PFC’s state-of-the-art embryo storage tanks

Freezing Sperm

1.Labeled vials	2. Cooling Tank	3. Storage tray	4. Storage drawer	5. Nitrogen tank

Sperm can be frozen on demand and tolerate freezing more readily than embryos. Most of the sperm samples that we freeze are for patients who have had sperm collected surgically. Freezing provides for future IVF cycles without the need of further surgery. Occasionally we will freeze sperm for men who cannot attend on the day of their partners' egg collection procedure, or for men who are anxious about producing a sample.
The sperm are stored in carefully labeled vials (1) and placed in a cooling tank (2). The vials are then placed in a tray (3), slid into a labeled storage drawer (4) and lowered into a nitrogen tank (5).

Freezing and storing human embryos
2004 marks the 20th anniversary of the first successful birth that resulted from human embryo cryopreservation. Since 1984, thousands of babies have been born worldwide after having undergone embryo cryopreservation. Freezing of excess good quality embryos allows for the transfer of fewer embryos in the stimulated IVF cycle and therefore ensures fewer high-order (triplets or more) multiple births. This process provides patients with a "back-up" should the initial fresh embryo transfer not result in a pregnancy, at a much lower cost than starting IVF all over again and often with minimal medications. Frozen embryo transfers have allowed many of our patients to achieve more than one pregnancy from a single cycle of ovarian stimulation.
In 2002, 343 babies were born as a result of assisted reproduction procedures performed at Pacific Fertility Center. Of these babies, 98 or 28.6%, were babies conceived after having been stored as a frozen embryo

Freezing is a very traumatic procedure for an embryo and not all embryos survive. It is normal for one or more of the cells in the embryo to die as a result of the freezing and thawing procedure. However, provided that the majority of the cells in an embryo survive, the embryo still has a chance to establish a pregnancy.

How are embryos frozen?

Step 1.	Step 2.	Step 3.	Step 4.

Embryos can be frozen at different times after fertilization. Most typically, embryos are frozen 1, 3 or 5 days after the sperm and egg were put together. Freezing is a stressful process for an embryo, and only embryos that are growing well in the laboratory will tolerate the freezing procedure.

Step 1. Before an embryo can be frozen, all the water that it contains must be removed. Since water expands in size as it turns to ice, water inside the embryo would burst (kill) the embryo if we simply placed it in the freezer. To prevent the embryo from shriveling as the water is extracted, we replace the water with antifreeze. Antifreeze is a solution that does not expand in size when it freezes. The embryo is cooled to room temperature as the water is replaced with antifreeze.

Step 2. When most of the water has been removed the embryo is inserted into a carefully labeled vial, or more typically a small straw, and placed in the cooling chamber of a controlled rate freezer.

Step 3. The embryo is then cooled very slowly at -0.30C per minute. Slow cooling like this allows the embryologist to have precise control over the freezing process, to maximize water extraction from the embryo and to prevent formation of large ice shards that could pierce the embryo.

Step 4. The cooled straw is placed into carefully labeled metal canes and lowered into the tank with other frozen embryos. The entire process takes several hours and the embryo(s) are stored frozen at –1960C in liquid nitrogen. Liquid nitrogen is a safe and effective coolant, which is easy to work with in the laboratory.

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 many ways like a large thermos flask. It is vacuum lined . Each patient has a designated storage space within a tank, where his or her embryos are kept. The straws that contain the embryos are color coded and labeled with precise and unique identifying information. At a minimum, this information includes the patients full name, their date of birth, their social security number, the number of embryos in the straw, the stage at which the embryos were frozen and the date on which the freezing was performed.

The tanks that contain frozen embryos are monitored 24 hours a day, 7 days a week, 365 days a year. Each tank gets a physical inspection twice a day, looking for problems or signs of wear. 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 once or twice a week, since it continuously evaporates at a slow rate (if a tank was not filled regularly, the nitrogen would evaporate entirely in about 6 weeks).

Electronic tank monitoring uses 3 different sensors to ensure that tanks perform to specifications. A probe attached to the tank lid, actually sits in the nitrogen with the embryos. The probe will detect a rise in temperature within the tank, or a drop in the level of liquid in the tank. The laboratory also has an oxygen alarm that will detect when nitrogen is evaporating at a high rate and displacing oxygen from the air. All 3 of these sensors are connected to a telephone system that will alert staff to an alarm condition.

The telephone alert system is a complicated monitoring device. It requires that 8 people be contactable at any given time, and calls and recalls each person in turn until somebody enters the laboratory and cancels the alarm. The alarm cannot be canceled remotely, and our protocol requires that an embryologist be in the laboratory no more than 30 minutes (day or night) after the alarm is set off. The alarm system is tested every day 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 1980's. This means that the longest time a human embryo has been stored is 12-15 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 10-12 years and the embryos have been thawed successfully. Beyond this time frame, we don't know how long an embryo will remain viable.

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 SurTransferSM 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.

The embryos coming out of the freezer (at –1960C) are warmed to room temperature in 35 seconds. This rapid thaw method minimizes damage to the embryo from ice shards. 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 30 minutes, the embryo is stepped through 4 different solutions, until finally the antifreeze is gone and all the water has been replaced.

The thawing procedure is performed at room temperature, and once complete, the embryo is warmed up to body temperature (370C). It can be ready for transfer in as little as 40 minutes after leaving the freezer.

Can freezing damage my embryos?

While every care is taken to protect the embryos during the process, some embryos will have one or more burst cells after they have been thawed. In the photo above, the arrow is pointing to 1dead cell in 4-cell embryo. This cell loss results either from puncture by tiny ice shards around the embryo or from rupture as water rapidly enters the cell during thawing. At PFC, on average, 2 out of every 3 cells in an embryo tolerate the thawing process. We consider an embryo with 50% or more of their cells surviving as having a normal chance of implanting after transfer. If an embryo thaws with fewer than 50% of its cells alive, we usually recommend thawing another embryo if one is available.

The first baby resulting from a frozen-thawed embryo was born in Australia in 1984. The embryo had 8 cells when frozen, but 2 cells died during thawing. Even though the transferred embryo had only 6 living cells, it was still capable of developing into a normal baby.

We consider that any embryo that survives thawing, even with only one cell intact, has the potential to establish a pregnancy. However, the chance for pregnancy will depend on how well the embryo survives. If an embryo survives with all cells intact, it will have a better chance for pregnancy than an embryo that loses half of its cells. Embryos with less than half of their cells remaining will have significantly lower chances of developing.

Am I more likely to have a child with a genetic or congenital abnormality because I'm pregnant after a frozen embryo transfer?

Even after 20 years, there are few studies in the scientific and medical literature concerning outcomes after embryo cryopreservation. However, the few studies that have been published are thus far reassuring. Children born from frozen embryos do not seem different from children born from embryos that had not been frozen. Even if an embryo loses one or more of its cells during thawing, (see section above) this does not cause any abnormalities. Freezing does not cause or introduce genetic abnormalities. Go to Fertility FlashSM Volume 2 Issue 3 for more information.

What are the costs for keeping embryos in frozen storage?

The costs to Pacific Fertility Center are huge. Most of the cost is accounted for by the manpower required to maintain the tanks. Embryologists receive special training in handling liquid nitrogen and maintaining 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 embryos. Tanks are serviced, replaced and maintained according to a strict schedule and alarm systems are also maintained to a very high standard. Ask any embryologist, and they'll tell you that the real cost is in having to race to the lab at 4.00 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 weeks.

What are my options for using the embryos?

We hope that most couples will be able to use the embryos to have a healthy baby. Patients having frozen embryos transferred, make up about one fourth of the patients visiting our office. Some are thawing embryos after failing to become pregnant during their IVF cycle, and some are using the embryos years after a successful IVF cycle, to have a second or third child.

You may be surprised to learn that there are a significant number of people who do not want to use their frozen embryos to become pregnant. These are typically people that have completed their families and are not interested in having any more children. Having embryos remaining creates a very difficult situation for these families. The embryos can be discarded as medical waste, but the decision to destroy the embryos is not made easily. Couples with children resulting from IVF treatment often view the frozen embryos as potential children and siblings for their existing children. Coming to terms with destroying the embryos can be impossible, and many couples avoid taking this decision by simply leaving the embryos frozen indefinitely. In the UK, the government has taken action against these couples by ordering the destruction of all embryos in frozen storage for more than 5 years.

Frozen embryos can also be donated for research studies. Embryos donated for research will be thawed and used in a scientific study, and discarded after a few days. A research study might look at new ways of freezing or thawing embryos, new ways of growing embryos in the laboratory or at the genetic make up of the embryos. The studies will not benefit the patient that donates the embryos, but the research may benefit other IVF couples in the future.

How are embryos destroyed when patients request disposition?

A formal request to destroy the embryos must be received in writing from the patients. The request must be signed by both partners and notarized or witnessed by a member of our staff. Once the laboratory has received the disposition notice no action is taken for 30 days. This gives the couple a cooling off period and an opportunity to change their decision.

When the 30-day waiting period has passed, two Embryologists take responsibility for carrying out the patients' wishes. They fill out a form indicating that they have checked the disposition request and are in agreement that the patient wants the embryos discarded. They locate the embryos in the storage tank and double check the identity with the paperwork. The embryos are then thawed and discarded as medical waste. The paperwork is complete when both embryologists sign, attesting that they performed and witnessed the destruction according to the patients' wishes. The paperwork is kept in the laboratory files and a copy filed in the medical record of the patient.

Can I donate my embryos to another infertile couple?

Yes, it is possible to donate embryos. This process is sometimes referred to as embryo adoption.

There are many advantages to embryo donation, including giving another couple the chance to have a child and avoiding having to discard the embryos that took so much effort to create. However, the process resembles adoption in many ways, as there are a series of requirements that need to be satisfied before the donation can be completed. These include legal contracts between the parties and psychological counseling. Also, matching donors and recipients can be very difficult as each couple will have specific demands and these can be difficult to satisfy. You might want to donate your embryos anonymously or you might ask that your children be allowed to have contact with any children that result from use of the embryos. The recipient couple may or may not want contact with you. However, they might insist on medical tests on you or information on your family that you won't necessarily want to give out. They may want a clause in the contract that gives them access to you if the child becomes severely ill. They could ask that you adopt the child if anything happens to them.

Since the matching of couples for embryo donation is complicated, Pacific Fertility Center do not typically get involved. Patients are required to find their own donor or recipient and if the legal contracts and psychological testing can be completed, we can help with medical tests and treatment. Often, the embryos will have to be moved to another part of the country, since you won't necessarily find a recipient in San Francisco. In this situation, shipping the embryos is the responsibility of the donating couple, and we can provide information on getting embryos sent to another IVF clinic.

Couples considering embryo donation can find information on this subject on the Internet at www.snowflakes.org and from RESOLVE, a national organization for childless couples at www.resolve.org.