The first human pregnancy from a frozen oocyte was achieved in Australia in 1985 by Dr. Christopher Chen (Lancet 1(vol 8486): pp 884-886, 1986), just one year after the first baby had been born from a previously frozen embryo. Since then we have come a long way and current reports suggest that preserved and then thawed oocytes can perform just as well as fresh oocytes. In a recent study using oocytes from young donors, Peter Nagy from Reproductive Biology Associates in Atlanta showed that frozen oocytes actually outperformed fresh oocytes from the same donors in a side by side study. 134 out of 153 oocytes survived after warming and fertilization, embryo development and pregnancy rates were normal, leading to 16 pregnancies among the 20 recipients in the study (Fertility and Sterility, 92: pp 520-6, 2009). Pregnancies were also achieved from frozen embryos derived from the frozen oocytes in that study.
As a young embryologist in London in the late 1980’s I remember meeting Dr. Chen, and the wide skepticism that surrounded his work made an impression on me. It hadn’t been possible to repeat his effort and his early success did not yield a method that could be reliably or consistently used to freeze oocytes. He had used a procedure called “slow-freezing” which worked and continued to work reasonably well for embryos, but which has been unreliable for freezing oocytes.
The human oocyte is a huge cell at about 100 µm in diameter. It is so big that under the right circumstances it can actually be seen with the naked eye, without the use of a microscope. All human cells are smaller than this, with skin cells measuring about 30 µm and a sperm head (the smallest human cell) measuring just 5 µm long and 3 µm wide. And this size has been one reason why oocytes have been so difficult to freeze successfully. Cell freezing is dramatically influenced by the cell’s surface area to volume ratio, and by its water content, and since the oocyte is so big, these dimensions are very different from all other cell types. One way to think about this is to compare the dimensions of the earth to those of the moon, which measures about 25% of the earth’s diameter. The moon’s surface area however, is only about 7% that of the earth and its volume is just 2%. Similarly, the dimensions of the human oocyte outstrip other cells when we look at these key measurements. The table below shows the key surface area to volume ratios for a sperm and an oocyte.
Other than size and water content, there is one other significant hurdle to oocyte freezing that is not a factor when freezing other cells. At ovulation, the oocyte is close to completing meiosis, a type of cell division that reduces DNA content by half in preparation for fertilization. While the ejaculated sperm has fully completed meiosis and is in a state of nuclear stability, the oocyte in contrast pauses at a very sensitive stage, metaphase II, and as a result is very sensitive to physical (e.g. being handled too much), chemical (pH) and environmental (temperature) changes, which can cause irreversible damage if not carefully controlled.
Chen’s method, and the traditional method for embryo freezing involves slowly dehydrating cells and then cooling them very slowly over a period of approximately 90 minutes, until they reach a temperature of between -30 and -400C. The oocytes or embryos are then stored in liquid nitrogen at -1960C until they are needed, at which time they are thawed and rehydrated. The removal of cell water is a critical part of the process as any water remaining inside the cell can freeze (turn to ice) that will rupture (kill) the cell. Dehydration is achieved with the use of permeating agents (called cryoprotectants) such as ethylene glycol, glycerol or DMSO which enter cells and draw water out by simple osmosis. Non permeating cryoprotectants such as sucrose, which will not cross a cell membrane by osmosis, are also used to further dehydrate the cell before freezing. Slow freezing uses minimal concentrations of cryoprotectants and is a slow and delicate process that aims to prevent ice formation inside cells.
In the 1990’s and 2000’s as IVF became a widely available technology with ever improving success rates, several countries around the world moved to regulate the industry. In Europe, Germany, Italy and Switzerland all established laws that limited the number of embryos that could be created and/or frozen in an IVF treatment cycle. The law in Italy required that any oocyte that had been incubated with sperm had to be transferred to the mother, even if that oocyte did not fertilize. Such laws limited the number of oocytes that could be inseminated, and since IVF usually generates multiple oocytes, research on improving oocyte freezing methodology became very important. The Italians with their prohibitive laws lead this effort and several promising papers resulted that brought improvements in the slow freezing method for oocytes. However, despite the considerable time and effort to develop a method for slow freezing oocytes, only about 300 babies were reported worldwide after about 2 decades of trying (Reprod Biomed Online, 18(6), pp 769-76, 2009). The slow progress of this method together with disappointing efficiencies, suggested that slow freezing was unlikely to ever be a useful method for oocyte freezing.
In the quest to improve oocyte freezing methods, and in the face of disappointing results with slow freezing, scientists began to look for an alternative method that would improve outcomes. In 1985, mouse embryos had been successfully preserved using a process called vitrification (Nature, 313, pp573-5) which prevents ice formation in cells by using higher concentrations of cryoprotectants and ultra-rapid cooling. Vitrification is a technically more challenging procedure and depends a lot on the skills of the embryologist in handling oocytes or embryos through the process. This handling skill (referred to as the “technical footprint”) is more critical since vitrification is an ultra-rapid procedure where the individual steps are timed to the second, and it requires higher concentrations of cryoprotectants that are extremely viscous and difficult to work with. It also uses smaller vessels to contain the oocytes compared to the large ¼ or ½ cc straws, or 1 ml vials that were used with slow freezing. The typical vitrification straw or container is just bigger than the oocyte itself, allowing for the maximum possible cooling rate when plunged into liquid nitrogen. In a typical vitrification procedure, the final step requires that the oocyte be placed in a solution that is composed of about 30-35% cryoprotectant by volume, then aspirated in as small a volume as possible into a tiny container before plunging into liquid nitrogen and getting to -1960C in 1-2 seconds. When we teach this procedure, practically all the emphasis is on the technical footprint.
By 2005, after years of experimenting on bovine and porcine oocytes (which are similar in dimensions to human oocytes) researchers led by Masa Kuwayama from Tokyo began publishing very exciting results from vitrified human oocytes (RBM Online, vol. 11, pp 300-8). Survival of oocytes was over 90% after warming, and fertilization, embryo development and pregnancy rates were similar to those achieved with fresh oocytes. Finally a reliable method was available and it could be learned and implemented by embryologists all over the world. Vitrification worked so well that it was quickly adapted for embryos, and is now the standard procedure for preserving surplus embryos after IVF treatment. The number of IVF clinics that still freeze embryos by the older slow-freeze methodology is constantly declining, and virtually all oocyte freezing is now done using vitrification. By 2009, the number of babies born from vitrified oocytes had already passed the total number conceived over more than 20 years with slow freezing (Noyes st al., RBM Online, Vol 18 (6), pp 769-76), and the babies were normal when compared to naturally conceived children.
Here at PFC, we quickly abandoned slow freezing as soon as vitrification was seen to be safe and readily available. We first vitrified embryos in March 2007 and have not regretted the move away from slow freezing which was completely phased out by June of that year. We also began vitrifying oocytes in late 2007 and continue to use the procedure for patients facing cancer treatment and women wanting to preserve their fertility. We constantly help other programs that are learning vitrification and have visitors from all over the world that come to watch us and learn the techniques. In addition, we helped run hands-on workshops for embryologists domestically in Houston, Boston, Virginia and Austin, and overseas in Europe and South America in 2011. Vitrification has finally allowed oocytes to be reliably preserved, and since it works so well, demand for the procedure is increasing steadily.
- Joe Conaghan, PhD, HCLD