Author name: Inside IVF

As part of the in vitro fertilization (IVF) process, you may have embryos cryopreserved through freezing for later use. Here is what you should know about the freezing and thawing of embryos. How are embryos frozen? Embryos were traditionally frozen using a ‘slow-freezing’ method where the temperature is gradually reduced using a specially designed machine. However, the survival rates of frozen embryos have skyrocketed since the development of a new freezing technique called vitrification. Vitrifying embryos means that they are frozen at such a rapid rate that there is no time for ice crystals to form inside the cells, which is the main cause of damage, often leading to embryo death. Vitrification Instead, the water inside the cells freezes at -20,000°C per minute into a glass-like state. Because no ice is formed, technically vitrification isn’t freezing; it is referred to as ‘cooling.’ This super-fast rate of cooling is achieved by holding the embryo in extremely small volumes of liquid and plunging it into liquid nitrogen which keeps the embryo perfectly preserved at -196°C. Cryoprotectants Before embryos can be frozen, they need to be gradually dehydrated to remove the water in and around the cells. They are moved individually through different liquids which vary in their concentration of cryoprotectant (solutions that replace the water and protect the cells). Although cryoprotectants protect the embryos from ice damage, they can also be toxic. This means that the embryos can only be in contact with the media for very specific amounts of time to ensure the cells are sufficiently preserved, without excessive exposure which can do more harm than good. The water in the cells is gradually drawn out and the cryoprotectant is slowly transferred in. It is standard protocol for labs to artificially collapse blastocyst before freezing. It is routine for many labs to collapse a blastocyst using a laser because it contains a central cavity filled with water that needs to be removed before freezing. Read: 6 Days in the IVF Lab How are embryos thawed? When the embryo is ready to be used for transfer it can be thawed out. Although because the embryo has been vitrified into its glass-like state and there is no ice involved, technically the embryo isn’t thawed; it is ‘warmed.’ The process of warming embryos is essentially the reverse of cooling them. They need to be gradually rehydrated by moving them through solutions designed to draw out the toxic cryoprotectants and replace the water in the cells. Embryo Warming Survival Rates Embryos are actually very resilient and they handle the freeze-thaw process very well. If you have more than one embryo cryopreserved, they will be thawed in order of their quality with the highest grade embryos being chosen first. (Read more about embryo and blastocyst grading.) The survival rate should be around 95% if the embryo is handled correctly. However, immediately after the embryo has been warmed it is often collapsed and looks squashed so it is difficult to tell whether it is still viable. Many clinics leave the embryo in an incubator for a few hours or even overnight after warming so it can re-expand and be assessed fully to get a better idea of its potential before transfer. Other clinics may thaw the embryo immediately before transfer because they believe that the embryo should be given the opportunity to re-expand inside the uterus which might be a more favorable environment for it to recover in. Can you refreeze a thawed embryo? It is possible to refreeze an embryo after it has been thawed. For example, this might be done if the parents want the frozen embryo to be genetically tested. In this case, it will be thawed and biopsied then refrozen while we wait for the results. Read: From Eggs to Blastocysts: Understanding IVF Attrition What can go wrong with embryo freezing? The most common problem is that the embryo doesn’t survive the freezing/thawing process. This may be due to: Technician Error Technician error can occur when the embryo is exposed to toxic cryoprotectants for longer than it should be. Issues with Freezing Process The embryo may not be frozen correctly which causes damaging ice to form inside the cells. Storage Embryo degeneration (failure to survive) could also be due to damage caused by many accidental mini freeze-thaw cycles during storage. This can happen if lots of embryos are stored in the same place; all the embryos have to be taken out of the freezer (dewar) to find a specific patient and it causes the temperature of all of those embryos to increase ever so slightly. Although the change in temperature is only brief, the embryo is frozen in such a tiny volume of liquid that it can have a substantial effect on the embryo’s survival. Membrane Permeability Permeability of the egg or embryo's plasma membrane to water and cryoprotectants, the tolerance of the cell to osmotic swelling, and membrane shrinkage are all factors that affect how well cells survive the freezing and thawing process. If the plasma membrane of the cells does not function well to let water or cryoprotectants flow through then ice crystals can form inside the cells and lyse (break) the cells open. Also, the cryoprotectants are chemically toxic and need to be removed from the cells through the membrane. Lastly, the freeze and thawing process can cause osmotic swelling and volume changes of the cell that may not be tolerated well. Genetic makeup Alternatively, the embryo may not survive simply due to its genetic makeup. The embryo may be low grade and perhaps should not have been frozen in the first place. However, even high-grade embryos can degenerate after freezing for no known reason and through nobody’s fault. This is one of the most frustrating aspects of working under biology’s control. Partial Survival Sometimes, the embryo can just partially survive. This means that some of the cells look healthy and some less so since each cell's membrane functions independently. An embryo is often considered suitable for transfer if