Equipment of slow programmable freezing. (Eugonia archive)

Cryopreservation is the preservation of cells, tissues or even organs at very low temperatures, with the intention of future use. At the temperature of -196oC, metabolism stops and the cells enter a state of suspended animation making it possible to store them for a long time. To achieve such a low temperature we use liquid nitrogen. The first successful cryopreservation was performed in frogs in 1945. The first animal sperm banks operated in 1949. The first successful inseminations I the human were reported in 1953 (preservation in dry ice, -70oC) and in 1964 in liquid nitrogen. Embryo cryopreservation was first performed in laboratory animals in 1972 and in human in 1984.

Today, cryopreservation is a routine method, allowing the storage of gametes and embryos for an extended period.

Embryo cryopreservation gives couples the opportunity to use frozen embryos for a future pregnancy without requiring ovarian stimulation, oocyte retrieval or sperm collection, but only cycle monitoring and embryo transfer. These embryos can be used in case that the couple wishes to have one more child. Otherwise, if the first IVF has failed, the embryos can be thawed and transferred in a following cycle.

Internationally, success rates of pregnancies with cryopreserved embryos are slightly lower than those with fresh embryos. This is because usually the best embryos have already been selected and transferred, and also because all embryos may not survive after thawing. In the case of elective cryopreservation (in which only Class I or II embryos are cryopreserved), pregnancy rates are higher and tend to be closer to those of fresh embryos.

Concerns and objections that arise from cryopreservation of embryos mostly have an ethical basis. It is true that cryopreservation means a temporary suspension in the life of an embryo. During this time the conditions may change in the lives of the parents.

According to the Greek legislation (Ν. 4737/2020 (ΦΕΚ Α’ 204/22-10-2020) cryopreserved embryos can remain in storage for up to 5 years. The duration of cryopreservation can be extended for a maximum period of twenty (20) years with a written request of the couple every five (5) years.

In Greece, law 3089/02 describes the potential fate of cryopreserved embryos in case the parents disagree on their use. IVF Units with a good level of organization should have special cryopreservation consent forms that must be signed by the two partners the fate of regarding supernumerary embryos.

The procedure of cryopreservation is considered to be safe for children born. All relevant epidemiological studies on thousands of babies born from cryopreserved embryos are reported to be healthy without a statistically important increase of congenital abnormalities. Similar results are reported for children born after the use of frozen donor sperm.

The transfer of frozen embryos is scheduled so that the uterine environment is favorable to receive the embryo, and have an optimal implantation period. The preparation of the endometrium happens either during a natural cycle, or a pharmaceutical cycle. The embryos are usually thawed a few hours before the embryo transfer.

After thawing, the number of embryos and blastomeres that have survived are evaluated. Embryo quality is assessed and the number of embryos for transfer is then decided.

Not all embryos have the same ability to survive the freezing-thawing process and about 20-30% of their cells are destroyed. However, it has been proven that embryos that successfully survive after thawing have the same implantation potential as fresh embryos, and sometimes even higher, due to a better implantation window.

Frozen-thawed Day-3 embryos with excellent survival.

The damage of one or two blastomeres in a day 3 embryo does not impair successful implantation. At this stage the cells are still totipotent, i.e. they have not been differentiated yet. There are reports of live births following transfer of thawed embryos that had lost 7 out of 8 cells.

In addition, there are reports of live births in cases when frozen-thawed embryos, were re-frozen as supernumerary, showing that cryopreservation does not seem to affect the chromosomal status of an embryo.

During vitrification, embryos are exposed in special cryoprotective solutions, then loaded onto special vessels and plunged directly in liquid nitrogen. In this way the formation of glass is induced in the inside of the cells, avoiding the formation of ice crystals that are detrimental for embryo viability.

Vitrification is a more recent method of freezing, and has been successfully applied on all embryonic stages, as well as oocytes. Especially for freezing of oocytes, vitrification is now the method of choice as it is related with higher survival and pregnancy rates compared to slow freezing. Also, vitrification does not require the use of a special machine, and is much faster than slow freezing, which last for 2-3 hours.

However, vitrification requires experienced embryologists as it is a matter of seconds during freezing and thawing if the embryos will retain their viability or not.

Before freezing, embryos are first equilibrated in special solutions containing cryoprotecting agents. These agents protect the embryos from intracellular ice formation, which would be detrimental to their viability. The embryos are then placed in special straws, sealed and put in a machine with an integrated computer. This machine, the programmable freezer, lowers the temperature in a slow controlled manner until -196oC. There are several protocols of slow freezing, depending on the stage of the embryos and the type of cryoprotectant solutions used. The embryos are then plunged in liquid nitrogen and are stored until used.

Usually 1-3 embryos are placed in each straw. In this state, embryos may be preserved for a very long period of time. Embryos can be frozen at the 2PN stage (Day 1), cleavage stage (2-8 cells; Days 2-3), or blastocyst stage (Days 5-6 post oocyte retrieval).

Cryopreservation in liquid nitrogen at a temperature of -196oC does not require electric power. The only requirement is the replenishment of liquid nitrogen in the tanks containing the embryos.

Procedure of embryo thawing. The embryologist locates and retrieves the straw containing the frozen embryos from the liquid nitrogen bank (Eugonia archive).

During an IVF cycle it is very common to have surplus embryos of good quality after the performance of the embryo transfer. These embryos can be frozen and stored in an IVF laboratory, either using a programmable freezer or using a rapid freezing method called vitrification. The embryos are stored in large dewars with liquid nitrogen (-196oC).

Cryopreservation of embryos offers us several possibilities:

• The storage of surplus embryos of good quality
• Postponing embryo transfer is special cases like poor endometrium, or severe OHSS.
• The preservation of reproductive potential of a woman prior to chemotherapy, radiotherapy or ovariectomy.

Cryopreservation complements other ART methods and allows the increase of cumulative pregnancy rates, reducing hormone administration and the total cost.

If the measurements of b-hCG are found negative (b-hCG levels < 5 units), the drugs are discontinued following instructions from the doctors and menstruation (period) will follow a few days later.

We are aware of the unpleasant feelings that stem from a failed attempt. The only way to deal with this situation is to examine, together with the couple, all the parameters that may have lead to a negative result.

The interval between two consecutive IVF cycles must not be smaller than 3 months to give the ovaries time recover, and to yourselves time to recuperate psychologically.

It is important not to lose hope in the face of failure. We are here to help and support you in this difficult phase.