VITRIFICATION (ΥΑΛΟΠΟΙΗΣΗ in GREEK):

About 20% of all offspring born worldwide from IVF cycles are from oocyte and embryo cryopreservation.


Ice crystals should never be allowed to appear and grow inside the cells of the embryo and two basic approaches were developed:

A) Controlled slow-rate freezing
B) Vitrification protocol

Main Principles of Cryopreservation

Initial exposure to cryoprotectant (intracellular water has to be removed)
Cooling* (slow / rapid) to subzero temperature (-196°C)
Storage
Thawing or Warming (slowly or rapidly)
Dilution and removal of the cryoprotectant agent and replacement of the cellular and intracellular fluid at precise rate
Return to a physiological environment

*Embryos can be successfully cryopreserved by being cooled slowly (0.3°C/min) or rapidly (³ 10,000°C/min)


Vitification is not a new technique and practised widely in other parts of the world but only now is it gaining popularity in the west.

Vitrification, or achieving a glass-like state (-130°C for water), was first described as a potential alternative to slow freezing in 1985. Since chilling injury to oocytes is time dependent, the rationale is to prevent ice formation and injury by cooling at a rate fast enough to solidify the intracellular water before it can crystallize.



Human eggs have high water content and are therefore extremely fragile and sensitive to all freezing procedures.
Controlled rate slow-freezing methods do not always succeed because they allow some intra-cellular ice crystal formation that causes some damage to embryos, and severe damage to most eggs, render them unusable. However, vitrification avoids the damage caused by crystal formation inside the cell increasing dramatically their survival rate.
Healthy children have already been born after insemination of vitrified oocytes.
Another advantage of vitrification is that it is possible to observe the entire procedure in the laboratory.

Read more on freezing in ivf…..



Freezing of human sperm, oocytes and Embryos

Human eggs


Unfortunately, the technology to freeze human eggs is still being developed. Due to the low success rates of this procedure, our centre does not routinely offer egg freezing but only in specific cases.

Sperm freezing


Sperm can be frozen on demand and tolerate freezing more readily than Embryos. Its small water content and tightly packed DNA head make freezing sperm relatively easy. Since1953 human semen has been successfully cryopreserved, with pregnancies thereafter.If sperm can be found in testicular tissue from azospermic men this too can be frozen. We can freeze sperm for men who cannot attend on the day of their partners' egg collection procedure, or for men who are anxious about producing a sample or for men due to undergo therapy and a loss in fertility may occur.
The sperm are stored in carefully labeled vials and lowered into a nitrogen tank.

Freezing and storing human embryos


Since 1984, thousands of babies have been born worldwide after having undergone embryo cryopreservation.Freezing of excess good quality Embryos allows for the transfer of fewer embryos in the stimulated IVF cycle and therefore ensures fewer high-order (triplets or more) multiple births. This process provides patients with a "back-up" should the initial fresh embryo transfer not result in a pregnancy, at a much lower cost than starting IVF all over again and often with minimal medications. Frozen embryo transfers have allowed many of our patients to achieve more than one pregnancy from a single cycle of ovarian stimulation.Freezing is a very traumatic procedure for an embryo and not all embryos survive. It is normal for one or more of the cells in the embryo to die as a result of the Freezing and thawing procedure. However, provided that the majority of the cells in an embryo survive, the embryo still has a chance to establish a pregnancy.
How are Embryos frozen?Embryos can be frozen at different times after fertilization. Most typically, Embryos are frozen 1,2,3 after fertilization but also recently at the blastocyst stage.Embryos are frozen with cryo-protectant, placed in special straws and stored in liquid nitrogen.

Do not be disheartened if there are no embryos capable of being frozen, the best embryos are always transferred-we do not wish to jeopardise the chance of a pregnancy by freezing the best embryos. Since freezing is a stressful process for an embryo, and only embryos that are growing well in the laboratory will tolerate the freezing procedure we only freeze if the embryo is of good quality.

how is slow freezing done

See video link opposite on YOUTUBE

Step 1. Before an embryo can be frozen, all the water that it contains must be removed. Since water expands in size as it turns to ice, water inside the embryo would burst the embryo if we simply placed it in the freezer. To prevent the embryo from shrivelling as the water is extracted, we replace the water with antifreeze. Antifreeze is a solution that does not expand in size when it freezes. The embryo is cooled to room temperature as the water is replaced with antifreeze.

Step 2. When most of the water has been removed the embryo is inserted into a carefully small straw, and placed in the cooling chamber of a controlled rate freezer.

Step 3. The embryo is then cooled very slowly. Slow cooling like this allows the embryologist to have precise control over the freezing process, to maximize water extraction from the embryo and to prevent formation of ice crystals that could harm the embryo. The freezing of the straw is manually induced at the end of the straw to prevent damage to the embryos.

Step 4. The cooled straw is placed into carefully labelled metal canes and lowered into the tank with other frozen Embryos. The entire process takes several hours and the embryo (s) are stored frozen at –196 C in liquid nitrogen.

read this in GREEK: http://www.kosmogonia.gr/40_04_freezingnew.htm