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.

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 out of 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 at least half of the cells have survived.

Blastocysts are made up of hundreds of cells and when they are first thawed they look shrunk and crumpled which makes it difficult to grade accurately. Because of this, the embryo will be left to re-expand in the incubator and the embryologist will assess how well it rehydrates back to its normal size which usually happens in 2-4 hours.

Some studies have found that blastocysts which re-expand more quickly have a higher implantation potential than those which have not fully rehydrated after several hours.

Read: 5 Things Your Embryologist Wants You To Know About Your IVF Cycle

What are the benefits of embryo freezing?

With such amazing improvements to embryo freezing techniques and great survival rates, many clinics now opt to do 100% frozen embryo transfers (FET) and there are many reasons why.

Most importantly, it gives your endometrium a chance to develop to the optimal thickness so it can be in perfect synchrony with the embryo. This aims to mimic the process of natural conception to give the embryo the best chance of implanting.

Freeze Time

Freezing embryos also freezes time. The age you are when you start your IVF cycle is one of the biggest factors which determines success and it rapidly declines with every year your pregnancy is delayed.

Freezing your embryos gives you the peace of mind that your success statistics are locked in which means you can take your time with as much breathing space as you need — assisted reproduction is a very stressful process after all.

Single Embryo Transfer

It is also important in encouraging single embryo transfers. Having multiple embryos transferred significantly increases the chance of twins and even triplets which increases the risk of serious complications for both the mother and the babies.

Now that embryos can be frozen with great success, embryos can be transferred one at a time which optimizes the chance of a positive outcome. The aim will always be to produce a single, healthy live birth.

Every patient is unique

Remember that embryo freezing and thawing are very intricate processes and the complexities cannot be fully explored here. There are advantages and limitations in all situations which need to be carefully considered as every patient and every embryo is unique.

Trust your clinician and embryologist but don’t be afraid to ask for more information if you feel you would benefit from it—they are your embryos.

Embryo Freezing & Thawing: What You Need to Know