Embryology Laboratory Services

 

Cryopreservation of Embryos

Why is cryopreservation (freezing) of embryos important?

During the in vitro fertilization process, a physician stimulates the patient’s ovaries with fertility medication in order to make several follicles grow.  After this stimulation has been completed, some of these follicles produce healthy, mature eggs while other follicles produce immature, broken or atretic (degenerate) eggs.  There are also follicles that are aspirated that do not yield eggs.  The eggs in these follicles may be immature and not ready to break loose from the follicle wall.  

When the eggs are exposed to sperm, either through insemination or by injection (ICSI), not all of the eggs will fertilize.  The average is 70% to 80%, but varies greatly.  As the fertilized eggs grow into embryos, some develop into healthy embryos while others grow slowly or abnormally.

By the time of transfer on day 3 or on day 5, some embryos will have developed the expected number of cells and appear very healthy.  Other embryos will be growing slowly, will have developed into abnormal embryos or simply will have stopped growing.

There is always a decrease in the yield from the number of growing follicles seen at the last scan, to the number of mature eggs retrieved, to the number of eggs that fertilize, to the number of healthy embryos that develop.  This decrease in number occurs in every patient and varies greatly from one person to the next. 

Because of this decrease in numbers, retrieving and fertilizing as many eggs as possible is a good plan.  However, this sometimes results in the production of more embryos than a patient should transfer.  This is when cryopreservation is very important.  It allows a patient and a physician to transfer the number of embryos that is ideal for the patient’s age, medical condition and plan for family size.  The remaining healthy embryos can be cryopreserved for use at a later time.

The cryopreservation process, no matter what stage of embryo growth or what method is used, always involves the removal of water from the cells of the embryos and the replacement of that water with a cryoprotectant substance.  This substance is usually some form of alcohol in combination with sucrose.

What has been learned over the years is that this exchange of water with cryoprotectant takes place more quickly and thoroughly if the cells of the embryos to be frozen are small, such as in the blastocyst stage.  Also, once this freezing process has been completed and the embryos are in liquid nitrogen, their viability after they are thawed does not lessen with the number of months or years that they remain in storage.  In this program, embryos have been thawed and produced a healthy baby after being in storage for more than nine years. 

This does not mean, unfortunately, that if embryos are cryopreserved, they will always be viable when they are thawed.  There is a percentage of embryos that do not survive the freezing or the thawing process.  When embryos were frozen at the 8 cell stage, their survival rate was around 67%.  Now that blastocysts are frozen (containing smaller cells), their survival rate is over 90%.

What is the difference between slow-freezing and vitrification?

When embryos are cryopreserved, they are taken from the incubator where they have been growing in an atmosphere and temperature that mimic conditions within the reproductive tract of the woman.  They are then transferred into a medium containing an alcohol product, such as glycerol, and sucrose.  This cryoprotectant medium is important because it replaces the water that is in the cells of the embryo.  It is introduced into the cells of the embryos in precisely timed stages according to exact formulas that have been determined through the work of researchers over several years.

During slow-freezing, embryos are placed in the different concentrations of cryoprotectant medium for 5 to 10 minutes each.  This results in a slow diffusion of the cryoprotectant into the cells of the embryos.  The embryos are then loaded individually into a vessel, such as a straw-like device.  They are then placed into a cryopreservation freezing machine which lowers their temperature in two different stages over the course of two hours.  This gradual lowering of temperature results in a gradual increase in the concentration of the cryoprotectant media and a gradual increase in the amount of cryoprotectant that enters the cells of the embryos.   At the end of the two hours, they are removed and placed into labeled holders and immediately plunged into the liquid nitrogen storage tanks.

During vitrification, embryos are placed into much higher concentrations of cryoprotectant media for very short periods of time.  This results in a very fast movement of water out of the cells of the embryos.  As the water leaves the cells, the high concentration of cryoprotectant enters the cells very quickly.  The embryos are moved through the drops of cryoprotectant rapidly, loaded into a labeled vessel in a micro droplet and immediately plunged into liquid nitrogen.  They are placed into a labeled holder and transferred into the liquid nitrogen storage tanks.

The slow-freezing method has been the standard in IVF for twenty-five years, but vitrification has been gaining in the number of clinics that employ it as their primary freezing method.  Currently, the only question with vitrification at Austin IVF is not whether we will be using it as our primary freezing technique for eggs and embryos.  Instead, the question is which of the many vessels available we will decide to use for the process.  This will be finalized within the month.  Meanwhile, vitrification will be our standard for patients by March 2010.

When a patient returns for her cryopreserved embryos, the uterine lining must be properly prepared so that the embryo(s) will implant on the lining and continue to develop.  The lining may be prepared in one of two ways; by the use of a natural cycle, whereby the patient detects her ovulation and allows the uterine lining to develop on its own, or by using Estrace to build the uterine lining.  Whichever method is used, it is important to synchronize the age of the embryo with the uterine lining.  This is done by first looking at the number of days after the egg retrieval that the embryo was frozen.  For example, let’s say that the embryo was frozen on the fifth day after the patient’s egg retrieval.  If the patient is using her natural cycle, she must determine the day of her ovulation by monitoring her level of luteinizing hormone (LH).  Once the day of ovulation has been determined, the five day old embryo will be placed into the uterus on the fifth day after the patient’s ovulation.  If Estrace is used, there is an established protocol of the number of days that this oral medication is given.  Before the embryo is thawed and the transfer is performed, the uterine lining is checked to see if it is appropriately thick.  Both of these methods are equally successful and the choice of which to use is determined by the patient’s physician.