Oocyte and Embryo Vitrification
Vitrification is a technology that is used in the embryo and egg freezing process so that they can be stored for later use. It is a technology that has many uses outside of fertility care with egg and embryo freezing, as it allows something with a crystalline structure to be converted into something very smooth. The classic example is the creation of glass using sand (which is crystalline) as the main ingredient. Other examples include the manufacture of china plates and cups (also using sand), cotton candy from sugar (also crystalline) or ice cream which is smooth to the taste and contains no ice crystals despite its frozen state.
When we freeze cells in a lab, the main focus of the process is avoiding ice crystal formation as the fluid in the cell cools to subzero temperatures. Ice crystals pose 2 significant and deadly problems for cells. First, despite its beauty, an ice crystal is razor sharp and will readily shred any cell membrane, killing the cell. Second, as water in the cell turns to ice, it expands in volume, rupturing (killing) the cell. As a result, processes must be developed that allow cells to be frozen while avoiding the formation of ice. This science, called cryobiology, has come up with 2 methods that work well with human embryos, slow freezing and vitrification.
Human embryos were first successfully frozen and used to establish a pregnancy in Australia in 1984. The procedure used to preserve the embryos was aptly called slow freezing since the embryos were cooled at a rate of just 0.3ºC/minute until their temperature had passed below -30ºC. They were then stored in liquid nitrogen (at -196ºC) until the time of thawing. Ice crystal formation was avoided by incubating the embryos in chemicals called cryoprotectants, which draw water out of the cells in the embryo before the embryos freeze. By removing much of the water and replacing it with cryoprotectants, ice crystal formation is limited or avoided inside the cells.
Cryoprotectants work by drawing water out of a cell by simple osmosis, and then crossing the cell membrane to replace that lost water. When an embryo is placed in a mild solution of cryoprotectant, it initially shrivels slightly as it loses water, and then returns to normal size as the cryoprotectant works its way into the cells. The water molecule is small and leaves the cell very quickly, compared to the larger cryoprotectant molecule that takes longer to cross the cell membrane. Cryobiologists have successfully used many cryoprotectants to preserve embryos, but the most commonly used during slow freezing are ethylene glycol, glycerol and propylene glycol.
When slow frozen embryos are thawed, the water is allowed back into the cells and the cryoprotectant is drawn out. During thawing, water again crosses into the cell before the cryoprotectant can leave so there is a risk that cells can over expand and rupture. Thawing is therefore performed slowly and in small steps to try to prevent cells rupturing and dying during the process.
Slow freezing has been used successfully as a method for preserving human embryos for just about 30 years now and many hundreds of thousands of babies have been born after spending time in the freezer as an embryo. The process is considered extremely safe and it works reasonably well for embryos at all stages of development.
One limitation of the slow freezing process was that it never worked very reliably for freezing unfertilized eggs. Cryobiologists worked on trying to develop a method for egg freezing for 25 years, and despite some individual successes, a repeatable and reliable process was never worked out. The human egg is a huge cell, the largest cell in the human body by some distance, and it has stubbornly resisted most attempts at slow freezing. Because of its size, it has an unusually large surface area to volume ratio, and this significantly affects the rate at which it can be dehydrated or re-hydrated during freezing and thawing. The egg is also in a very delicate state at ovulation as it is in the process of reducing its own DNA content in preparation for the arrival of the sperm which brings some new DNA. The egg has been found to be ultra-sensitive to temperature changes and to chemical (cryoprotectant) exposure, and because it is such a large cell, these exposures needed to be longer and cooling also took more time than for embryos. Unfertilized eggs did not tolerate these insults well and worldwide results with egg freezing were poor.
Cryobiologists eventually gave up on slow freezing eggs and looked at another technology, vitrification as a possible method of preservation. Vitrification of mouse embryos was first demonstrated in 1985, but the procedure never became popular, perhaps because it used much higher concentrations of cryoprotectants than slow freezing. However, 20 years later, in the mid 2000’s the technique was revitalized and shown to work well for freezing unfertilized eggs. The technique worked so well in fact, that it has now become the preferred technique for freezing embryos too.
Over the last 5 years there has been a dramatic worldwide shift to vitrification from slow freezing. At PFC, we began learning the vitrification process in 2006 and were so impressed that we have not slow frozen an embryo here since 2007. Our computer controlled slow freezers were placed in storage and we have not looked back.
Vitrification is an ultra-rapid process. If the starting material is a solid (e.g. sand or sugar crystals) then vitrification is achieved first by heating (to melt the substrate) and then ultra-rapid cooling to prevent the crystalline structure from reappearing. If the starting material is already a liquid (e.g. the contents of cells), then vitrification is achieved with ultra-rapid cooling alone. And by ultra-rapid, we mean thousands of degrees per minute. A typical embryo vitrification process cools the cells in the embryo at rates close to 5,000 degrees per minute.
The process also depends on much higher concentrations of cryoprotectants that that used for slow freezing. Typically, embryos that are vitrified are exposed to 5-10 times more cryoprotectant than slow frozen embryos. The actual concentration depends on how fast the embryo can be cooled, and the faster the rate of cooling, the less cryoprotectant that has to be used. Of course biologists everywhere are sensitive to keeping cryoprotectant exposure to an absolute minimum; so much effort has gone into developing faster and faster cooling methods. This effort has yielded many storage devices that are only marginally bigger than the eggs or embryos being vitrified, to allow the fastest possible rate of cooling while keeping the eggs/embryos safely contained. The straws that we use to hold embryos during vitrification and storage are absolutely tiny compared to the large ½ and ¼ cc straws that we used to use with slow freezing. The amount of fluid surrounding a vitrified embryo is 100-200 fold less than what would surround a slow frozen embryo.
So, the process of vitrification has 3 critical components. First, eggs or embryos are exposed to high concentrations of cryoprotectants to allow rapid dehydration of cells. Second, the eggs or embryos are loaded into tiny storage devices (usually straws) that will facilitate ultra-rapid cooling, and third, the straws containing the eggs/embryos are cooled as fast as possible, typically at thousands of degrees per minute.
In practice, this means that eggs and embryos are vitrified very quickly in the laboratory. A typical embryo vitrification protocol is complete in about 10 minutes. Embryos are removed from the incubator in the laboratory and exposed to an equilibration solution for 8 minutes to begin the dehydration process. This solution contains about 15% cryoprotectant, and in an effort to avoid exposure to high levels of individual cryoprotectants, the solution actually contains 7.5% of 2 different cryoprotectants instead of 15% of just one. After the 8 minute time in equilibration solution has elapsed, the embryos are moved into a vitrification solution that contains 30% cryoprotectant (15% each of two individual cryoprotectants) and are exposed to this solution for just 60 seconds. Then they are quickly loaded into straws and plunged into liquid nitrogen at a temperature of -196°C. The tiny straw will cool from room temperature (about 25°C) to -196°C in 2-3 seconds giving a cooling rate of 4420 to 6630°C/minute.
This high cooling rate combined with the use of high concentrations of cryoprotectants allows the contents of the straw (embryos plus surrounding fluid) to turn to a glass like substance instead of ice. Avoiding ice formation in this way successfully protects the embryos from damage and allows them to be warmed later giving survival rates consistently above 90%.
For egg vitrification, the process is similar, but the exposure to the equilibration solution is slower, usually about 15 minutes, and it is broken up into 4 steps (7.5% cryoprotectant, then 10%, then 12.5% and finally 15%). This slow stepwise approach is designed specifically for the egg and achieves the same level of dehydration as can be obtained with embryos in a shorter time. These steps however are critical to the survival of the eggs and must be followed exactly. Moving the eggs into vitrification solution (with 30% cryoprotectant), loading and cooling the straws is the same as is done for embryos.
When patients return to use their vitrified embryos or eggs, the vitrification procedure described above is reversed to allow warming back to room temperature and then 37°C, and to allow for rehydration. Since the eggs/embryos are not technically frozen (no ice was formed), we do not use the term “thawing” for this process. The procedure “warms” the eggs/embryos in just 20 minutes and they are placed back in the incubator at 37°C in the laboratory. Embryos can be transferred back to the uterus immediately and eggs can be injected with a single sperm 3-4 hours later.
Procedures associated with vitrification
The cryoprotectants used in the vitrification process cause hardening of the zona pellucida, the glycoprotein shell that surrounds the egg and embryo. Since the embryo will have to escape from within the zona after it has been warmed and placed in the uterus, we will make a hole in the zona prior to transferring the embryo. This hole is made with a fine laser and it simply allows us to overcome the zona hardening that is characteristic of the vitrification process. The zona is a non living part of the embryo, so no damage is caused by cutting a small escape hole.
- Artificial collapse. Embryos frozen at the blastocyst stage have a large fluid filled cavity or cyst. When the embryo is placed in the equilibration or vitrification solutions, penetration of the cryoprotectant into the cells of the embryo can be slowed by the fluid in the cavity. The fluid in the cavity dilutes the cryoprotectant making it harder to expose cells inside the cavity to the full cryoprotectant concentration. To get past this issue, it is normal to make a small hole between two outer cells in the embryo to allow the fluid to escape from the cavity. As the fluid escapes, the embryo collapses in on itself giving the procedure its name. It is also known as assisted collapse or artificial shrinkage. This procedure is only performed on blastocysts that have a good sized cavity. See pictures below.
Image 1: A small hole is made between 2 outer cells (5 o’ clock position in this picture) to allow the fluid in the blastocyst cavity to escape
Image 2: The blastocyst collapses in on itself and towards the hole
Image 3: When the embryo is fully collapsed, it is ready to be frozen. After thawing, the embryo will re-inflate in about 2 hours
- Assisted hatching. The cryoprotectants used in the vitrification process cause hardening of the zona pellucida, the glycoprotein shell that surrounds the egg and embryo. Since the embryo will have to escape from within the zona after it has been warmed and placed in the uterus, we will make a hole in the zona prior to transferring the embryo. This hole is made with a fine laser and it simply allows us to overcome the zona hardening that is characteristic of the vitrification process. The zona is a non living part of the embryo, so no damage is caused by cutting a small escape hole.
- ICSI. Intra cytoplasmic sperm injection (ICSI) is a procedure used to inject a single sperm into an egg. Normally this procedure is only used for patients that have poor sperm quality and where we suspect that sperm may not be able to fertilize eggs on their own. However, due to the hardening of the zona that occurs with vitrification, all vitrified eggs are fertilized using the ICSI procedure. The changes in the zona prevent sperm from penetrating in the normal way, and each egg has to be individually injected with a single sperm.