1. Embryos are stepped through increasing (freeze) or decreasing (thaw) antifreeze concentrations in a 4-well plate.	2. Once saturated with antifreeze, the embryos are loaded into small straws.	3. The straws are placed in a controlled- rate freezer.
4. Once frozen, the embryos are placed in state-of-the-art, computer controlled storage tanks.	5. The straws snap into labeled metal canes. They are barely visible through the mist of the cooling nitrogen gas.	6. After thawing, it is not unusual to see one or more dead cells in an embryo. Arrow is to 1dead cell in 4-cell embryo.

To successfully freeze, or cryopreserve any cell, its water must first be removed. Otherwise, the water expands into ice crystals as it freezes, and this process bursts or kills the cell. To maintain embryo viability during cryopreservation, the embryo is bathed in antifreeze, which draws the water out of the cells while the antifreeze enters. (1) Made with propylene glycol supplemented with sucrose, the antifreeze is not harmful in any way, provided the embryos are kept cool.

After this first step, the embryos are loaded into small straws (2) and placed in a controlled-rate freezer, which cools the embryos at a rate of -0.3°C/minute until they reach a temperature of -38°C. (3) Then they are placed in, and stored in liquid nitrogen at -196°C. (4,5)

To thaw the embryos, the straw(s) are first warmed to room temperature and the embryos unloaded into a Petri dish. Then they are stepped through decreasing concentrations of antifreeze until eventually all the antifreeze has been removed and the embryos have been rehydrated. (1)

There is tremendous variability in how well embryos tolerate the freeze/thaw procedure, although surprisingly, it has little to do with the quality of the embryos at the time of freezing. A poor quality embryo will endure the process just as well as a good quality one. However, since poor quality embryos have a very low possibility of implanting, they are often not worth freezing.

While every care is taken to protect the embryos during the process, some embryos will have one or more burst cells (6) after they have been thawed. This cell loss results either from puncture by tiny ice shards around the embryo or from rupture as water rapidly enters the cell during thawing.

In 2003, over 80% of embryos survived freezing and thawing with one or more cells intact. On average, 2 out of every 3 cells in an embryo tolerated the process, and we consider an embryo with 50% or more of their cells surviving as having a normal chance of implanting after transfer. If an embryo thaws with fewer than 50% of its cells alive, we usually recommend thawing another embryo if one is available. At PFC 66% of embryos thaw with 50% or more of their cells intact.

Tags: Embryo Freezing, IVF - In Vitro Fertilization, Lab

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