Freezing

Preservation by freezing

In women of reproductive age numerous diseases can occur and their treatment may affect the activity of the ovary. The egg producing capacity of the ovary can be irreversibly stopped by medicines (chemotherapy) used in treating malignant tumor diseases (mammal tumor, melanoma, leukemia, cervical cancer, ovarian cancer in early stages etc.),but at the same time, due to current efficient therapy, the patients can be provided with improved survival rate and quality of life. Some gynecological diseases can appear, but not necessarily be malignant (great size ovarian cysts, endometriosis, tumor of the germinal epithelium etc.) requiring surgical treatment, causing lower cell productive function of the ovary. It may happen that the ovaries stop their function long before the age of fifty and the so-called ovarian failure occurs.

Preservation of the ovule

Nowadays, the method, still in an experimental stage a few years ago, has already become a routine procedure. Its advantage lies in the fact that preservation by freezing can be solved in the absence of he partner. The difficulties of freezing, preservation and use of the ovule are also proved by the fact that although the first fetus conceived with a thawed frozen egg was born in 1986, the number of successful interventions has been very low worldwide. The ovule is a relatively big size cell, with a high content of water, due to which it is fragile. Conservation solutions (the so-called cryoprotective materials) and special freezing methods were developed to improve the outcomes, but the cells can suffer damages due to ice crystal formation. As a result of the so-called slow freezing – followed by a rapid thawing the survival rate is about 50%, but the pregnancy rate barely reaches 10%.

Currently a completely new method for freezing ovules and pre-embryos is spreading, the so-called vitrifying, which guarantees a significantly higher survival rate (64-69%) and pregnancy rate. The freezing of the immature ovules can also be considered a new still developmental method. Immature cells have a smaller volume and, consequently, a lower content of water, and thus during the cryopreservation ice crystals formation could produce less lesions. After thawing, mature ovules can be created after artificial ovulation, suitable for fertilization. The other also developmental procedure differs from the previous one by the order of the work steps: immature cells obtained from the ovary will be first subjected to ovulation and then mature cells freezing takes place. Another advantage of both methods is the fact that it is not necessary to stimulate the ovaries and thus no time is wasted before beginning the causal therapy, the procedure being achieved even with ovules from the second part of the cycle. We must not forget that even if the partial results are very encouraging, very few pregnancies have been reported after thawing, fertilizing and transferring eggs obtained through the above described methods.

Preservation of the pre-embryo

The preservation by freezing fertilized ovules is the most complex and detailed procedure, considering its technique. The survival rate of the pre-embryos preserved this way is of 35-90%, the implantation rate calculated per embryo is between 8-30%, while the cumulative pregnancy rate, calculated after several implantations overcomes 60%.

Of course, the method also has difficulties which cannot be neglected. One of the criteria implies the patient to have a husband/life partner.

Another disadvantage is the fact that ovarian stimulation must be used (this is why it cannot be used at a very young age), which delays by a few weeks the beginning of the irradiation therapy/chemotherapy. We mustn’t overlook either the fact that multiple ovulations significantly increase the level of female hormone, an aspect which has to be considered in the case of tumors sensible to hormones (e.g. mammal cancer). The embryos preserved in liquid azote at minus 196 °C, basically preserves its viability for an unlimited period of time.

To review: before beginning the fertility jeopardizing treatments, patients can select from different available methods through which their fertility capacity can be preserved. A significant part of these procedures is elaborated precisely from a technical point of view; the used solutions provide stability, while the freezing devices are computerized. For the recommended method which will be applied, the age of the patient, the stage of the tumor,its possible hormonal dependency, the state of her relationship with the partner must be considered. Informing the patient about the expected efficiency is indispensable, but it is also important to permanently inform her about the fertility preservation possibilities and methods, which have been dynamically updated.


Freezing the fertilized egg (Cryopreservation)

If more than three ovules are fertilized, there is a possibility of freezing the extra ones, if they can be preserved and subsequently transferred into the uterus in an untreated cycle.
This is particularly useful when the embryo transfer right after egg retrieval does not produce any results. The frozen pre-embryos can be also preserved for the second or third pregnancy.


Beginnings of cryopreservation (freezing)

Creating more and better embryos after the drug therapy of the ovary, besides improving the pregnancy rate, it also raised professional and ethical issues: what happens to the extra embryos remained after the transfer?

Cryopreservation has its roots back in the 1940’s, when freezing and the preservation of the sperm was solved, the method being initially applied in veterinary medicine. Freezing and conservation at minus 196 degrees of the fertilized ovules proved to be a much more difficult task. For the development of the appropriate materials which facilitate the preservation (cryoprotective substances), very many experiments were necessary, from manual freezing to creating computerized freezing devices, and to creating preservation recipients. In 1983 the efforts turned out to be successful for the first time, when the Lancet newspaper announced the first pregnancy which resulted after freezing and thawing of an octocellular embryo. However the first successful birth occurred only after another year and the success from 1984 belongs to the Rotterdam work group, Zielmaker and his colleagues. Nowadays cryopreservation has become a routine procedure, the law imposes every clinic performing IVF to freeze and preserve the extra embryos. Even after a period of several years following the freezing of embryos there is a good chance that out of the thawed and transferred embryos healthy children to be born.

Vitrification

Using high quality culture media results in a large number of viable embryos. This, associated with a treatment plan which recommends the transfer of a single embryo emphasizes the crucial importance of the vitrification of the excess embryos.

Vitrification is a rapidly evolving cryopreservation technique of the gametes and embryos. The definition of vitrification is “an instant solidification of a solution brought about by an extreme elevation in viscosity during cooling without ice crystal formation”. Vitrification is a fast cryopreservation procedure with high survival rates, due to low risks of damages to the cells from ice crystal formation.

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