Sperm and Embryo Freezing
Freezing of human sperm, eggs and embryos
Within the laboratory at PFC we have a tissue bank where we freeze and store oocytes (eggs), sperm and embryos for our patients. The bank has a full time manager under supervision of the lab director, and is licensed by the State of California. All tissue in the bank is stored frozen in liquid nitrogen at a temperature of -1960C in vacuum lined tanks that are computer controlled and monitored 7 days a week with a dedicated alarm system. The embryologists are responsible for maintaining the bank and no other PFC employee has access.
How are frozen embryos stored and monitored?
The air that we breathe contains a gas called nitrogen. This gas makes up about 78% of the air around us. If nitrogen gas is cooled, it becomes liquid at -1960C. This liquid is very stable and easy to work with. In the laboratory we have large tanks filled with liquid nitrogen in which we store frozen embryos. Each tank is in effect like a large thermos flask since it is vacuum lined. All patients have designated storage spaces within a tank, where their eggs or embryos are kept. The straws that contain the embryos are color-coded and labeled with precise and unique identifying information.
The tanks that contain frozen embryos are monitored 7 days a week. Each tank gets a physical inspection daily, looking for problems or signs of problems. The quantity of nitrogen in the tank is assessed as a means of monitoring for a possible slow leak or an impending tank failure. The nitrogen in the tank is topped up daily, since it continuously evaporates at a slow rate.
Electronic tank monitoring uses different sensors to ensure that tanks perform to specifications. Probes attached to the tank detect a rise in temperature within the tank, or a drop in the level of liquid in the tank. Sensors are connected to a telephone alarm system that will alert staff to an alarm condition outside of normal working hours
The telephone alert system is a complicated monitoring device. It requires that multiple people be contactable at any given time, and calls and recalls each person in turn until somebody responds to the alarm. We aim to have an embryologist in the laboratory no more than 30 minutes (day or night) after the alarm is set off. The alarm system is tested weekly and continues to run on battery power in the event of a power failure. The alarm system can also be checked remotely. The status of each individual tank can be ascertained by telephone at any time.
How long can embryos be stored?
No one knows what the maximum storage period might be. Procedures for human embryo freezing were developed in 1984 and only went into widespread use in the late 1980s. This means that the longest time a human embryo has been stored is 25-30 years and, typically, patients that have left embryos in storage for this long are not coming back for them. Some patients have come back after 10-15 years and the embryos have been thawed successfully and created healthy babies. Beyond this time frame, we don't know how long an embryo will remain viable, but it is possible that, kept in liquid nitrogen, an embryo may be viable indefinitely.
How are embryos thawed?
The process of embryo freezing has already been explained. Thawing the embryos is simply a reversal of the freezing procedure.
Thawing includes our SurTransferTM protocol. When an embryologist removes embryos from the freezer, a second embryologist is required to witness the act, and verify the identity of the embryos before they can be thawed. Under no circumstances can a lone embryologist remove embryos from the freezer without another embryologist to confirm correct identification.
The embryos coming out of the freezer (at –196°C) are warmed to body temperature in 2-3 seconds. This rapid thaw method minimizes damage to the embryo from ice that could form during warming. The embryologist has to remove the antifreeze from the embryo and replace the water that was removed at the time of freezing. This is done by incubating the embryo in decreasing concentrations of the antifreeze, and increasing concentrations of water. Over a period of 20 minutes, the embryo is stepped through different solutions, until finally the antifreeze is gone and all the water has been replaced.
The embryo can be ready for transfer in as little as 40 minutes after leaving the freezer.
What are the costs for keeping embryos in frozen storage?
The costs to Pacific Fertility Center are significant. Most of the cost is accounted for by the personnel required to maintain the tanks and manage the inventory of stored embryos. Only embryologists handle liquid nitrogen and maintaini the frozen embryo bank. Although nitrogen liquid is relatively stable, it can cause severe frostbite, rapid suffocation and death if mishandled. We even have a special permit from the city just to have the liquid in the building and the emergency services have to be kept informed of our activities.
We buy several hundred liters of nitrogen each week to keep the tanks filled and to use for freezing of new eggs and embryos. Tanks are serviced, replaced as necessary and maintained according to a strict schedule and alarm systems are also regularly tested. Ask any embryologist, and they'll tell you that the real cost is in having to race to the lab at 3:00 am on a Saturday morning only to find a false alarm.
Would the storage tanks survive a major earthquake or other disaster?
Probably not. While the tanks are secure and robust, they could be crushed or severely damaged by falling masonry. Any catastrophe that would collapse the building would almost certainly destroy the tanks.
The storage tanks require no power and would not be impacted by a power failure or blackout. They are made of metal and would probably survive a small or moderate fire. If the tanks were not physically damaged or knocked over in a disaster, they should survive intact. Even if no one was able to physically check the tanks, or if we were unable to obtain liquid nitrogen, the tanks should still hold their temperature for several days.
Other than for running the computer, the tank does not need electricity and the liquid nitrogen can be topped up manually at any time by an embryologist. However, the computer is monitoring and maintaining the liquid levels most of the time in the same way that the autopilot on an airplane does most of the flying. The computer will alert us if there is a problem or a power failure. The automated phone based alarm system calls the on-call embryologist for physical inspection before the alarm can be shut off. The tanks have aninternal reservoir for liquid nitrogen as well as a connection to the bulk supply. Furthermore, we conduct a manual inspection of the tanks at the end of each day, 7 days a week, to ensure that everything is in full working order. The alarm system and the back-up power generator are each tested in a frequent and periodic manner.
The frozen tissues at PFC are housed in a stable, safe and modern environment with daily monitoring and upkeep. As far as we know, there is no limit to how long cells remain viable in the frozen state. We have had some patients return to thaw embryos after more than 10 years and the embryos were no different that when they were frozen. The temperature of liquid nitrogen is so cold that scientists think that all biological activity is stopped and that there are no issues with very long term storage. But for most patients, shorter tem storage should be the goal. We are happy to counsel patients on their options for storage or disposition of their frozen biological tissue and help them make the appropriate decisions for their family.
The laboratory itself is equipped with an array of highly complex equipment. Since the laboratory is a clean-room with access restricted only to embryologists and physicians, maintenance and housekeeping activities are handled by the lab staff themselves, including keeping the equipment fine-tuned and in excellent working order. When it is necessary to have visitors in the laboratory, they are escorted by laboratory staff but only after putting on white "bunny" suits. Any outside objects that enter the lab are cleaned with alcohol.
All embryo straws are labeled and a file is maintained for every patient who has embryos in storage. This extremely important aspect of storage is taken very seriously. A thorough inventory is maintained in multiple places. Patient addresses are kept up-to-date and confirmed annually when the invoice is sent or when patients notify the center of an address change.