction from the University of Valencia along with the Valencian Infertility Institute (IVI). Postgraduate course in Medical Genetics. More information about Andrea Rodrigo
Dr. Paloma de la Fuente Vaquero
Dr. Paloma de la Fuente Vaquero
MD, PhD, MSc
Gynecologist
Bachelor's Degree in Medicine from the Complutense University of Madrid, with a Master's Degree in Human Reproduction and a Doctorate in Medicine and Surgery from the University of Seville. Member of the Spanish Fertility Society (SEF) and the Spanish Society of Gynecology and Obstetrics (SEGO), she performs as a gynecologist specializing in assisted reproduction in the clinic YES! Reproducción. More information about Dr. Paloma de la Fuente Vaquero
License: 4117294
 Sara Salgado
Sara Salgado
BSc, MSc
Embryologist
Degree in Biochemistry and Molecular Biology from the University of the Basque Country (UPV/EHU). Master's Degree in Human Assisted Reproduction from the Complutense University of Madrid (UCM). Certificate of University Expert in Genetic Diagnosis Techniques from the University of Valencia (UV). More information about Sara Salgado
 Zaira Salvador
Zaira Salvador
BSc, MSc
Embryologist
Bachelor's Degree in Biotechnology from the Technical University of Valencia (UPV). Biotechnology Degree from the National University of Ireland en Galway (NUIG) and embryologist specializing in Assisted Reproduction, with a Master's Degree in Biotechnology of Human Reproduction from the University of Valencia (UV) and the Valencian Infertility Institute (IVI) More information about Zaira Salvador
License: 3185-CV
Adapted into english by:
 Romina Packan
Romina Packan
inviTRA Staff
Editor and translator for the English edition of inviTRA. More information about Romina Packan

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