Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.
The oocyte vitrification and the warming procedures are essential for fertility preservation. Following this protocol as demonstrated allow a user to maximize the effectiveness of these procedures. Fertility preservation for oocyte vitrification is today an established and ethically acceptable approach.
It allow to achieve effective and consistent clinical outcomes and overcome the moral and legal limitation associated with embryo cryopreservation. To achieve proficiency with the vitrification process, it is important to become familiar with all the critical points of the protocol, paying particular attention to the exposure time of the samples to the cryoprotectants. Within 38 hours of retrieval and immediately after denudation, label all of the plastic supplies with the patient's name, ID, and type of solution, and ask a witness to confirm the patient's information on the cryo device.
When all of the supplies have been labeled, carefully invert each vial of oocytes several times to mix and place a 30 microliter drop of HEPES buffer medium supplemented with human serum albumin and a 30 microliter adjacent drop of equilibration solution on a petri dish. Use the stripper pipette to place the oocytes in the first drop and create a bridge of medium to a 30 microliter drop of equilibration solution to obtain a gradual increase in concentration of the cryoprotectants. After three minutes, add a second 30 microliter drop of equilibration solution to the plate and use the pipette to create a medium bridge between the drops of solution.
While the oocytes are equilibrating, add one 30 microliter drop of equilibration solution for each cryo device to be used onto the plate. After three minutes, move the oocytes into the pure equilibration solution for six to nine minutes to allow the oocytes to return to their initial size. When the oocytes have recovered, prepare a central well IVF dish containing one milliliter of vitrification solution.
Transfer the oocytes to the dish in as little medium as possible and carefully move them to remove any excess equilibration solution. Approximately 10 seconds before the end of the incubation, place a cryo device labeled with the patient's information under the microscope and adjust the focus on the tip of the cryo device. Place the oocytes on the cryo device beside the tip in the minimum amount of vitrification solution and move the stripper pipette away from the oocytes to remove any excess vitrification solution, leaving the oocytes covered with a thin layer of medium.
Quickly plunge the cryo device into liquid nitrogen and rapidly shake the device to remove any air bubbles from its surface. Holding the protective cap of the cryo device with tweezers, fill the cap with liquid nitrogen before inserting the device into the cap while keeping the propylene strips in liquid nitrogen. Then store the cryo device in a visotube labeled with the patient's information and update the laboratory sheet.
For oocyte warming, carefully invert each vial of thawing, dilution, and washing solution warmed to room temperature, and add one milliliter of thawing solution to the central well of a petri dish for an at least one hour incubation at 37 degrees Celsius. Label all of the plastic supplies with the patient's name and ID and the type of solution, and ask a witness to confirm the patient's information on the cryo device. For each device to be warmed, add 200 microliters of dilution solution to the first well of a six well plate and add an equal volume of washing solution to the second and third wells of the plate.
Add PBS to the area on the outside of the wells to prevent solution evaporation. Place the dish of thawing solution under the stereo microscope and adjust the focus to the center of the dish. Carefully twist to remove the protective cap of a cryo device while keeping the propylene strips in liquid nitrogen and transfer the cryo device from the liquid nitrogen into the thawing solution as quickly as possible to avoid the risk of devitrification.
After one minute, focus on the tip of the cryo device to locate the oocytes and eventually use a stripper pipette to gently release the oocytes from the device. Use a 170 micron diameter stripper pipette to transfer the oocytes and a small volume of thawing solution into the dilution solution in the first well of the six well plate. After three minutes, move the oocytes into the first well of washing solution for five minutes.
At the end of the incubation, transfer the oocytes to the second well of washing solution and incubate at 37 degrees Celsius for one minute before transferring the oocytes into an appropriate volume of pre-equilibrated IVF culture medium for one hour and updating the laboratory sheet. Over a 12 year period, 285 women underwent at least one oocyte retrieval entailing the vitrification of the whole cohort of mature eggs collected. Most of these women underwent a single retrieval.
35 underwent multiple retrievals. The reasons for undergoing oocyte retrieval for egg vitrification were generally characterized as medical other than cancer, cancer, nonmedical, and other. Patients with a medical reason for egg vitrification and patients undergoing fertility preservation because of cancer were younger and showed higher antral follicular counts than patients with nonmedical or other reasons.
However, as the mean maturation rates were slightly lower in the medical reasons patients, the number of oocytes vitrified on average was similar between the groups. Approximately half of the patients with medical reasons other than cancer, and the majority of patients with other reasons for egg vitrification actually returned for warming. Conversely, very few patients who underwent fertility preservation for cancer or nonmedical reasons used their vitrified oocytes for IVF.
Despite differences in the time elapsed between vitrification and warming, the survival rate of the oocytes was similar between patient groups, confirming the efficacy and safety of the oocyte vitrification and warming protocols. Moreover, the survival rate was independent of the vitrification and warming operator's experience. The most crucial steps that may affect the consistency of the results are the one related with warming.
Therefore, it is really important to carefully control the volume and the temperature of the thawing solutions. Ovarian tissue cryopreservation the only strategy currently under investigation as an option for pre-pubertal patients. Although promising, this approach has important limitation which limited its application.
