Embryos are placed into straws or vials containing anti-freeze or cryoprotectant solutions. These are transferred to a programmable biological freezer which is used to achieve a controlled slow rate of cooling. During cooling, cells dehydrate and as the temperature is reduced, more ice forms and water is removed gradually from the cells. Slow cooling is continued to ~ -35°C at which point embryos are rapidly cooled by plunging into liquid nitrogen (-196°C). Embryos are kept in storage tanks of liquid nitrogen until thawing is performed.
Embryos can be frozen at any preimplantation stage between one-cell (one day old) to the blastocyst stage (5-6 days old). To maintain control of excessive numbers only embryos only at the blastocyst stage are preserved. The embryos are contained within special indelibly labeled plastic vials, or straws, that are sealed prior to freezing. Once frozen, they are placed inside labeled tubes attached to aluminum canes and stored in numbered canisters within the liquid nitrogen dewar. Site and label designations are stored in three separate file systems to avoid confusion and misidentification of cryopreserved embryos.
When it comes time to thaw the embryos, all available identifiers of the stored specimen must match and be confirmed before thawing commences. The embryos are thawed out at room temperature, which takes about one to two minutes. However, the most critical element of the thaw procedure is not the timing but the careful dilution of the cryoprotectant fluid to return the embryo to its favored culture medium. This permits resumed growth and development in vitro. Once this is done, the embryo is assessed for cryodamage to determine if it is suitable for transfer. Experience has shown that if the embryo survives 50% or more intact, it is worthwhile to replace it. Embryos can accommodate such levels of cellular damage and still establish healthy pregnancies. All thawed embryos routinely undergo assisted hatching prior to transfer. The zona pellucida, which surrounds the embryo, has been shown to suffer a certain amount of hardening during cryopreservation. This can be overcome by artificially making an opening in the outer embryo shell.
There always remains a possibility that there may be no embryo survival after thaw occurs, and no transfer is possible. If many early embryos are frozen, it is possible to thaw all of them and culture them for several days to allow selection of the best for transfer. When too many embryos are available for transfer in this circumstance, then extra embryos of sufficient quality may be refrozen for later use. This course of action has produced healthy offspring, proving the efficacy of double freezing of embryos.