Oocyte cryopreservation procedure comprises several different stages:
> The first stage entails stimulation of the ovary(s) to obtain multiple follicle growth and egg production (IVF). During the stimulation phase, follicular grow this carefully monitored with transvaginal ultrasound and testing for estradiol and LH, the hormones produced by follicles during their growth phase, in order to determine the appropriate time to harvest the eggs.
> When a sufficient number of follicles have achieved satisfactory growth, the final, follicular maturation phase is induced with a trigger shot of hCG.
> The next stage is the retrieval of the eggs from the follicles. This is done by needle aspiration through the vaginal wall under ultrasound guidance. The procedure is performed under intravenous anesthesia. Usually the patient is discharged approximately 30-60 minutes after the procedure.
> Several hours after the procedure, oocytes that show a proper grade of maturation (metaphase II) are cryo-stored by vitrification (rapid freeze) and stored in liquid nitrogen.
> So, the process of vitrification has 3 critical components. First, eggs or embryos are exposed to high concentrations of cryoprotectants to allow rapid dehydration of cells. Second, the eggs or embryos are loaded into tiny storage devices (usually straws) that will facilitate ultra-rapid cooling, and third, the straws containing the eggs/embryos are cooled as fast as possible, typically at thousands of degrees per minute.
> In practice, this means that eggs and embryos are vitrified very quickly in the laboratory. A typical embryo vitrification protocol is complete in about 10 minutes. Embryos are removed from the incubator in the laboratory and exposed to an equilibration solution for 8 minutes to begin the dehydration process. This solution contains about 15% cryoprotectant, After the 8 minute time in equilibration solution has elapsed, the embryos are moved into a different vitrification solution that has different percantige of the components and stay exposed to this solution for just 60 seconds. Then they are quickly loaded into straws and plunged into liquid nitrogen at a temperature of -196°C. The tiny straw will cool from room temperature (about 25°C) to -196°C in 2-3 seconds giving a cooling rate of 4420 to 6630°C/minute.
> This high cooling rate combined with the use of high concentrations of cryoprotectants allows the contents of the straw (embryos plus surrounding fluid) to turn to a glass like substance instead of ice. Avoiding ice formation in this way successfully protects the embryos from damage and allows them to be warmed later giving survival rates consistently above 90%.
> When patients return to use their vitrified embryos or eggs, the vitrification procedure described above is reversed to allow warming back to room temperature and then 37°C, and to allow for rehydration. Since the eggs/embryos are not technically frozen (no ice was formed), we do not use the term “thawing” for this process. The procedure “warms” the eggs/embryos in just 20 minutes and they are placed back in the incubator at 37°C in the laboratory. Embryos can be transferred back to the uterus immediately and eggs can be injected with a single sperm 3-4 hours later.
