In the first two segments of this series, we discussed embryo grading and cryopreservation (1 – Do these embryos make the grade? and 2 – Embryo Cryopreservation: An Easy to Understand Review). This final segment in the series will examine the process of thawing or warming of embryos for transfer.

In Review…

Let’s begin with a short review of the previous blogs. You will recall that we discussed two methods of embryo cryopreservation, slow freezing and vitrification. Both methods require that the cells of the embryo have as much water removed as possible and that cryoprotectants, materials that protect the cells during freezing, be forced into those cells to reduce ice crystal formation. Slow freezing uses a controlled rate freezing instrument that slowly lowers the temperature to well below freezing to avoid the formation of ice crystals. Vitrification is a method in which embryos are plunged directly into liquid nitrogen and immediately turned into a glass-like substance, essentially cooling the embryos so quickly that ice crystals do not even have time to form.

Cryopreservation Storage Containers

One topic not covered in the freezing blog was the type of containers used to hold the embryos. Slow freezing, in most cases, uses straws or vials to hold the embryos in a small amount of solution called media. Vitrification uses many different storage devices to hold the tiny embryos, which cannot be seen without a microscope. These devices commonly include various types of straws, nylon loops or electron microscope grids, just to name a few.

How Often do the Cryopreserved Embryos Survive?

It is expected that about two-thirds of embryos cryopreserved via a slow freeze technique will survive thawing (Son and Tan, 2009) and the embryos that do not survive are most likely genetically abnormal. For embryos that are vitrified, the rapid cooling/warming process seems to work better, with about 80-90% of the embryos surviving. For this reason, vitrification is becoming more popular for cryopreserving both eggs and embryos.

How Many Embryos Should Be in Each Container?

Ideally only one or two embryos should be packed into each storage container. By packaging one embryo per container, the embryologist can thaw or warm only the exact number of embryos to be transferred without having any left over. For example, if a patient wants to have two thawed cryopreserved embryos transferred and they have a total of five embryos each frozen in separate containers, the embryologist will thaw/warm one container at a time until just two healthy embryos are recovered. If, however, the same five embryos were packaged in two containers with three embryos in one and two in another, the embryologist would probably first begin thawing/warming the container with three embryos. Should all of the embryos survive, there is one excess embryo that would need to be transferred, refrozen or discarded. While recent studies have indicated that freezing embryos a second time can result in a pregnancy, particularly when using vitrification, it is ideal to only thaw the exact number of embryos that we want to transfer and not one embryo more. (Kamasko, Y. et.al., 2009).

How are the Embryos Thawed/Warmed?

Ice crystal formation is also the enemy when thawing embryos, just as when cryopreserving them,. As embryos warm from -196°C (-321°F) toward 0°C (32°F), ice crystals may again form and damage the cells. We use the following techniques to rapidly warm/thaw both slow frozen and vitrified embryos in an attempt to avoid ice crystal formation:

1. For embryos frozen by the slow freeze method, the straws or vials are removed from the storage tank and held at room temperature for 30-60 seconds, allowing the embryos to warm only slightly. The storage container is then plunged into 37°C water, completing the thawing process quickly enough so that the ice crystals don’t have a chance to form.
2. Vitrified embryos are taken out of liquid nitrogen, with the container then plunged directly into warming media that is either at room temperature or 37°C, depending on the method used to vitrify the embryos. Once again, the ice crystals simply do not have enough time to form.

Once the embryos have been thawed/warmed, the cryoprotectants that replaced the water in the cells must be removed and balanced solutions placed back into the cells. To accomplish this, embryos are moved from small bath to small bath with varying concentrations of water and other substances. As the cryoprotectants are removed, the cells fill with water containing the nutrients and growth factors needed for cellular recovery. Once the embryos have been thawed/warmed, they are placed in the incubator in supporting culture media for two to twelve hours to allow the cells to continue equilibration prior to transfer. If we are thawing embryos that were frozen early, we may even grow the embryos for a few days so that we transfer the fewest healthy embryos we need to achieve a successful pregnancy.

Are There Other Factors That Influence the Survival of Cryopreserved Embryos?

While ice crystals play a big role in the survival of embryos, they are far from the only concern. Embryo survival rates vary for many reasons including;

1. Embryo quality: Poor quality embryos freeze and thaw poorly. Poor early development of an embryo suggests that the embryo is in the process of dying and such embryos should probably not be cryopreserved.
2. Freezing media and cryoprotectant “recipes“: Some recipes are potentially better than others and each laboratory has to find which works best. What works well for one, may not be the best for others.
3. Laboratory techniques: Laboratories must have an excellent quality control program to assure they are following the steps involved in freezing/cooling and thawing/warming the cryopreserved embryos precisely.
4. Embryologist variability: As with all techniques that involve humans, some seem to do a better job than others. There is no substitution for careful training and experience.

In Closing…

We hope this series has given you a glimpse of what is involved in grading, freezing/cooling and thawing/warming your precious embryos. Cryopreserved embryos give patients the option to build their families in a cost effective manner. Without cryopreservation, embryo donation would really not be possible.

Corey Burke, B.S, C.L.S.
Laboratory Supervisor
Embryo Donation International
CBurke@EmbryoDonation.com

References:

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

Son, W.Y., and Tan, S.L. Comparison between slow freezing and vitrification for human embryos. Expert Rev. Med. Devices 2009:6(1),1-7