Embryo Cryopreservation: An easy to understand review

Why are Embryos Cryopreserved?

Infertility patients invest so much time, effort, money and emotion into each IVF cycle. After the fresh embryos are transferred, about 30-40% of our patients will have excess high quality embryos available for freezing, also known as cryopreservation, to be used for future cycles. Frozen embryo transfers are a cost-effective way to pursue further treatment, regardless of the outcome of the fresh embryo transfer cycle.

Patients are often curious about the methods their laboratory uses to freeze their embryos.   Cryopreservation is a delicate process requiring extensive expertise to prevent damage to the embryos during the freezing and thawing procedures.

Preventing Potential Damage During Freezing

There are three major types of injury that can occur to cells during the cryopreservation procedure (Jain JK, et al., 2006):

  • (i) Exposure of cells to ice crystal formation during freezing and/or thawing;
  • (ii) Damage to the embryos from the solutions used to prepare the embryos prior to freezing; and
  • (iii) Damage to the embryos as water and electrolytes shift in and out of the cells that make up the embryos.

    Water being removed from embryos and replaced with "antifreeze".

Damage from ice crystal formation is overcome by removing as much water from within the cells making up the early embryo and replacing it with fluid containing cryoprotectants. To remove the water, embryos are placed in a series of solutions using increasing concentrations of salt to draw the water out of the cells. Once this is accomplished, the embryos are then placed into additional solutions containing the cryoprotectants, which enter the cells taking the place of the water.

Removing the water from the cells is relatively simple while replacing the fluids with cryoprotectants is a bit more difficult and damage to the embryos can occur if not done correctly. Cryoprotectants can be toxic to the embryos, so the cells can only be exposed to them for a very short time before they are frozen. Once the cells have been filled with cryoprotectants, cryopreservation needs to take place fairly rapidly.

Slow Freezing and Vitrification Techniques

The two most common cryopreservation methods used to freeze embryos are slow freezing and vitrification. The slow

Embryos being prepared to be placed in storage container.

freezing method has been around for decades. Slow freezing involves loading the embryos into special straws or vials and placing them in a separate container, which is surrounded by a liquid nitrogen bath. The container controls the rate of temperature drop and freezes the embryos slowly over a few hours, preventing most ice crystal formation. The freezing device lowers the temperature gradually to -38° C. Once the embryos have reached this temperature, the vial or straw is plunged into a bath of liquid nitrogen to reach a final temperature -196°C (-321°F). They will remain at this temperature until they are thawed.

With vitrification, embryos are rapidly cooled to -196°C (-321°F) almost instantly. This instant cooling does not allow ice crystals to form. Vitrification comes from the Latin vitreum, meaning the transformation of a substance to glass. Please note the use of the word “cooling” is used rather than “freezing” when referring to vitrification. Freezing actually involves transitioning a liquid to a solid through crystallization. No crystals are actually formed during vitrification. To understand the difference better, take a look at an ice cube from your freezer. While it is mostly clear, you will notice it is somewhat cloudy due to ice crystal formation and not as clear as glass. Water that is vitrified appears absolutely clear because ice crystals never had the chance to form and a glass-like substance is created.

Vitrification still requires the replacement of water with cryoprotectants but uses a different recipe. In fact, the cryoprotectants used for vitrification are at a higher concentration and potentially even more toxic to the embryos. Once the embryos are exposed to the high concentration cryoprotectants, they must be frozen very rapidly. Whereas the slow freeze process takes place over hours, vitrification takes place over minutes.

Embryos in storage containers ready to go into larger storage tank.

Is One Cryopreservation Technique Better than the Next?

The process of embryo vitrification is relatively new compared to the slow freeze method; however, great advances have been made over the past decade.

There is evidence that vitrification is a slightly better system than slow freezing. In most studies, the embryo survival rates are better for vitrification than the slow freeze technique. A recent study by Kaskar, K. et.al, also showed that the pregnancy rates with vitrified/warmed embryos (64%) were significantly higher than those that were frozen/thawed by the slow freeze techniques (47%). With higher survival rates and higher implantation rates, vitrification is slowly replacing the slow freeze technique, especially for the storage of eggs and embryos.

Success with Re-vitrification

Vitrification may allow embryos to be warmed, re-vitrified, and warmed again with successful pregnancies reported. While the pregnancy rates are somewhat lower that that of embryos vitrified only once, the results are far better than embryos slow frozen, thawed, re-frozen, and thawed yet again. A 2007 study by Kamasko, Y. et.al, showed no significant differences in implantation rates between once vitrified embryos and twice vitrified embryos. The reason this is important is that embryos may be frozen/cooled in groups larger than we want to transfer. For example, if a patient succeeded with a twin pregnancy with two fresh embryos transferred and only wants one frozen embryo transferred for a future child, having two cryopreserved embryos survive that were stored in a single vial would present a problem. In this case, we would possibly transfer more embryos than we wanted to, discard the extra embryo (not at all desired) or refreeze/re-vitrify the extra embryo. Using vitrification, the embryo may indeed survive a second stage of warming for an additional pregnancy in the future.

Your Embryos Are Very Important

As embryologists, we take our responsibilities to care for your cryopreserved embryos very seriously so that you will have the opportunity to use them for future treatment cycles. Rest assured they will be available when you need them, having been cryopreserved with the most state-of-the-art techniques at our disposal.

Thawing/Warming Your Embryos
If you would like to learn about how embryos are thawed/warmed, be sure to watch for our upcoming blog on this topic!

Kaskar, K., et.al. Comparison of clinical outcome of blasocyst vitrification with slow freezing and fresh embryo transfer. Fertil Steril 2010;94:113-114

Jain JK, Paulson RJ. Oocyte cryopreservation. Fertil Steril 2006;86(Suppl 3):1037-46.

Kamasko, Y. et.al. The efficacy of the transfer of twice frozen-thawed embryos with the vitrification method.  Fertil Steril 2009;91:383-386

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