Cryopreservation

• Slow Freezing
• Vetrification
• Sperm
• Egg
• Embryo
• Overian Tissue

Cryopreservation and Vitrification

It is possible to cryopreserve sperm and embryo at -196º C in liquid N2 by computerized slow cooling method. Subsequent cryothaw cycles treatment becomes cost effective and patient does not require undergoing active cycle management. Embryos and sperms can be kept cryopreserved for years. Slow cooling cryopreservation needs computerized cryo machine. Recently developed vitrification technique uses rapid cooling of embryo. Human egg cryopreservation was difficult because of damage done to spindles. With successful cryopreservation of ovarian tissue it is of immense help to young cancer patients undergoing chemotherapy or ionizing radiation therapy for future fertility.

Kutluk Oktay and colleagues took a strip of ovarian tissue from a 30-year-old woman with breast cancer before chemotherapy. Six years later, after the patient had entered menopause, the tissue was re-transplanted back underneath the skin of her abdomen. After some three months the tissue regained normal function and started to produce viable eggs which were fertilized in vitro, resulting in one promising embryo. This latest clinical development indicates that endocrine function and fertility may be restored by long-term cryopreservation of ovarian tissue re-transplanted back into post-menopausal women.

Cryopreservation of Human Embryos

Egg retrieval under ultrasound guidance and subsequent fertilization and embryo culture are carried out according to our current procedures. If there happens to be a surplus of embryos following selection for fresh transfer (usually between one to four embryos are transferred to the uterus), then embryos of sufficient quality may be considered for cryostorage. While embryos can be frozen at any preimplantation stage between one-cell (one day old) to the blastocyst stage (5-6 days old), in an attempt to minimize the freezing of excessive numbers of "spare" embryos and to help pre-select the most potentially viable embryos, we generally choose to cryopreserve only at the blastocyst stage. In certain cases where all embryos need to be frozen without a fresh transfer (e.g., when a woman may be at risk from ovarian hyperstimulation that might be complicated by pregnancy), we generally freeze all embryos the day after egg collection at the one-cell stage.

Techniques of controlled-rate freezing are utilized that slowly cool embryos in cryoprotectant fluid ("anti-freeze" solution) from body temperature down to -196°C, at which temperature they are stored in containers of liquid nitrogen called dewars. The embryos are actually contained within special indelibly labeled plastic vials, or straws, that are sealed prior to freezing. Once frozen, they are placed inside labeled tubes attached to aluminum cans and stored in numbered canisters within the liquid nitrogen dewar. Site and label designations are stored in three separate file systems to avoid confusion and misidentification of cryopreserved embryos. When it comes time to thaw the embryos, all available identifiers of the stored specimen must match and be confirmed before thawing commences. The embryos are thawed out at room temperature, which takes about one to two minutes. However, the most critical element of the thaw procedure is not the timing but the careful dilution of the cryoprotectant fluid to return the embryo to its favored culture medium. This permits resumed growth and development in vitro. Once this is done, the embryo is assessed for cryodamage to determine if it is suitable for transfer. Experience has shown that if the embryo survives 50% or more intact, it is worthwhile to replace it. Embryos can accommodate such levels of cellular damage and still establish healthy pregnancies. All thawed embryos routinely undergo assisted hatching prior to transfer. The zona pellucida, which surrounds the embryo, has been shown to suffer a certain amount of hardening during cryopreservation. This can be overcome by artificially making an opening in the outer embryo shell.

Varying strategies may be applied according to how many and which embryos are thawed prior to transfer. It should be noted that not every couple undergoing IVF will need to worry about embryo freezing/thawing, since not every couple will have sufficiently large number of "surplus" or non-transferred embryos available for freezing. Indeed, most couples have only one or two embryos frozen, so that all are thawed and any surviving are replaced. In the event that there are more than two or three embryos frozen, thawing is usually undertaken until two to three healthy appearing embryos are recovered. In some cases, this may mean that all the cryopreserved embryos are thawed, in others just two or three. There always remains a possibility that there may be no embryo survival after thaw occurs, and no transfer is possible. If many early embryos are frozen, it is possible to thaw all of them and culture them for several days to allow selection of the best for transfer. When too many embryos are available for transfer in this circumstance, then extra embryos of sufficient quality may be refrozen for later use. This course of action has produced healthy offspring, proving the efficacy of double freezing of embryos.

During a medication-prepared frozen/thawed embryo transfer cycle as a patient, you will follow a treatment schedule using Lupron, estrogen (pills) and progesterone (suppository) in order to achieve appropriate endometrium (uterine wall lining) for embryo transfer. Following embryo transfer, estrogen and progesterone will be administered daily until the 7th to 8th week of pregnancy or until a negative pregnancy test.

Considerations and Riska

The Ethics Committee of the American Society of Reproductive Medicine (ASRM) has published guidelines for ethical consideration of human embryo cryopreservation. Possible advantages of cryopreservation of embryos suggested by the Committee include

• Reduction of the risk of triplets or quadruplets by cryopreservation of embryos exceeding an optimal number for transfer to an individual patient
• Possibly increasing pregnancy rates by replacing thawed embryos during spontaneous ovulatory cycles or cycles in which the estrogen and progesterone hormone levels do not exceed that which occurs naturally
• Possibly decreasing the number of stimulated ovary drug treatment cycles needed for the attainment of pregnancy

The primary concern with the use of cryopreservation techniques is the possible loss of embryos to cryoinjury, meaning some healthy embryos may not survive the stress of freezing & thawing. The exact number of embryos lost to cryoinjury varies, but it is very likely that freezing will cause loss of some embryos, perhaps as many as 25-50% of those cryostored. One interpretation of this is that cryopreservation may even act as a "selection gate" for the more viable embryos, though this has never been proven.

Another concern with cryopreservation is the potential risk of birth defects in children produced from frozen/thawed embryos. In the domestic animal industry, large-scale freezing and transfer of embryos has not resulted in increased birth defects. Studies to date on those human offspring arising from thawed embryos have not shown any significant increase in abnormalities when compared to pregnancy outcomes in the rest of the population.

To optimize the likelihood of successful embryo cryopreservation, the mechanical processes of human embryo cryopreservation will be strictly controlled to minimize the chances of technical failure. A back-up freezing system is always available to decrease the risk of interruption in the freezing process, as well as generator back-up power in the event of a power shortage. Individual embryos are placed in permanently labelled storage containers and identified according to origin, developmental stage, and date frozen. Permanent records are kept for each individual's embryos. Liquid nitrogen dewars are connected to alarm systems to monitor the liquid nitrogen levels and prevent premature thawing. However, even with all these safeguards, the possibility of technical failure leading to loss of stored embryos following natural disaster cannot be totally and completely eliminated.

Sperm Banking

There are many myths and misconceptions regarding cryopreservation or sperm banking. Hopefully, this reading material will clarify some of those issues and help you to better understand the concept of freezing sperm, or "banking."

Many patients who have malignant tumors will have a decrease in the ability of the testicle to produce sperm. That is, often when a patient has a tumor, the testicle will not produce sperm as well as it does normally. This tumor may be Hodgkin's disease, lymphoma, sarcoma, a testicular malignancy, or a variety of other malignancies. Therefore, it is important to recognize that at the time you present to our clinic for sperm banking, your testicles may not be working as well as they might normally. Although this decreased function is not directly related to the stage of your tumor, it appears that the better you feel at the time you present to our clinic, the better the results of your semen specimen may be.

Many patients who have developed tumors such as Hodgkin's disease and testicular cancer have already fathered children. This shows us that, in general, their testicles have the potential to work quite well. If you are in this group, we would expect that your semen analysis would be quite good. Again, however, it does depend somewhat on how you are feeling at the time you come to see us, since this may reflect how the malignancy has affected your general health.

Most chemotherapeutic drugs will damage the testicle at least temporarily. Some will have permanent effects on the testicle. The testicle performs two functions, the first of which is to produce male hormone. None of the chemotherapeutic drugs seem to impair the testicle's ability to manufacture testosterone (the male hormone) either while you are receiving or after you have finished your chemotherapy. The second function of the testicle is to produce sperm. Almost all chemotherapeutic drugs will shut down the production of sperm to some degree while you are receiving the drugs. Certain chemotherapeutic medications damage the machinery of sperm production to such an extent that the testicle will never recover and will never again produce sperm.

In general, extended treatment for Hodgkin's disease will permanently damage the testicle's ability to make sperm in approximately 80%-90% of patients. It is important to remember that although this is a significant side effect of the therapy for Hodgkin's disease, the cure rates for Hodgkin's disease have been improving over the last several years, and your doctors want to make sure that you are cured of your disease.If you have a testicular tumor, you should know that the chemotherapeutic medications that are used to treat these specific types of tumors may not permanently damage the testicle as much as the ones that are used in a prolonged fashion to treat Hodgkin's disease. Approximately 30%-40% of patients who are treated for lymphoma or leukemia will regain normal sperm counts by 2 years after the chemotherapy ends. Approximately 50%-70% of patients who are treated with the standard chemotherapeutic medications for testicular cancer will regain some sperm production 2-3 years following their chemotherapy.

It is important that you understand that sperm banking before you receive chemotherapy is like an insurance policy. If you are one of the lucky patients who regains normal sperm counts after chemotherapy, then you should consider yourself fortunate that you do not need the services of the sperm bank. However, if you are one of the patients who does not regain normal sperm counts after chemotherapy, then to put sperm in the sperm bank before your chemotherapy was a very intelligent decision.

Cryopreservation

For patients with testicular cancer, some of you may not have good function in your remaining testicle even before chemotherapy. This is simply due to non-malignant changes in the testis itself. Therefore, before and after chemotherapy, the remaining testicle simply may not work as well as it should.

Sperm banking is performed by first analyzing your semen to determine the sperm count and also looking at the movement of the sperm. Depending on these factors as well as the volume or amount of the sample that you have provided us, we then place the semen sample in individual plastic vials for the freezing process. The number of vials that we are able to obtain is dependent upon the sperm count, sperm movement, and volume of your ejaculate. We generally recommend that 2-3 samples obtained for sperm banking. The more sperm that you are able to leave in the sperm bank, the better your chances are to have children in the future if you are one of those patients who does not regain normal sperm counts after the chemotherapy. Your physician may be anxious to start chemotherapy as soon as possible. In this case, we will simply have to get as many samples as we can; however, you must remember that your chemotherapy and your ultimate good health are our top priorities!

After you have given your samples, the laboratory will let you know how many vials they were able to obtain. The sperm will be frozen for 5 years. At the end of that 5-year time period, they will contact you to see if you would like them to store the sperm for another 5 years. If you have regained normal sperm counts and are doing well, then at that time you may make the decision to discard your stored sperm. If you have not regained normal sperm counts, then they mayl continue the storage for another 5 years.

If at any time you would like your spouse to become pregnant and you have not regained an adequate sperm count, then your frozen sperm can be used in association with one of the modern assisted reproductive techniques. These include intrauterine insemination (IUI), in vitro fertilization (IVF), and micromanipulation (placing one sperm microscopically into each egg). The quality and quantity of cryopreserved semen determines which options in assisted reproduction are available to you. Patients with several vials of good-quality semen may attempt IUI, whereas patients with smaller amounts of cryopreserved semen, or semen of poor quality, may opt for routine IVF, or IVF with micromanipulation. Pregnancies have been achieved with all of these methods utilizing cryopreserved semen. It is crucial to understand that any sperm that fertilize the egg and produce an embryo are likely to be normal. That is, any sperm that are produced by your testicles, even after chemotherapy or after x-ray therapy, as long as you have waited 1 year, or any sperm that are frozen and then thawed and used for insemination, will not have a higher likelihood of producing a deformed child. Your chances of having a normal baby are as high as that of the general population.

In summary, the key points to remember are:

• Before your chemotherapy or radiotherapy, your sperm counts may or may not be as good as they normally were.
• Depending upon the type of disease that you have, the chemotherapy may or may not render you permanently sterile.
• If you do regain normal sperm counts after your chemotherapy, then please contact the laboratory, and they will discard your sperm.
• If you do not regain normal sperm counts after chemotherapy, please contact them any time you would like to use your sperm in order to achieve a pregnancy. They will work closely with gynecologist.
• Any child who is conceived after either chemotherapy or radiotherapy is completed and a "rest" period passed, or any child conceived with sperm from the sperm bank, will have as much chance to be normal as a child born to normal parents.

Sperm Banking

When a specimen is processed for cryopreservation:

• A semen analysis is performed on each ejaculate. This includes a complete seminal fluid analysis quantitating sperm motility, forward progression, sperm density, and morphology.
• All specimens are stored in liquid nitrogen storage tanks (-196_C).
• There does not appear to be any increased risk of birth defects using frozen semen.
• There is no guarantee that any given specimen will necessarily produce a pregnancy; however, recent studies indicate that the overall success rate following the use of cryopreserved semen is from 40%-50%.