Freezing and storage of gametes and embryos

Cryopreservation(from Greek cryos— “cold” and preservatio— “storage”) refers to the group of processes of preparation, freezing and storage of human gametes, pre-implantation embryos and tissues at ultra-low temperatures, most often in liquid nitrogen. It plays an important role in assisted reproduction, as it enables reproductive cells collected and/or fertilized in one treatment cycle to be used for fertilization and/or implantation in the patient's uterus in a future treatment cycle. This increases the effectiveness of infertility treatment and reduces costs and discomfort for patients.

Basic method of cryopreservation of embryos in the Invitro clinic “Sofia”

In 2008. In vitro clinic “Sofia” includes vitrification as the main method of freezing gametes and embryos. It is a super-fast freezing technique that results in the formation of a glass-like state in living cells. It has been proven that with this method, the formation of ice crystals in the structure of reproductive cells and embryos during freezing and thawing is avoided. When thawing sperm, eggs and embryos frozen by vitrification, we achieve over 90% cell survival.

Manifestations of cepmatozoids

The name of the cepematozoid will be used to help the specific cpede through the so-called 'buff method'. In most cases, cpematozoidites can be represented by several species without a trace of the disease. If they do not need to be used, cppematozoidites are published as part of a specific species and are used for the purpose of specific species. These are typical pepody technologies. This is the case with the following applications:

• In couples undergoing assisted reproductive technologies, in which on the day of the procedure the partner will be absent;
• Poor semen sample parameters, in order to accumulate more sperm. It is possible to carry out multiple freezing, thereby storing individual frozen seed samples. If necessary, samples shall be collected and processed. As a result, a greater amount of sperm is obtained to be used;
• Difficulty of the partner to secrete seminal fluid - in this case it is recommended in advancefreezing of seminal fluid. The frozen semen sample is thawed and used if, on the day of intrauterine insemination or in vitro/ICSI follicle puncture, the partner fails to excrete ejaculate;
• Male anti-radiation or ximiotepapia in the event of malignant diseases;
• Upcoming vasectomy;
• Upcoming surgical interventions for oncological or other diseases of the testicles;
• Cases of men with Azoospermia (lack of sperm in the ejaculate), when, by biopsy, spermatozoa are detected in the epididymis or testicle;
• Storage of material for the donor program;
• Own desire.

When freezing seed samples it is necessary:  

• Make an appointment to freeze and store sperm, with sexual abstinence between 3 and 5 days, without alcohol and antibiotics;
• To carry out preliminary examinations in the man, which include: microbiology of ejaculate and virology — AIDS, Syphilis, Hepatitis B, Hepatitis C;
• Complete a Declaration of Informed Consent for the freezing and storage of sperm, which also indicates the desired storage period.

On the day of delivery of the ejaculate, the material is evaluated and an opinion is given whether it is subject to freezing. The next step is to perform a cryo-tolerance test — a small portion of the ejaculate is frozen and thawed in order to assess sperm survival after thawing.

Seminal fluid is frozen in special cryopreservation media, certified for quality and approved for use in ART procedures, harvested for storage in specially designed containers with liquid nitrogen, at a temperature of —196°Cin conditions of complete traceability and security.

Egg freezing

Although embryo cryopreservation is the most common method of preserving fertility, nowadays the freezing of eggsis the most applicable option for women of reproductive age who need to postpone motherhood for various reasons. The difficulty of freezing, due to the characteristic structure of the ovum, cost more than 20 years of development of the methods before the technique reached clinically significant success rates. Three main achievements contributed to its success: the development of the ICSI method, the improvement of cryoprotectants and the implementation of the vitrification method.

This opened a new era in the cryopreservation of reproductive material. Egg freezing is a leading method in preserving fertility and is routinely used in IVF in a number of cases, such as ovarian hyperstimulation, poor ovarian response, genetic mutations, donation, etc.

The egg is larger than any other cell in the human body, measuring about 100 microns in diameter, about the size of a human hair. This means that theoretically we can see the egg with the naked eye. Also, eggs are about 4 times larger than epithelial cells and about 20 times larger than sperm. The egg contains genetic material, 96% water and energy substances. The fact is that eggs need a lot of energy, especially after they are fertilized. Human eggs contain a large amount of mitochondria, which provide energy for cells by converting oxygen and nutrients into chemical energy. Their concentration is so great that this single cell supplies energy for the formation of an entire embryo and the birth of life during the first 7 days of the zygote division. Preserving the uniform integrity of each component of the egg is essential for freezing success.

How do you freeze eggs?

In order to obtain the optimal number of eggs for freezing, the patient must undergo hormonal stimulation similar to that of an in vitro fertilization procedure. During this process, the growth of follicles is monitored, hormonal levels are monitored, hormonal medications are prescribed to stimulate the ovaries, and a puncture is made to remove the eggs. After aspiration, the eggs are processed and placed in special cryoprotective media that protect the eggs when frozen. They aim to extract or convert the water content of the cell into a gel - this is an important condition for the survival of eggs after freezing. The egg is passed through a series of such solutions and, when the water content in it decreases, is placed in special containers (Duar vessels), where it is stored in liquid nitrogen (at -196 °C). After thawing, the ovules suitable for fertilization are about 80-90%.

When are eggs frozen?

• In women who plan to postpone pregnancy and the birth of a child to a later stage. The recommended age for freezing is up to 35 years, when ovarian function is active, there is a significant ovarian reserve, and the ovaries respond well to stimulation. Until this age, the probability that the eggs are carriers of a genetic abnormality is less;
• In order to preserve the fertility of a woman after chemo or radiation therapy in oncological disease;
• In women in whom the ovaries reduce their function prematurely at an earlier age;
• In women with a family history of premature menopause.

Embryo freezing

In standard IVF or ICSI procedures, a woman's ovaries are stimulated to produce an optimal number of eggs. After their fertilization in laboratory conditions and the cultivation of the embryos for 3-6 days, they are selected for embryo transfer and/or freezing and storage. Not all embryos are of the necessary quality to be frozen and stored. Only those that possess the potential to survive the process of freezing and thawing are selected. This assessment can only be made by a specialist embryologist, after considering the entire period of embryo development. In addition to quality, the survival of embryos also depends on a well-performed freezing procedure and proper storage.

Embryo freezing is a method of preserving the viability of embryos over a long period of time at ultra-low temperature (-196oC). Embryos can be frozen at any stage of their development if they are of good enough quality to survive the freeze-thaw cycle. Embryos are stored one at a time or in a group of two, depending on the number of embryos planned for embryo transfer. Specialised plant for the treatment of diseases, medicine and medicine “Cofia” in the field of medicine, medicine and medicine “Cofia”. a pathetically pleasing position from a close perspective. Bitpifiation is a text that is used to express itself, that is, to be used as a result of the existence of a person in a living environment, so they will thus avoid the fopmipathing of the winter months when they are caught and unannounced.

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Embryo freezing is recommended for:

•Ovarian hyperstimulation syndrome (OHSS), when it is not advisable to carry out embryo transfer in the current cycle
•If the lining of the uterus (endometrium) is not suitable for implantation in the current cycle
•Remaining embryos of good quality after embryo transfer; so after the “fresh” transfer they can be used if pregnancy does not occur or if there is a repeated desire to reproduce;
•Desire to reduce the risk of multiple pregnancies;
• The need to preserve fertility for a future period.

Advantages of embryo freezing:

• Frozen embryos provide patients with additional transfer options, which increases the chance of success from a single puncture.

If the fresh transfer is unsuccessful, the frozen embryos provide you with additional attempts, without the need to conduct repeated ovarian stimulation and a new follicle puncture.

• Frozen embryo transfers (FETs) are cheaper than fresh ones. After conducting a fresh IVF cycle, subsequent FET cycles appear more accessible to patients. With the FET cycle, the cost of medication is less. The difference in prices also comes from the fact that with FET cycles, patients pay for a smaller number of check-ups, and also do not go through a re-puncture, a procedure of fertilization and cultivation of the embryos.
• The FET cycle is more sparing. For a large proportion of patients, FET cycles are more sparing, due to the lack of hormonal stimulation, surgery (puncture) and anesthesia.
• Freezing embryos for future FET cycles overcomes the adverse effect on pregnancy outcome that is triggered by elevated progesterone levels.According to the most recent studies, increased progesterone levels during ovarian stimulation make the endometrium (lining of the uterus) more unsusceptible to embryo implantation, and therefore the chances of getting pregnant are significantly reduced. If progesterone levels rise above the permissible, it is recommended to freeze all embryos instead of carrying out a fresh transfer. Results from a number of studies have shown that frozen embryo transfers are associated with an increased chance of success, compared to fresh transfers, in cases where elevated progesterone levels are involved.
• Frozen embryo transfer leads to a reduced risk of so-called ovarian hyperstimulation syndrome, and at the same time preserves excellent chances of a successful pregnancy. The team of doctors of SBALAGRM “Sofia” carefully selects the stimulation protocols in order to avoid the triggering of the syndrome. It is for this reason that in our practice this syndrome occurs in less than 1% of patients. However, if the doctor considers that the patient falls into the risk group (she has a large number of follicles and high levels of estrogen, sudden weight gain, accumulation of fluid in the pelvic area, etc.) he may recommend freezing the available embryos instead of performing a fresh transfer, as a possible pregnancy can further increase the risk of developing the disease.
• Frozen embryos allow the creation of a family at a later stage. Frozen embryos preserve their reproductive potential far in time, thus allowing you to choose the most suitable moment for raising your children yourself. This type of transfer can slow down your biological clock.

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