The first human pregnancy from an embryo that had been frozen and thawed was achieved in Australia in 1984, 6 years after the birth of the first IVF baby in the UK. The method used to preserve that embryo is called "slow freezing" and it is still the preferred method for preserving embryos throughout the world today. Slow freezing is a reliable and established technique that has served the IVF community well for over 20 years. The procedure has been refined throughout those years and it works, with slight modifications, for freezing all embryo stages and for sperm. However, despite many years of trying, slow freezing has never worked very successfully with oocytes. Frustrated by years of failures, scientists turned to an alternative procedure called vitrification in their quest to preserve oocytes. This approach is relatively new, but appears as through it will be preferentially used for oocyte preservation as we go forward. Vitrification kits are just beginning to get FDA clearance following scientific trials, and embryologists are being trained in the use of the new technology.

The main concern during the freezing of any cell is the removal of water without actually killing the cell. Since water expands in volume as it freezes, ice formation inside a cell would cause the cell to rupture and die. Therefore, cell water is traditionally replaced with a cryoprotectant (antifreeze) prior to cooling of the cell. This is achieved by sequentially incubating the cell in increasing concentrations of cryoprotectant. The cryoprotectant draws water out of the cell and itself enters the cell, all by osmosis. Once most of the water has been removed, the cell is cooled at the very slow rate of -0.3° C/minute until it has been cooled to below -30° C and is therefore fully frozen. Thereafter, storage of frozen cells is in liquid nitrogen (-196° C), which is a simple and practical storage medium.

Vitrification still requires the use of cryoprotectants and the cell is also ultimately stored in liquid nitrogen, but the journey from the incubator (at 37° C) to the nitrogen (-196° C) is much faster. The word "vitrum" in Medieval Latin means "glass" and the process turns the cell contents to a glass like substance instead of ice. Since no ice forms, the risk of rupturing the cell is eliminated. For glass to form instead of ice, the rate of cooling must be thousands of degrees per minute instead of the 0.3 degrees/minute that we use in slow freezing. Therefore, the process is sometimes referred to as ultra-rapid freezing, although the word "freezing" is really inappropriate here since the cell is not really frozen (i.e. no ice is created).

One of the big stumbling blocks during oocyte freezing was the sheer size of the cell (the oocyte is the largest human cell by some margin) and therefore its high water content. Just getting the cell to survive, (an oocyte has only one cell), was a huge stumbling block. Studies where 50-60% of the oocytes survived were considered groundbreaking, and still today there are few studies that have done better. Vitrification as a technique had been largely ignored by the IVF community as it was technically more challenging and used much higher concentrations of cryoprotectants. Cryoprotectants were thought to be toxic to cells. Today we know that they are safe and effective and do not contribute to cell death. It is possible that cryoprotectants may have deleterious effects on cells if they are metabolized, but virtually all freezing protocols utilize them at room temperature or below, where cell metabolism is significantly slowed or stopped. So, with success rates using traditional slow freezing failing to improve, vitrification has been given serious consideration as an alternative. In the few years since its introduction, vitrification has shown promising and excellent results in clinical studies (see Oktay et al., Fertility and Sterility, 2006, Vol 86(1), pages 70-80 a comparative review of slow freezing and vitrification results with human oocytes).

Making the transition from slow freezing to vitrification has been a challenge for the IVF community. As already stated, it is a technically challenging procedure, and training of embryologists in the technique has been slow. With slow freezing, embryos are placed in relatively weak solutions of cryoprotectant for as long as 15 minutes at a time. Then, they are usually moved on through slightly stronger solutions before being placed in large straws or vials which are then loaded into a computer controlled freezer for the long journey to -30° C. The embryologist can spend about 30 minutes with a set of embryos from the time that they come out of the incubator until they go into the controlled rate freezer. After 2 or more hours, the embryos can be placed in liquid nitrogen and the process is complete.

During a vitrification procedure, where typically only one oocyte or embryo can be worked on at a time, the transition from incubator to nitrogen takes only a few minutes. The embryo is stepped through solutions containing high and then higher concentrations of cryoprotectants where it shrivels and swirls in the extremely viscous medium. In the final stage, which is measured in seconds, the embryo is placed in an extremely concentrated cryoprotectant solution and then quickly loaded up into a tiny straw that is barely larger than the embryo itself. The straw is then sealed at both ends and plunged immediately into liquid nitrogen. The straw is so fine that it freezes in an instant, an important part of the vitrification process. The loading of the straw occurs at room temperature (25° C in the IVF lab) and it is cooled to -196° C in one or two seconds, giving a cooling rate of 6000-13000° C/min. The faster the straw can be cooled, the more successful the procedure. Performing this final step too slowly or too quickly can be the difference between success and failure and therefore requires extensive training.

At Pacific Fertility Center, we have been working on vitrification for over 2 years. Our initial interest was in oocyte freezing, but we were also interested in extending the technique to be used with embryos, and in particular to blastocyst stage embryos where slow freezing has not always worked well. Slow freezing has served us well over the years for embryos being frozen 1, 2 or 3 days after an oocyte retrieval, but blastocysts (5 or 6 day old embryos) did less well. With an industry wide transition to blastocyst stage embryo transfers, we looked at vitrification as an alternative method of preservation for these precious embryos.

A blastocyst is an embryo that has developed to the stage where it is ready to implant in the uterus. Instead of having a small number of loosely associated cells characteristic of earlier embryonic stages, it has 2 defined cell populations and a fluid filled cavity (or cyst). The cells that surround the cavity will form the placenta, and the cells within the cavity will develop into the embryo proper, or fetus and some of the extraembryonic membranes, such as the yolk sac. It is these interior cells that cause trouble during freezing since they are on the inside and difficult to expose to cryoprotectant. Slow freezing relies on cryoprotectant traveling through the outer placental cells, then the cavity, and finally into the fetal cells while water travels in the opposite direction. Fully dehydrating these fetal cells has always been a challenge and an embryo where these cells do not survive freezing and thawing will not result in a viable pregnancy. And with slow freezing, embryos tend to collapse in on themselves during dehydration, making it difficult to assess survival after thawing.

After investing heavily in vitrification training and implementing a successful oocyte vitrification program, PFC began working on blastocyst vitrification in January of 2007. By March we had a program established and were delighted by how easily blastocysts seemed to tolerate the procedure. Often, blastocysts looked no different after vitrification when compared to how they looked before the procedure. This result was in stark contrast to slow freezing where blastocysts always look shriveled and deflated after coming out of the freezer. By July 2007, we had switched completely to vitrification and currently we are enjoying the successes that it is bringing to our patients and us.

Our vitrification team consists of 3 embryologists: Mariluz Branch, the team leader, with Erin Fischer and Liz Holmes. Because of the technical challenges involved, we have to be cautious with involving other embryologists. So one of the three team members must be on duty every day (our lab is open 7 days a week). I am grateful to this team for their flexibility in accommodating our needs. By the end of the year we expect to have 2 more embryologists on the team, and then the final 3 in 2008.

Vitrification has been an exciting and challenging technique which we have embraced and conquered in 2007. We look forward to the gradual elimination of slow freezing and the successes that vitrification will bring us in the future.