Vitrification: The Process of Freezing Eggs & Embryos

Thanks to Vitrification, a relatively recent advancement in biotechnology, it is possible to preserve human embryos, sperm, and unfertilized human oocytes (eggs) with little to no decline in quality. Since the 2000s, there has been an overwhelming shift in fertility preservation practices worldwide, replacing slow freezing technologies with the rapid freezing process of vitrification. CNY Fertility has been utilizing vitrification as its sole method of freezing embryos since 2009.

Vitrification uses a flash-freezing methodology in which the eggs and embryos are cooled at an approximate rate of -12,000°C/min. This rapid freezing prevents the formation of ice crystals (that would cut and destroy said egg or embryo) and instead instantaneously turns the molecules making up the embryo into a glass-like lattice structure. Vitrification, as it so turns outcomes from the Latin vitreum, which means “glass”.

Slow Freezing:

Before we dive deeper into vitrification, let’s take a step back and examine the previously used slow freezing technique.

Slow freezing was successfully used to preserve human embryos for nearly 30 years, beginning in Australia in 1984.  But slow freeze technology faced a major challenge:  the need to avoid the formation of intracellular ice crystals in the fluids in the cells as the water in them turned to ice. These razor-sharp ice crystals can be deadly to cells for two reasons: (1) they can kill cells by shredding the cell membranes, and (2) as water turns to ice, expanding its volume beyond the capacity of the membrane, it can rupture the cell walls.

To solve this problem, embryos were run through different solutions of media to dehydrate the cells of water and replace it with cryoprotectants such as ethylene glycol and glycerol. Then the cryoprotected embryos were individually labeled and stored in cryopreservation straws, which were put in special freezers.

These freezers slowly (at a rate of -0.3° Celsius per minute) cooled the embryos to -35° Celsius using liquid nitrogen. Embryos were then stored in liquid nitrogen (-196° Celsius). At that extremely cold temperature, cellular activity is brought to a halt, allowing the embryos to remain viable indefinitely. By replacing the water in the cell with cryoprotectant, this “swap out” avoids or limits much of the formation of ice crystals in a cell.

Slow freezing works reasonably well for embryos and sperm, but was not as successful for eggs.  Eggs have a much larger volume of water than embryos. The large water component of an egg increases the formation of intracellular ice crystals during the freezing process, which causes degeneration.

Vitrifying eggs reduces the occurrence of intracellular ice crystals, which improves freeze, thaw, and pregnancy outcomes.

Vitrification (aka Fast Freezing):

Vitrification is a relatively new process for cryopreserving embryos, eggs, and sperm. Through vitrification, the water molecules are removed and replaced with a higher concentration of cryoprotectant than in the slow freeze method. The embryos are then plunged directly into liquid nitrogen. This drastic (-12,000° Celsius per minute) freezing changes the egg or embryo into chemical glass in milliseconds, so quickly that there is no chance for any significant ice crystals to form, thus decreasing the degeneration of cells upon thawing for embryo transfer.

Embryo Vitrification:

Patients who undergo IVF generally have several eggs collected. The eggs are then fertilized with a sperm and checked for fertilization. Fertilized eggs are called embryos. A patient may have multiple high-quality embryos available for transfer back to the uterus. A certain number of embryos can be chosen for fresh embryo transfer (usually one and not more than two), and the surplus of high-quality embryos are able to be cryopreserved for future use.

Before freezing, vitrified embryos are exposed to 5-10 times more cryoprotectant than embryos that are slow frozen.  Cryoprotectants cause hardening of the shell surrounding an embryo or egg. Once thawed and placed in the uterus, an embryo will have to escape from the shell. Assisted hatching is a procedure in which a fine laser is used to make a hole in the shell prior to transferring the embryo. This procedure helps avoid complications associated with hardening of the shell. Since the shell is a non-living part of the embryo, assisted hatching does not damage the embryo.

Many studies show survival rates of vitrified embryos to be far higher than survival rates of slow freeze embryos. According to 2019 research, pregnancy and live birth rates are better with eggs, sperm, and embryos that were frozen with vitrification .

While embryo freezing is still the preferred technique for fertility preservation (as opposed to freezing unfertilized eggs), women who don’t want to use a sperm donor may want to preserve their healthy eggs in preparation for motherhood at a later time giving them the opportunity to pursue education or careers until they’re ready to start a family.

Oocyte Vitrification:

Oocytes (unfertilized eggs) are exposed to high concentrations of cryoprotectant in a somewhat slower, multi-step approach designed so oocytes can survive freezing as well as human embryos do. The eggs are bathed in a series of cryoprotectant solutions with increasing concentrations, and when the eggs are placed in the last cryoprotectant solution, they are instantaneously frozen.

Using Frozen Embryos & Eggs

When patients decide to use their cryopreserved embryos to try for a pregnancy, the embryos are removed from the liquid nitrogen, warmed and run through solutions of media to remove the cryoprotectant and rehydrate the cells with water.

This same process is used for eggs.  Once thawed, the eggs must be fertilized using ICSI, where a single sperm is injected directly into the egg.  Regular IVF is not possible because the freezing process hardens the eggs outer membrane making it impenetrable to sperm.

Fresh vs. Frozen:

While couples once faced difficult a decision about transferring multiple fresh embryos and risking the chance of twins, triplets, or more or freezing extra embryos and risking them not surviving the freeze/thaw process, vitrification has eliminated this quandary.  With the advent of vitrification, survival rates similar to fresh oocytes and embryos can now be obtained. This has resulted in two major developments in fertility treatment:

Freezing Unfertilized Eggs:

• Clinics now have a reliable way of freezing unfertilized eggs so women can bank their eggs and thaw them for future use when they decide to have a family. This allows women to effectively stop their biological clocks, giving them time to pursue education, careers, and have a family when the time is right. It also allows women with low ovarian reserve or a cancer diagnosis to preserve eggs for future treatment as well.

IVF with Delayed Frozen Embryo Transfer:

• Pregnancy rates from frozen embryo transfers are now at the point where the success rate of frozen cycles exceeds the success rate of fresh transfers when a woman is stimulated and supplies eggs for her own IVF cycle.  The downside of stimulation in an IVF cycle is that medications used to optimize egg development can often cause a hormone imbalance that interferes with endometrial receptiveness which may make successful implantation unlikely. Doctors have found that freezing the embryos and switching to a frozen embryo transfer (FET) gives the woman’s body the chance to recover from any negative effects of the stimulation drugs, upping the odds of successful implantation and pregnancy.

What are the Risks of Egg, Embryo, and Sperm Freezing with Vitrification?

Even though vitrification is a more reliable freezing method than slow freezing with much higher survival rates, it’s still important to remember that not every frozen egg or embryo survives the warming process. Not every thawed egg will fertilize, nor will every embryo continue to develop. Just as every fresh egg or embryo does not always develop as expected, frozen eggs and embryos face the same natural limitations.

There has been some concern about the level of cryoprotectants used in vitrification and that they may have some effect on frozen cells. Researchers are always searching from ways to decrease this risk, but frozen embryos come with the advantage of being able to screen for certain genetic mutations and chromosomal abnormalities, such as trisomy 21, which causes Down’s Syndrome, and others. The ability to de-select abnormal embryos has the potential to improve live birth rates even more.