Every year, several Pacific Fertility Center professionals participate in ASRM’s national meeting. They evaluate the research and share their findings with PFC and Fertility Flash.

Among those attending the conference from PFC were Dr. Philip Chenette and Dr. Isabelle Ryan and Peggy Orlin, MFT. Their reviews cover the following topics: Update #1: Ovarian Stimulation Techniques, Update #2: PGD and Aneuploidy Screening Techniques, Update #3: Egg Freezing, Update #4: Acupuncture, and Update #5: Men and ART.

ASRM Update #3: Egg Freezing

Oocyte cryopreservation is the storage of the female gamete, the egg, prior to fertilization. Preservation of fertility for single women that must undergo cancer therapy or surgery, or that must delay or choose to delay childbearing, and donated oocyte banking are all applications of oocyte cryopreservation. The need for this technology is clear, but reports of success with oocyte cryopreservation have been limited.

Highly successful oocyte cryopreservation is now attainable. New studies are showing pregnancy rates with oocyte cryopreservation that are equal to traditional IVF techniques.

The key to this technology is oocyte vitrification – an ultrarapid cryopreservation technique. Researchers from Atlanta described their experience with vitrification. Out of 11 patients with transfers, nine conceived, with an implantation rate of 65%.

Pregnancies after oocyte cryopreservation have developed normally. An Italian study of 105 children born after oocyte cryopreservation showed no problems. A Chicago study of the genetics of oocytes, embryos, and children born after oocyte cryopreservation was reassuring. No increase rates of aneuploidy or malformations were reported, and normal development was found in post-natal follow-up.

These results are similar to those we have previously reported from our own research at Pacific Fertility Center (see December 2007 Fertility Flash). Oocytes are now cryopreserved with high success rates. Oocyte cryopreservation technology has matured, and we look forward to providing these techniques for our patients.

Philip Chenette, MD

Question: I am 35 years old and single, but am still hoping to find my life partner. I am getting a little concerned as my gynecologist has asked me about my plans for having children. She mentioned that I might want to consider freezing my eggs for future pregnancies. Is this something I should do?

Answer: Vitrification is a very new process for preserving unfertilized eggs. As noted in this month’s lead article, PFC has successfully been vitrifying oocytes from proven egg donors. Our first birth from this process occurred in October. Three additional pregnancies from this trial are ongoing. PFC undertook this vitrification trial in order to develop expertise with the technology of oocyte vitrification. For this reason, our study population was confined to donor eggs from healthy donors in their mid-twenties who had successfully completed conventional egg donation.

Why do we want to freeze eggs? For the many single young women diagnosed with cancer and facing fertility-threatening chemotherapy, egg vitrification will provide a fertility preservation option. This group of women has a compelling reason to consider undertaking the procedures and costs involved with in vitro fertilization. The potential threat to their ability to have their own biological children in the future may justify the unknowns that are involved with preserving their eggs in this manner. These unknowns include whether their eggs will survive the vitrification process and whether egg vitrification will ultimately prove to be as safe as conventional in vitro fertilization and embryo cryopreservation. The answers to these questions may not be answered until the patient’s eggs are warmed, fertilized and implanted, which may be years later.

We recognize that a much broader spectrum of the population will look upon oocyte vitrification as a way for women to preserve their fertility. Single women, such as you, who have not yet met their life partner, may be particularly interested in this option. In addition, it may also become an option for women in their 30’s who wish or need to delay their childbearing.

Many questions remain unanswered. Will eggs from women in their 30’s do as well as eggs from proven egg donors in their 20’s? Logic suggests older eggs will not do as well, but will the differences be significant? How many eggs would a woman need to preserve in order to have a reasonable chance for one or two children in the future? How many IVF cycles will that take? Is it safe to rely on these preserved eggs? Would having preserved eggs change a woman’s approach to reproductive planning in her life?

These are not trivial issues. They are important, life-changing concerns. For these reasons, we are not encouraging single women to prematurely jump on the egg vitrification bandwagon. Stay tuned. This area is changing rapidly.

Dr. Carolyn Givens

In late October of this year, our first patient who underwent embryo transfer with embryos created from vitrified and warmed donor oocytes has successfully delivered. The baby was born at term and appears to be perfectly healthy.

Three other pregnancies are ongoing and are expected to deliver in 2008. We congratulate our new parents and the parents-to-be who have participated in this ground breaking program.

PFC has ended enrollment of patients into this program, but expects to continue research efforts with respect to oocyte vitrification.

While it has been possible to preserve sperm for many years (the famed Dutch microscopist Anton von Leeuwenhoek allegedly cooled and then recovered sperm using snow and ice in the 17th century), reliable methods for oocyte preservation have been elusive.

We previously discussed some of the problems with oocyte freezing (see Fertility Flash, January 2005, Volume 3, Issue 1), and now report our success in overcoming some of the obstacles.

Traditionally, preservation of sperm and embryos has been achieved with the use of a technique called slow freezing. This process incubates the sperm or embryos in low concentrations of cryoprotectants (antifreeze) to draw water out of the cells. After this incubation, they are cooled very slowly to sub zero temperatures. Typically this slow freezing technology just works for cells that exist individually (such as sperm), or together in small numbers (embryos), as the water must be extracted from every cell. Tissues, which are made up of many hundreds of thousands of cells, cannot be dehydrated successfully and therefore cannot be frozen intact. Cells in the tissue can burst when the water remaining in the cells expands as it turns to ice. For example, it is not possible to freeze a whole ovary, but some success has been achieved with ovaries that were cut into tiny pieces.

Frustrated by the lack of progress with slow freezing, scientists have more recently moved towards a technology called vitrification for oocyte preservation. Vitrification, which was described in detail in September’s Fertility Flash (Volume 5, Issue 8 ) works by using higher concentrations of cryoprotectants and much faster cooling rates. Cells are typically cooled in tiny straws (see article heading). This process allows us to achieve cooling rates of several thousand degrees per minute.

When vitrification straws and cryoprotectants were first approved by the FDA for human embryos, PFC began the process of adapting the technology to oocytes. Our embryologists attended training courses and became proficient with the technology by practicing on mouse and hamster oocytes and embryos. Even though we have been handling oocytes and embryos for many years, this technology provided many new challenges, mainly due to the tiny size of the straws and the speed at which the cells had to be cooled. Once we became proficient with the procedure, we began to freeze high quality oocytes from donors who had proven fertility. In this way, we knew that if the procedure did not work, it would be the vitrification technology and not the oocytes that were to blame. In addition, we satisfied ourselves that the technology was safe by looking at the exhaustive work by Dr. Gary Smith at the University of Michigan, which showed that vitrified/warmed oocytes were both physically and genetically normal and that the resulting pregnancies and babies were healthy.

We recruited five oocyte donors and vitrified all of their oocytes immediately after their oocyte retrieval procedures. We then offered the oocytes to individuals who were on our waiting list to accept donated embryos. Typically, these individuals were unable to get pregnant with their own oocytes or financially unable to proceed to an egg donor cycle. The availability of the vitrified oocytes was a great alternative to accepting donated embryos as it allowed couples to choose their own sperm source. Furthermore, the immediate availability of vitrified oocytes was an attractive alternative to what may be a very long wait for donated embryos.

Pacific Fertility Center had immediate success with the first recipient. We had vitrified 16 oocytes from the first donor, and for the first recipient we warmed only 7 of these. Four hours later we injected a single sperm into each of the 6 oocytes that appeared alive and healthy (1 oocyte had not come through the process successfully). The next morning, 3 of the oocytes fertilized normally. After 2 more days, we had 3 nice embryos for transfer. The positive pregnancy test 11 days later, and a singleton pregnancy confirmed by ultrasound at 7 weeks were great rewards for our efforts and thrilling news for the recipient. Our second recipient used a different donor and although her pregnancy started out well, she miscarried in the first trimester. Our disappointment over this loss was compounded when we discovered the oocytes from 2 of the donors did not survive well when warmed. In these particular donors, we recovered high numbers of oocytes (each had close to 40) and for unknown reasons their oocytes were overly sensitive to vitrification. The next three donor cycles proceeded well and resulted in pregnancies. These 3 pregnancies are all ongoing at the time of writing. We will update readers with their outcomes at a future date.

Although we were warming relatively small numbers of oocytes (typically 6 or 7), we began to have more embryos than could be safely transferred to recipients. Our first pregnancy had been achieved after transferring 3 embryos. It is more typical, however, to transfer only 1 or 2 embryos when donor oocytes are used. Even when using only 2 embryos, multiple pregnancy rates were unacceptably high. Understandably, few patients are willing to risk a decreased chance of conceiving by transferring only a single embryo. In order to avoid high multiple pregnancy rates in a typical IVF cycle, embryos are usually cultured for 5 days to determine which embryos in a cohort have the best chance of establishing a pregnancy. However, if a patient has only a few embryos, the benefits of extended culture are less, and the transfer is typically done after only 3 days growth. With our recipients of the vitrified oocytes, we began by doing 3-day transfers. Once high success rates were evident, we elected to implement day-5 transfers, in an effort to decrease high order multiples. The last 2 pregnancies both resulted from day-5 transfers of 2 embryos each, and they are both twin gestations.

In summary, we have had 7 out of 10 embryos implant after transfer (excluding the 2 failed donors with the high oocyte numbers). This implantation rate (70%) is comparable to the implantation rates that our patients have when using fresh embryos from donor oocytes.

We are moving forward cautiously with our oocyte vitrification program and hope to use the remaining oocytes soon. While these results are encouraging and have brought great joy to a small number of our patients, there are more issues to resolve before we declare complete success. The 70% success rate was obtained with the use of the highest quality oocytes from young donors who were known to be fertile and healthy. We have already seen that some oocytes are less tolerant of the procedure, as evidenced by the results from the 2 donors with high oocyte numbers. We also fully anticipate that the results for older women using their own oocytes will be worse, as they are for these same patients using a fresh IVF cycle. In fact, at this time, we do not have any idea if the oocytes from women in their 30’s will be able to tolerate vitrification.

Going forward, we will offer oocyte vitrification unconditionally to women with cancer who are likely to be left sterile by their treatment. For these women, and for others who elect to vitrify oocytes for social reasons, we will exercise great caution in our estimates of future pregnancy potential with the warmed oocytes. Until we have more data with oocytes from a variety of women, we will have no way of telling if there is any hope from anything other than donor oocytes. That data will accumulate more slowly because women who elect to preserve oocytes are not likely to be using them for some time. For now, until there is more data, we continue to believe that embryo freezing has the greatest potential for those wishing to preserve future fertility. However, for those who are single and in their late 30’s, we will be reluctant to recommend oocyte vitrification as a reliable fertility preservation method. Hopefully, they will find Mr. Right before we have objective data.

Joe Conaghan, PhD, HCLD

Introduction

Sara (a hypothetical patient) found a breast lump. 36 years of age, she was a single active professional, otherwise healthy, careful about her diet, and carefully evaluating her options after a diagnosis of breast cancer. Along with the discussion on surgery, chemotherapy, and radiation therapy came the question “Were you planning to have children?”

A diagnosis of cancer presents many decisions that must be made quickly. Confirming the diagnosis and planning therapy will be the primary concerns, but the implications of therapy on long-term quality of life must be assessed. One of the primary issues facing women with a diagnosis of cancer is future fertility.

Candidates

Cancer treatment can interfere with future fertility. Toxicity varies by treatment. Cyclophosphamide, an alkylating agent used in many chemotherapy regimens, is highly toxic to sperm and eggs; methotrexate and 5-flouro-uracil (5FU) are not. Medications used for longer time intervals create a higher risk of fertility problems than shorter time intervals; effects on women in older age groups are more severe than younger. Radiation therapy, in high doses, can have effects on eggs and sperm. Surgery and anesthesia are not known to have direct effects.

It is difficult to give specific fertility risks for chemotherapeutic regimens, since studies are not yet definitive. Among the more toxic treatments are stem cell transplantation for leukemia in which total body irradiation and cyclophosphamide are used, beam radiation to a field that includes the ovaries, and extended chemotherapy of up to 6 cycles using cyclophosphamide in combination with other agents. After conventional chemotherapy for breast cancer for women under 40, the chance of infertility is roughly 50%, in older women the risk is over 80%.

Treatment options

What are the options for fertility in patients diagnosed with cancer? The best choices are available to those that have not yet initiated treatment and involve cryopreservation. During treatment, the risk of problems rises, and after treatment, there may not be adequate recovery of fertility to achieve pregnancy.

Cryopreservation allows cells to be stored with great stability for long periods of time. The record time from sperm cryopreservation to pregnancy is 28 years; there probably is no real limit to the time that cells can be stored. To store cells requires technology that reduces the formation of ice crystals, which disrupt cells, and prevents the rapid rise in salt concentration that occurs as water freezes. Cryopreservatives and management of temperature changes (slow freeze or vitrification) are used to reduce the risk of these problems.

Male

The option for fertility preservation in men is straightforward, cryopreservation of sperm. Sperm is obtained by masturbation and frozen in multiple vials in liquid nitrogen. 2-3 sperm samples can be obtained per week, with 2-4 vials stored per ejaculate; two weeks worth of donations could yield 8-24 vials of sperm. Costs vary widely, but would range from $1500-$3000 for processing and 3 years of storage.

Testicular sperm extraction is an option for individuals with azoospermia. Testicular tissue cryopreservation remains a theory that has not yet produced a human pregnancy. It has been proposed as an option for preservation of fertility in children, but has yet to be proven in clinical practice.

Female

Women have the option of cryopreservation of oocytes or embryos. For women without a partner, oocyte cryopreservation holds promise as a means to preserve fertility potential without committing to a specific sperm source or partner. For women with a partner or sperm donor, embryo cryopreservation is a proven technology.

To create cryopreserved oocytes, Follicle Stimulating Hormone (FSH) is administered over a ten day time period to stimulate ovarian follicles. The oocytes are retrieved under sedation with a needle guided by ultrasound and then stored in liquid nitrogen.

Newer techniques of oocyte vitrification secure good pregnancy rates for those with good oocyte quality. Traditional oocyte cryopreservation is performed using a slow freeze technique, but more rapid vitrification procedures optimize results. The trick with cryopreservation is to lower the temperature while avoiding ice crystals that disrupt cell membranes and proteins. Vitrification, an ultrarapid freezing process utilizing a minimal fluid volume, reduces the risk of these problems and optimizes cell quality.

For those women with a partner, or that are willing to commit to a specific sperm donor, embryo cryopreservation is an excellent option. After stimulation and retrieval, oocytes are inseminated and cultured in an incubator for 1-5 days, followed by cryopreservation. The embryos can be thawed and transferred at a later date, after clearance from the oncologist. Embryo cryopreservation is the best established of the fertility preservation techniques, with years of experience in its applications. Good pregnancy rates can be anticipated.

Ovarian tissue cryopreservation, the cryopreservation of whole pieces of the ovary, as opposed to cells, remains experimental. Complex tissues are more difficult to cryopreserve than cells, though rare success has been reported.

Cancer recurrence

Is there risk to the use of fertility drugs in patients with cancer? It does not appear in studies to date that breast or ovarian cancer risk is affected by use of fertility drugs. Studies indicating an increased risk are balanced by other studies indicating a reduction in risk. Studies to date have been limited, and treatment decisions still must be individualized.

Does pregnancy increase the risk of cancer recurrence? In theory, certain types of cancer could be aggravated by the hormones of pregnancy, but studies have not confirmed an overall risk. Certain types of cancer are less common in women that have delivered a pregnancy. Treatment decisions must be individualized, as future studies gather more information.

Pregnancy

Certain cancer treatments create organ toxicity that must be evaluated in considering patients for pregnancy. Heart output is limited in patients that have received doxorubicin. Uterine irradiation is associated with miscarriage and pre-term labor.

Children

Children born after fertility preservation procedures do not carry any increased risk for birth defects. There are hereditary syndromes that can be associated with cancer that could be transmitted to children, but there does not appear to be any other increased risk for cancer or genetic disease in children of cancer survivors.

Patients contemplating conception must consider life span expectations as part of their decision on whether to conceive. Such considerations are not, however, a reason to withhold treatment, and are ultimately the individual and family should decide.

Philip E. Chenette, MD

Resources:

www.fertilehope.org Fertile Hope

www.livestrong.org Lance Armstrong Foundation

www.cryobank.com California Cryobank

www.PacificFertilityCenter.com Pacific Fertility Center

Many patients receiving medical care for infertility will use cryopreserved (frozen) sperm, oocytes and/or embryos at some time during their treatment. Here in the PFC laboratory, we routinely cryopreserve sperm and embryos. We also receive specimens from sperm banks nearly every day. All of these specimens are stored on-site in our secure tanks with continuous monitoring. All specimens are stored in liquid nitrogen at -196ºC. Movement in or out of the tanks only happens when specimens are transferred post freezing or retrieved for thawing or shipping. We store sperm and embryos for our patients for an annual fee as long as we are able to maintain yearly contact with them and the annual storage agreement is renewed.

The shipping of tissues that are frozen and stored at such a low temperature is not easily accomplished. The liquid nitrogen in which they are stored is not toxic in any way, but it is extremely dangerous and can cause serious injury and even death if not handled properly.

In attempting to transport tissues that are normally stored in liquid nitrogen, we have to use a device that will keep the tissues in their same deep frozen state. This is accomplished using a “Dewar” which resembles a large thermos. A Dewar is a vacuum insulated container, mostly filled with an absorbent lining that soaks up liquid nitrogen. The Dewar is “charged” prior to use by filling it with liquid nitrogen over successive days until it will absorb no more. Once saturated, the excess liquid is poured off and the Dewar is then ready for use. Specimens are loaded into the hollow core and they are maintained in their frozen state by the cold nitrogen vapor evaporating from the surrounding absorbent layer. The Dewar holds an appropriate temperature for as long as nitrogen remains inside. Loss of nitrogen by evaporation happens continuously. Typically a fully charged Dewar maintains temperature for between 7 and 30 days depending on its size, how often it is opened and how well it was charged before use. With any Dewar however, loss of refrigeration occurs after a certain period of time, unless more nitrogen is added. In addition, dropping the Dewar or otherwise damaging it in any way can crack the container and this will result in instant failure of the vacuum seal with subsequent loss of nitrogen and thawing of the contents.

When we receive a shipment of sperm from a bank, there is always a risk that the Dewar was damaged or that there was a shipping delay that was longer than the life of the liquid nitrogen in the tank. If the specimens have thawed, typically the sperm bank will replace them at no cost. However, their liability is limited to replacing the sperm, and if you just lost the last 3 vials of your favorite donor, you’ll have to choose a new donor.

Shipping of embryos is a much more risky proposition. Embryos can’t be replaced in the same way that a sperm sample can be replaced, if they can be replaced at all. The major shipping companies such as FEDEX, UPS and DHL will not knowingly accept embryos for transport and therefore would not have any liability for loss. At PFC we discourage shipment of embryos due to the risks involved. We will not ship embryos from our laboratory on your behalf, however you can come and collect your embryos in person and ship them yourself. We will ask you to sign papers releasing us of any liability once the embryos leave our office. We cannot accept any liability for embryos that are being shipped in from elsewhere; it is a practice that we discourage.

If you absolutely must ship embryos, we suggest that you contact a company that has the expertise and the experience to make this type of shipment as safe as possible. Locally, we recommend “Swift Stork Courier” (www.swiftstork.com) who will arrange collection and delivery of the embryos and ensure appropriate and safe handling during transport. For long distance shipments, we put patients in contact with “Kynisi Courier Systems” (email: kosta@kynisi.com), a company based in the UK that specializes in shipping embryos. If you want to send your embryos from

San Francisco to Detroit, or Dublin or Dubai, Kynisi is the only company we know that can get embryos on airplanes without being x-rayed in security. They also get advance clearance to make sure that embryos don’t get delayed in customs as they cross international borders. Kynisi can also arrange for an embryologist to travel with your embryos, and they can organize for the embryos to travel in the passenger cabin of the aircraft, as opposed to being thrown in the luggage compartment with the other cargo. This is important, as a Dewar left lying on its side will lose nitrogen more rapidly than when upright. Kynisi’s services aren’t inexpensive, but considering that the embryos are priceless, there really isn’t a good alternative.

For those patients considering moving their frozen tissues to a facility that offers long-term storage at reasonable costs, we recommend “ReproTech” (www.reprot.com) in Reno, NV. ReproTech is experienced and knowledgeable, and gives great customer service. They too can arrange safe movement of your tissue from us to them, and back again with minimal inconvenience. They often take the extra precaution with embryos by splitting them into 2 groups that are then shipped separately. ReproTech shares the PFC philosophy of thinking of embryos as irreplaceable, and they take every known precaution to ensure a safe and efficient shipment. However, despite the good work of ReproTech, Kynisis and others, I recommend that you do not ship your embryos. The risks are too great.

Joe Conaghan, PhD

The American Society for Reproductive Medicine’s (ASRM) annual meeting was held in New Orleans. It is the largest meeting for reproductive medicine specialists and scientists in the world. From our practice, Dr.s Givens, Schriock and Conaghan attended, as well as embryologists Jean Popwell, PhD and Jennifer Andres. Also, PFC nurse Allison Chamberlaine and PFC’s Marriage and Family Therapist Peggy Orlin attended. In addition, the genetics counselor with whom we work closely, Lauri Black from California Pacific Medical Center, was an attendee and active participant.

PFC’s embryologists attending ASRM’s research poster session Jean Popwell, PhD (left) and Jennifer Andres (right).

Single-Embryo Transfer: Minimizing Risks & Maximizing Outcomes
Dr. Givens attended a post-graduate course entitled “Moving Toward Single-Embryo Transfer: Minimizing Risks and Maximizing Outcomes.” This two-day course dealt with a pressing issue in assisted reproduction: the high incidence of multiple gestations. With the ever-increasing success of in vitro fertilization and the significant improvement in embryo implantation rates, the incidence of twin and higher-order pregnancies has risen dramatically in this country. Many countries now regulate the maximum number of embryos that can be transferred into the uterus at one time. The course topics included a summary of optimal medication protocols, several lectures on pre-cycle evaluation and testing and embryo transfer techniques.

Oocyte Freezing, PGS & Blastocyst Embryo Transfers
On the laboratory side, there were several talks on evaluation of eggs and embryo selection techniques, embryo freezing technology, including a debate about the usefulness of pre-implantation genetic screening (chromosome analysis of embryos) embryo selection. The combination was a fascinating mixture of new ideas, refinements in current technology, as well as a welcome opportunity to network and discuss with others the latest developments in reproductive science. Topping the list of presentations in New Orleans were those concerning the continuing refinements in oocyte freezing technologies, the more selective use of preimplantation genetic testing and the ongoing scrutiny of blastocyst stage embryo transfers.

Slow-freeze vs. Vitrification
The traditional slow-freeze technology used so successfully with embryos for many years, has essentially stalled with oocyte freezing. It appears the slow-freeze technology has finally met its successor: a process called vitrification. Slow freezing has had very limited success with oocytes due to their large size, high water content and their extreme sensitivity to cryoprotective chemicals and to changes in temperature and pH.

Vitrification, a technology that cools cells so rapidly that ice does not form, has been such a success for oocyte freezing that many labs are now abandoning slow freezing altogether. Here at PFC, we have been developing protocols for oocyte vitrification throughout 2006 and are actively working on blastocyst vitrification. It was reaffirming to see that this technology has gained wide acceptance, and is showing excellent results.

Preimplantation Genetic Screening (PGS)
While vitrification is on the rise, it was interesting to see that another technology, Preimplantation Genetic Screening (PGS), was lacking in new improvements or viable alternatives. Embryos have been screened for extra or missing chromosomes for over 15 years now, but the technology has not advanced significantly over that time. It is still possible to count only 12 chromosomes in an embryo. Although the error rate per chromosome is very low, the accumulated error rate becomes significant as we count more chromosomes. PGS was “under the microscope” in several presentations in New Orleans and it appears PFC’s limited use of genetic screening is well justified. Specifically, PGS does not improve embryo selection and pregnancy rates in younger patients. Its use is limited in older patients because there are often too few embryos available to justify testing. The patients who benefit most from PGS are the younger patients who experience recurrent miscarriages. However, unless there is evidence that previous pregnancies were genetically abnormal, PGS may provide limited benefit to this group.

Blastocyst stage embryo transfers
While younger patients (those under 35) don’t benefit from PGS, they are the patient population most likely to benefit from blastocyst transfers. Culturing embryos for 5 days to the blastocyst stage, instead of the more traditional day 3 embryo transfer, is one of the main ways in which the laboratory staff can help in selecting the “best” embryo for single embryo transfer (SET) patients. Blastocyst culture techniques are well refined now and support the commitment within the community to transfer fewer embryos at one time. Furthermore, the promise of vitrification can reassure patients that their remaining embryos can be stored indefinitely when preserved at the blastocyst stage. Several presentations showed that blastocysts which were vitrified early, before their cavity (or cyst) had expanded too much, benefited most from the technology. In more advanced blastocysts, artificial reduction of the cavity gave superior results. It may not be long before vitrification is the procedure of choice for preserving all blastocysts.

2006 ASRM guidelines for numbers of embryos to transfer
The new 2006 ASRM guidelines for numbers of embryos to transfer were presented. See Tables 1 and 2 below.

The topic of whether or not federal or state legislation should regulate the maximum number of embryos to transfer was also discussed. Many people in the general public support such legislation but those of us in the field (and most patients) are opposed to the government regulating medical practice and arbitrarily setting limits on embryo transfer. In order to forestall such legislation, it is obvious that we must decrease the number of twin gestations (the number of triplet and higher-order gestations has already dramatically decreased in the last 5-7 years). At Pacific Fertility Center we have instituted a new emphasis on single embryo transfers and expect to significantly reduce the risk of multiples and achieve our goal of “optimal” pregnancy outcomes. (See From Us to You in this issue for a discussion of our 2006 statistics and please see Conception Health in this issue for a discussion of why it is important to try to achieve single baby conceptions.

– Carolyn Givens, MD and Joe Conaghan, PhD

For those of us with an interest in human reproduction, scarcely a day goes by without us hearing or seeing something related to oocyte freezing. The topic has generated a lot of hype and it is difficult to avoid the frequent magazine and newspaper articles, advertisements and TV features that generate excitement on the subject.

We have already discussed oocyte freezing in a previous newsletter article (Keeping Egg Freezing in Perspective; January 2005) and readers unfamiliar with the technology are encouraged to visit our website where they can read this in the newsletter archive. Having already discussed the methods for freezing, and their merits, we now address the achievements of oocyte cryopreservation on this, the 20-year anniversary of the first success.

There are two technologies used in oocyte freezing, and the primary aim of each is avoiding ice formation within the cell. The first is the slow freeze method (used so successfully with embryos) that dehydrates and cools the cells gradually, over three hours. The second is an ultra-rapid procedure that is performed so quickly that the cell contents turn to a glass-like substance. This latter method is called vitrification and it is gaining in popularity for oocyte and embryo freezing. And since no ice forms, the cells are technically not frozen, but “vitrified.”

In reviewing the scientific literature since the first success in 1986, the importance of oocyte freezing is apparent by the sheer volume of publications on the subject. For the purpose of this article, the many papers that report on the technique only have been excluded, and here we will only report on the pregnancy outcome data. However, even this is difficult since some patients may have become pregnant from the first few thawed oocytes, leaving us with no data on the many oocytes still frozen on their behalf. Also, even though there are reports that detail only one or two pregnancies, there are probably many other isolated successes around the world that have gone unreported in the scientific literature.

Most of the pregnancy outcome data has been pulled together in a single review paper by Dr. K. Oktay and colleagues at Weill Medical College in New York (Fertility & Sterility, 2006, Vol 86 (1), pages 70-80). The 47 papers reporting outcome data for slow freezing were analyzed and from these, only 26 provided sound usable data. The others were excluded either because sub-optimal procedures were used, the pregnancies had not yet delivered or the authors could not be reached to clarify the data. The 26 useful papers collectively documented the freezing of 4,564 oocytes from which 4,000 had been thawed in 397 patient cycles. Out of 95 pregnancies, 76 resulted in live births, and since some of these were multiple pregnancies, the total number of children born was 97. If we add in the excluded data, the number of pregnancies becomes 170, resulting in 106 live births and 11 ongoing pregnancies. Because of ambiguities in the excluded data, a final number of children is not stated. However, the data suggest that the number of children that are alive today as a result of 20 years of slow freezing of oocytes is approximately 200. Taking all the data into account, the clinical pregnancy rate per thawed oocyte was a mere 2.3%. The live birth rate in the 26 usable papers was 1.9% per oocyte thawed.

Unfortunately it is not possible to give rates per oocyte frozen since some papers are not complete, but more importantly because many oocytes are still in the freezer.

Vitrification, which is a technology that came late to oocyte preservation, is quickly gaining ground on the slow freezing method. By June of 2005 there were only 10 reported births following oocyte vitrification, but a year later the numbers reported by Oktay are 61 pregnancies from which 42 have delivered live infants and 7 are ongoing. With limited data, vitrification appears to be a more highly efficient preservation method than slow freezing. The latest numbers, based on admittedly limited data, shows that >90% of oocytes survive and about 90% of these fertilize. Overall, 50% of vitrified oocytes make blastocysts in culture which is as efficient as fresh oocytes. These numbers are reported by Masa Kuwayama at the Kato Ladies Clinic in Tokyo. Also, from 29 embryo transfers, 12 pregnancies have yielded 7 live infants with 3 not yet delivered at publication time (Kuwayama et al., 2005, Reprod Biomed Online, Vol 11 (3) pages 300-308). We can compare this data to the latest results with slow freezing where the experience of 20 years has been incorporated. Using sodium-depleted medium, in which oocytes are slow cooled and frozen, 59% of oocytes survived and 68% of these fertilized. Nine pregnancies were established in 28 thaw cycles from which 5 delivered and 1 was ongoing (Boldt et al., 2006, Reprod Biomed Online, Vol 13 (1) pages 96-100). For those women who want to rely on oocyte cryopreservation to postpone motherhood, these data should be sobering. While we don’t expect the technology to ever be 100% successful, it currently offers no guarantees.

Expecting too much from today’s procedures could leave many women very disappointed. Further, many of the pregnancies reported in these studies were achieved by preserving the oocytes from young women. Since oocyte quality declines as a woman ages, the success rates for older women are likely to be less than reported here. Women considering oocyte preservation will need careful counseling and a good understanding of the success rates before putting their eggs in this basket.

– Joe Conaghan, PhD

PFC continues to be at the forefront of pioneering research in assisted reproductive technology and was the recipient of the 2005 California Pacific Medical Center (CPMC) Foundation Wishes for Wellness Grant. Through this grant, PFC will embark on a research project assessing the efficacy of a new IVF egg freezing method, vitrification.

The CPMC Foundation selects outstanding CPMC physicians in the fields of obstetrics and gynecology and pediatrics to be honored at their event Wishes for Wellness. PFC’s Eldon Schriock, MD and Carl Herbert, MD were among those selected in 2005. These honored physicians have the privilege of identifying needs and/or directing purchases and programs which will be funded by the Wishes for Wellness Grants.

Egg freezing has been successful in creating a handful of pregnancies, but the process is still very inefficient. Many eggs do not survive the freezing process. While the technology for freezing sperm and embryos has been used for decades and is very successful, the technology for egg freezing is still emerging.

The key to successful egg freezing is determining a technique that will not damage the fragile chromosomes of the egg. The eggs in the ovaries are held in “suspended animation”, until they are stimulated to grow and ovulate. During this state, the chromosomes of the egg are vulnerable to damage, including damage from the exertion of the freezing and thawing process. Past freeze/thaw techniques have been very inefficient because of the chromosomal damage incurred. The vitrification freezing technique seems to be a gentler technique, and therefore leads to less chromosomal damage. This then improves efficiencies in the thawing, fertilization and embryo development steps; and ultimately better pregnancy rates.

Our study is designed to study whether vitrification can improve the efficacy of freezing eggs. The study is designed is such a way that results should be obtained in a timely manner. Egg donors who have had previous IVF cycles resulting in pregnancy will be recruited to have eggs frozen. The results of fertilization, embryo development, implantation and pregnancy rates using the embryos resulting from egg vitrification will be compared to the pregnancy rates obtained in previous cycles using embryos obtained from fertilized fresh eggs.

PFC is excited and honored to be involved in this research. The potential benefits of egg freezing are substantial and our research team looks forward to sharing results with you, as soon as they are available.

– Eldon Schriock, MD

Is the Future of Egg Freezing Here?
On the surface, it sounds remarkable that one can now shop for frozen oocytes through a start-up company called Cryo Eggs International, which offers single frozen oocytes for sale for $2,500 apiece via mail order. Based in Arizona, the company offers no guarantees whatsoever. The company claims that couples can save money and reduce their risk by choosing individual frozen eggs over the more involved and expensive process of working with a donor to produce fresh oocytes for fertilization.

Oocyte cryopreservation technologies have been evolving since 1986, and there is little doubt that there is a strong future in egg freezing. Young women are expected to have the choice of “banking” a cache of their genetic material for later use. Infertile women may indeed turn to a frozen egg bank, much the way that frozen sperm is marketed, to choose available eggs.

But is that time now?
A handful of infertility clinics are offering female patients a chance to undergo an IVF cycle and freeze their eggs for future use. Eventually PFC expects to offer this. Yet the majority of these clinics insist on prominently displaying the disclaimer that egg freezing technologies are still evolving and are highly “experimental”.

Indeed, as of early 2005, less than 1% of eggs that had been frozen and thawed had resulted in live born infants. (Keeping Egg Freezing in Perspective). Most certainly, egg-freezing technologies advancing cryopreservation of oocytes are evolving rapidly. (A Few Good Eggs). Yet the research community is still weighing the advances of different freezing mediums and methodologies, such as rapid vs slow freezing and thawing.

Responsible researchers/authors publishing their work in the global body of scientific literature are calling for several more years of studies with larger numbers of participants. Most of the current research is based on very small groups.

Cryo Eggs International attributes its success to the advances of Dr. Jeff Boldt, an associate professor of medical and molecular genetics and scientific director of Assisted Fertility Services at the Community Health Network, Indianapolis. He is also reportedly the scientific director at Cryo Eggs International. Yet Boldt’s primary published work in a scientific journal reported the results of a study that only involved 11 women. He is quoted in the media as having a larger number (33) of cycles from which results were comparable to standard IVF procedures, yet this study has not yet been published in a peer-reviewed science journal.

Can one tell if an egg is good or bad upon thawing?
Unlike sperm, of which mainly healthy ones are frozen, there is no sure way to determine quality control of a donor’s eggs short of conducting a DNA analysis of the resulting embryo. In this regard, Cryo’s customers are essentially asked to purchase single oocytes not knowing if they are viable.

Associated Costs with Frozen Eggs
After oocytes have been frozen they may have a thicker outer wall, otherwise known as the zona pellucida. This generally requires the embryologist to apply additional costly methodologies such as assisted hatching and ICSI.

Healthy Quarantine
The six months of freezing that is required before the frozen eggs can be released is no different than the six month testing requirement that a typical donor must go through to test for infectious diseases. In this regard, it is misleading for the Cryo Eggs International web site to claim that this process is any safer than conventional donor cycles.

Successful Approach
A donor cycle at Pacific Fertility Center has yielded a consistent 65% or greater success rate for many years. A key point here is this record has improved incrementally over the years after decades of experience and applying evolving technologies.

It is every physician’s wish for his/her infertile clients’ to have inexpensive choices to tackle their life dream of conceiving. It is also important for people to be as well informed as possible so their money may be spent for the most cost-effective and successful method for their particular situation.

– Carolyn Givens, MD