A human embryo

In 2007, PFC took the bold step of changing the way we freeze embryos. Traditionally, embryos are frozen using a “slow-freeze” protocol where they are exposed to weak concentrations of cryoprotectants before being cooled slowly (-0.3 °C/min) for 2-3 hours. This system has worked well over the years, but recent advances in an ultra-rapid freezing technology showed great promise. PFC began looking at a technology called vitrification in 2006. After seeing wonderful results from in-house trials we were able to phase vitrification into our practice in March of 2007. By June, we had stopped slowfreezing completely. In late 2008, after our 200th thawing cycle with vitrified embryos, we examined the data.

From our first 2 years of thaws, we recovered 94% (423/448) of embryos vitrified, and 94% (397/423) of these were alive when the thawing process was completed. The total number viable was 88% (397/44 8). These numbers compare well to those reported in the scientific literature, but we continue to improve the process and strive for even better results. Vitrification uses tiny straws called “cryotips” to house the embryos during the process, and uses higher concentrations of cryoprotectants than slowfreezing. These details make the procedure technically challenging, which may sometimes result in the loss of an embryo. The tiny straws can crack or break due to the extreme physical force that they endure during freezing and thawing. If this happens, the embryo in the straw cannot be recovered. This lack of recovery or survival is a complication of any freezing procedure. We continue to go to great lengths to minimize these losses, some of which are unavoidable.

Frozen embryos are stored in liquid nitrogen

We have completed 202 thawing cycles to date (A thawing cycle refers to a treatment cycle wherein a patient returns to use vitrified embryos and we thaw and transfer 1 or more to her uterus at the same time). Ninety-seven of these 202 cycles (48%) resulted in an established clinical pregnancy. The average number of embryos transferred per cycle was 1.9 and the implantation rate (embryos implanting out of embryos transferred) was 31%.

The vitrification procedure and materials continue to evolve. Irvine Scientific, the company that manufactures the cryotips, continues to improve their product. They are working extremely hard to eliminate defects that may lead to straw failure during cooling and thawing. At the same time, PFC continues to evaluate new ways to improve embryo survival and implantation rates. This year, we are investigating a process which artificially collapses blastocysts prior to vitrification. We will also be investigating the use of assisted hatching with thawed embryos. Be sure to watch these pages for exciting updates in the months to come.

This past summer, Dr. Herbert and I had the opportunity to travel to Barcelona, Spain for the annual meeting of the European Society for Human Reproduction and Embryology (ESHRE). Though largely attended by Europeans, this scientific meeting draws physicians, embryologists and scientists from around the world to discuss their research, attend courses and lectures, and discuss the latest topics in our field.

Here are some of what I consider the highlights of the meeting:

Outcome of 1267 Children after Frozen Embryo Transfer – Study from Denmark

Control group: Fresh IVF pregnancies

Only 14% were twins

They compared 957 frozen embryo singletons with about 10,000 fresh IVF singletons

No increase in neurological problems or malignant diseases on FET babies.

No differences were seen when IVF or ICSI-derived frozen embryos were compared.

Results similar to prior Swedish study showing better outcomes for FET babies.

Why a better outcome? The authors postulated that patients conceiving with FET were more likely to be good prognosis patients.

Three years of clinical application in human oocyte vitrification (freezing): high survival rate and healthy deliveries (from Rome)

3138 unfertilized eggs were frozen between 10/04 – 10/07.

They reported on 295 cycles with planned embryo transfer – all patients were less than 40 years old. The patients underwent programmed endometrial preparation using a GnRH agonist (like Lupron) and oral estrogen and vaginal progesterone.

770 unfertilized eggs were thawed, 98.9% survived the thaw. The eggs were injected with sperm 2 hours after thawing and the embryos were transferred on Day 3.

Results: Avg. # embryos transferred = 2.3

Clinical pregnancy rate = 27.8%

Implantation rate = 13% per embryo, 11.3% per thawed egg. That is, about 11% of the eggs thawed resulted in a viable gestation.

58 deliveries of 63 babies, mean birth weight = 2930 grams

They experienced no congenital malformations at birth.

Then, the most controversial paper presented by Dr. Norbert Gleicher, an RE from New York.

The title: “In contrast to prevalent opinion, twin pregnancies after fertility treatments are medically, ethically and economically desirable outcomes.”

His arguments to support this opinion:

Most couples want to have more than one child. Therefore, they will need to undergo two pregnancies of two separate singletons vs. one pregnancy of twins to have two children. He argued that twins born after ART have much better pregnancy outcomes (by 30-50%) than spontaneously-conceived twins. He also argued that the accumulated costs and risks to mother and babies are higher with two singleton than one twin pregnancy.

Despite these intriguing arguments, this paper was hotly debated and essentially disavowed by the European ART community. Europe has led the way in legislating for avoidance of twins. In fact, in Denmark, if a woman has twins after the transfer of more than one embryo using IVF, she incurs any neonatal costs out of pocket.

Corifollitropin: a modification of Follistim to make it a once-a-week injection.

As most people know, the medication we most commonly use for fertility treatment, Follistim, is pure human FSH, manufactured using recombinant DNA technology. The company that makes Follistim, Schering Plough, is working towards FDA approval of a modified version of Follistim, called Corifollitropin, that will make the drug very long-acting. It may be possible to only take one injection per week!

A symposium at ESHRE presented information from studies underway in Europe and USA. Corifollitropin is not in clinical use yet, even in Europe, but will be very soon.

For those of you interested in the details, Corifollitropin is the recombinant FSH molecule + 22 C-terminal peptides from beta-hCG, It does not bind to the LH receptor.

This modification lengthens the half-life of Follistim from 22-34 hours to 60-74 hrs for Corifollitropin. After injection peak levels are reached in 2 days then slowly levels decline. The recommended regimen will be one dose per week, starting at baseline, switch to daily recombinant FSH after that.

	Carolyn Givens, M.D. was the first in San Francisco to successfully initiate a pregnancy using intracytoplasmic sperm injection (ICSI). She currently co-directs the Bay Area Pre-Implantation Genetic Diagnosis Program (PGD) and is director of PFC’s PGD program.
	Carl Herbert, M.D. was instrumental in the development of one of the first assisted reproductive technology programs in the United States and has been performing IVF longer than any physician in the Bay Area.

Pacific Fertility Center launched it’s Educational Series on July 31 st with a presentation on the “Disclosure of Use of Sperm or Egg Donors.” The speaker was Dr. Bob Nachtigall, a local Reproductive Endocrinologist, who has done much research and published numerous papers on various fertility related issues. Dr. Nachtigall addressed the difficult decisions couples face, who attempt conception with donor sperm or donor eggs. These include when to abandon medical treatment using their own gametes, whether to conceive with donor gametes over other options such as adoption, and decisions related to the selection of a donor. Yet the final decision, whether to disclose to their children the circumstances of their conception, is one of the most challenging.

He and his team, conducted research which was based on interviews with 254 parents of children conceived with donor sperm or eggs, they found that 95% of study couples came to a united disclosure after discussions that reflected a wide range of contexts and influences that included: the sociopolitical environment of the community; the couples’ friendships and support network; counseling and professional opinion; religious and cultural background; extended and immediate family structure and relationships; the child’s appearance; and the couple’s individual personal and ethical beliefs. For those couples who decided to tell their young children about their use of a donor, no parent expressed regret or reported a negative outcome after having initiated disclosure.

Dr. Nachtigall will be returning to PFC, to present his findings from a research study he did on “Frozen Embryos.” The annual number of IVF procedures performed in the U.S. has increased from less than 2,500 in 1985 to over 120,000 today. Yet the rapid growth and availability of this advanced reproductive technology has had an unforeseen consequence – the accumulation of an estimated 500,000 frozen embryos that represent the unused “leftovers” of past IVF cycles.

His presentation will address the question of what to do with frozen embryos, which is complicated by the variety and disparity of their potential uses and fates: (1) they can be used by the couple in further attempts to conceive; (2) they can be “donated” to other infertile couples who wish to have a child; (3) they can be used in stem cell research; (4) they can be destroyed; (5) they can be stored indefinitely. Dr. Nachtigall and his team interviewed over 100 couples (many of whom were PFC patients) who had undergone IVF. The team found that ambivalence, uncertainty and most significantly, feelings of deep connection to a couple’s own embryos are several factors that cause difficulty in reaching a disposition decision.

The presentation on “Frozen Embryos” has not been scheduled at this time. However, please watch for dates and times in upcoming issues of Fertility Flash.

PFC Educational Series 2008

The PFC Educational Series are presentations held the last Thursday of each month from 4:00 till 5:30 p.m. in the PFC Education Center located at 55 Francisco Street, Suite 500. The presentations address various topics, which are open to PFC staff, as well as members of the medical community. The PFC physicians found offering programs of this nature would be an ideal way to increase knowledge regarding different topics. In addition, this is a great opportunity to “reach out” to other local physicians and their staff, by offering educational resources, that they otherwise may not have access. The presentations are offered at no charge and the topics will be published in the Fertility Flash, as well as on the website www.pacificfertilitycenter.com. If you are interested in attending this presentation, please contact our Development Department directly at 415-249-3656.

At Pacific Fertility Center we aim to help our patients build a healthy family. To build healthy families, maximum pregnancy rates are a goal, but maximum pregnancy rates must be balanced by consideration of risk, the chance of an adverse outcome. High pregnancy rates with minimal risk is PFC’s goal.

The risk of multiple pregnancy has increased as fertility therapy has improved. The wider use of gonadotropins in the 1990s to induce ovulation of multiple follicles, as well as the use of more effective laboratory and clinical IVF methods, resulted in production of more and healthier oocytes and more embryos, and increased the chances of multiple pregnancy. The very dramatic improvement in success rates over this time period resulted in many more children being delivered after fertility therapies, but also more twins, triplets, and higher order multiples.

Over the last twenty years, the incidence of multiple birth has increased nationally. According to the National Vital Statistics Report and the March of Dimes, the incidence of twins has increased by two-thirds, and the number of triplets and quadruplets has increased four-fold since 1980.

It is thought that about one-third of multiple pregnancies arise because women are waiting until later in life to conceive; age is a well-known risk factor for multiples. Another third arise from use of ovulation induction with gonadotropins (Pergonal, Follistim, Gonal-F, Repronex) alone. Less than one fifth of multiples are from assisted reproduction techniques (IVF and related procedures). Assisted reproduction in 2003 accounted for 18% of multiple pregnancies, 16% of twins and 44% of triplets ¹.

The risks to the children of multiple pregnancy are numerous. Low birth weight and very low birth weight are increased in children born as multiples. The chance of low birth weight (<2500g) is increased 8 times in twins. Cerebral palsy is increased 4 times, neonatal death risk by 7 times ^{2, 3}.

The risk to the mother from multiple pregnancy is also increased. Pre-eclampsia, high blood pressure, preterm labor, and premature rupture of membranes are all more common with multiple pregnancy ⁴ .

Multiple pregnancy is also expensive. It is estimated that twins alone cost the healthcare system some $600,000,000. There is clear evidence of an increase in parenting stress and divorce in families of multiples ^{5, 6} .

The need to assure our patients of the highest quality care requires that we bear this in mind – the healthiest pregnancy is a singleton pregnancy.

Pregnancy requires the cooperation of sperm and egg, accurate transcription of the early genetic code in the developing embryo, a fertile spot for attachment to the mother in the uterus, and a route for getting there. All other factors being equal, pregnancy rates almost double when two embryos are transferred instead of one, and increase again when a third and fourth embryo are added. The desire for high pregnancy rates has driven a desire for more embryos to be transferred ⁷ .

Improvements in insemination technique, embryo culture methods, and transfer efficiency have added substantially to pregnancy rates. Each embryo transferred today has a considerably higher chance of producing a pregnancy than an embryo transferred twenty years ago. Such improvements have enabled us to think about ways to reduce the risk of multiple pregnancy by transferring fewer embryos.

The development of blastocyst (day 5 embryo) culture techniques allows the selection of high quality embryos for transfer. The blastocyst stage requires advanced incubation techniques with low oxygen incubators and specialized culture media. A tight quality control system is also required. The blastocyst stage is a more advanced stage in which the genetic code of the embryo is fully activated and working. Only the healthiest of embryos can move to the more advanced stages, allowing selection of the best embryos for transfer.

In 2006 the ASRM published guidelines for number of embryos to transfer:

These guidelines encourage all of us to transfer ‘just enough’ embryos to achieve pregnancy.

Pacific Fertility Center has pioneered techniques of transferring fewer embryos. Last year, in 2007, our program of single embryo transfer in oocyte donation recipients produced a 66% pregnancy rate. The multiple pregnancy rate in this group was minimal. Utilizing a single embryo, two-thirds of patients were able to conceive a singleton pregnancy. This pregnancy rate was very similar to the overall pregnancy rates regardless of the number of embryos transferred.

Today half of our patients using oocyte donation elect to transfer a single embryo. Single embryo transfer is not always possible. Our criteria include age and embryo quality. A young woman (under age 35) with high quality blastocyst stage embryos and a healthy uterus can reliably transfer a single embryo and achieve high pregnancy rates. An older woman (over 40) may need to transfer 3 or more embryos to achieve a good pregnancy rate. Because of the higher number of embryos transferred, the risk of multiple pregnancy remains higher in these older age groups⁹ .

Pacific Fertility Center is very pleased to offer these techniques of single embryo transfer as some of the best and most advanced fertility treatment technology available. We are moving closer to our goal of growing families, one healthy baby at a time.   Philip Chenette, MD

1. Martin, Births: Final Data for 2003. National Vital Statistics Reports, volume 54, number 2, 2005
2. Scher, Ped Res, Vol. 52:671-81, 2002
3. Rutter, J Child Psychol Psych, Vol. 44:326-41, 2003
4. Pinborg, Human Reproduction, Vol. 18:1234-43, 2003
5. Griesinger, Hum Reproduction, Vol. 19:1239-1241, 2004
6. Glazebrook, Fertil Steril, Vol. 81:505-11, 2004
7. Paulson RJ, Fertil Steril., Vol. 53:870-874 , 1990
8. Fertil Steril, Vol. 85, Suppl. 4, 2006
9. Pacific Fertility Center 2007 IVF Statistics

For patients having their embryos transferred at the blastocyst stage, the grading procedure used to assess the embryos can seem complicated. However, we simply look to see that the embryos are developing normally, are not slowing down, and are preparing for implantation in the uterus.

In the 2 days following fertilization, embryos go through 3 rounds of cell division. The fertilized oocyte divides in 2, these cells each divide again to give 4, and then these divide to give 8. In the resulting 8-cell embryo, each cell should be 1/8 the size of the original oocyte since there is no growth in size, and each cell should be intact and symmetrical. When we assess embryos at this stage, we first count the number of cells and we then assign a grade based on how good the embryo looks. Embryos that have disintegrating or asymmetrical cells are assigned a lower grade.

At this early stage, the individual cells stay together because they are contained within a shell called the zona pellucida. However, as the embryo progresses past the 8-cell stage, dividing to 16 and then 32, the cells attach to each other and cooperate to form a tight ball called a morula. At the morula stage, the cells are pressed so tightly together that individual cells cannot easily be identified or counted. Once the attachments between cells are formed, the cells begin to pump fluid into the center of the ball, giving rise to a tiny fluid filled cavity or cyst. As long as the junctions between cells hold, no fluid can escape from the cyst, and the cyst grows larger as more fluid is pumped in.

These are critical days for the embryo. In addition to forming the central cyst, the embryo is also busy organizing its cells into two distinct populations. As the embryo moves beyond the 8-cell stage, some cells stay on the outside of the ball and some are pushed to the inside. In the typical 16-cell embryo, there are 12 outer and 4 inner cells. At the 32-cell stage, 22 of the cells are outer cells and 10 are inner cells. Creating more outer cells is deliberate, because these cells are needed to maintain the integrity of the cavity as it becomes larger. More importantly however, these cells will become the placenta, and having enough cells to establish the placenta is critical to successful implantation in the uterus. Once the placenta is established, it can feed the inner cells which become the developing fetus.

The appearance of the cyst at the center of the morula marks the next embryo stage, the blastocyst. In assessing the blastocyst, we look at the size of the cyst and the integrity of the outer and inner cells. Depending on the size of the cyst, the blastocyst is referred to as early, expanding or fully expanded. If the cyst has become large enough to cause the embryo to burst through its shell, we call it a hatching blastocyst. Occasionally, we even see fully hatched blastocysts. Hatching is a natural process that frees the embryo from its shell to allow implantation to occur. The more expanded the cyst has become, the more we favor the embryo for transfer.

In addition to looking at cyst expansion, the grade of the blastocyst is further determined by the integrity of the inner and outer cells. Embryos with more cells are better, and the best blastocysts are well expanded with distinct inner and outer cell populations. In poor quality blastocysts, there can be few cells in one or both populations, and/or the cavity can be small. And sometimes, even in embryos with beautiful outer cells, we cannot see any inner cells at all. These embryos are destined to fail since a full blastocyst with 32 cells is incapable of making inner cells if they do not already exist.

The embryos that are most difficult to assess are those where the cavity has just begun to open up, but has not expanded sufficiently to allow us to see inside. These early blastocysts are usually assigned lesser grades as we are unable to determine whether any inner cells are present. We often look at these embryos again several hours later to see if further expansion has revealed the presence of those critical inner cells. We would then re-grade the embryo, if appropriate.

All of this development, from fertilization to blastocyst expansion and hatching, normally follows a tight timeline that is independent of cell number. The embryo attempts to hatch from its shell approximately 5 or 6 days post fertilization, regardless of the number of cells it contains. If development is slow, and cell number is consequently low, the outer cells stretch to enclose the cyst and expansion continues. This is important, as the uterus waits only a few days for the embryo to implant. If the embryo takes too long to make the “right” number of cells for expansion and hatching, it may miss the implantation window. The practical result of this is that we still get high implantation rates even if only early blastocysts are available for transfer.

The above phenomenon is relevant to frozen embryo transfer cycles too, because many embryos lose one or more cells as a result of freezing and thawing. Such embryos still try to form blastocysts according to their original timeline, even though they may have less than the ideal number of cells. The consequences of arriving with plenty of cells but too late for the uterus are worse than having a chance to implant even with fewer cells. As a result, frozen-thawed embryos that have lost a cell or two are not assigned a lower grade since we still consider them to have high implantation potential.  

Question: We hope to have embryos left after transfer and need to consider storage. Can you help us understand what determines your storage fees?

Answer: Many Pacific Fertility Center patients have surplus embryos at the end of their IVF cycle. If you chose to freeze your embryos, you will need to consider how long you plan to store the embryos before being used for a frozen embryo transfer. Patients who are finished building their family, but are not interested in destroying the surplus embryos, may choose to freeze them, offer them for adoption or donate them to research. These options are included on the consent forms, which must be signed prior to transfer.

Once you choose to freeze embryos, you need to factor in the annual storage fee. Pacific Fertility Center strives for lower fees, but must be able cover the underlying costs of services. Storage fees include expenses from the following sources: storage tanks, liquid nitrogen, leased floor space, embryologists and staff hours, equipment maintenance, annual inventory, information dissemination, forms, billing, legal fees and liability.

Let’s begin with the storage tanks themselves. At PFC we have 3 state-of-the-art embryo tanks: two tanks hold a total of 1376 spaces each. Every one of these spaces can hold up to 5 straws of embryos and each straw holds 1 to 3 embryos. These two tanks are full. Recently, we purchased another, larger tank, which holds almost 1500 patient spaces. This tank is already almost half full.

Once these tanks are filled with liquid nitrogen, they are extremely heavy. Because of their weight, they cannot be clustered together in the same room, but must be strategically placed to spread out the weight over the center’s floor. In addition, they must be stored in a secure, locked location. Every time we add a tank, an appropriate new space must be located. With square footage at a premium, this is not an easy task.

Storage tanks must be monitored. Gauges and seals must be functioning and the temperature must be kept at the optimum level with the addition of liquid nitrogen. The tanks are fitted with an alarm, which sounds if there is a problem. This alarm automatically sends an alert to the embryologist on call 24 hour a day, 365 days a year.

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 completed every year. This is a time-consuming process as every straw must be located and identified. 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.

If patients fail to notify us of a move and/or abandon their embryos, we make every effort to locate them. When they repeatedly fail to pay their invoice, we may be forced to send their billing on to a collections agency. During this process, we continue to store their embryos. As a last resort, we will go before a judge, show proof that we are unable to contact the patient after multiple attempts over a reasonable period of time, and request permission to discard the abandoned embryos.

One of the most frequently asked questions is “When am I going to be billed?” You will be billed based on the month that your storage begins. Patients often forget they have a back-up sample of frozen sperm and are “surprised” when they receive an invoice indicating they must pay their storage fee.

PFC is always available to answer any questions you may have regarding the storing of your embryos and sperm. For disposition questions, please contact Alexis Von Austin, Tissue Bank Manager at (415) 249-3636. For questions regarding an invoice, please contact Rosemarie S. Tagle, Billing Supervisor at (415) 249-3651.

Fresh Embryo at Blastocyst Stage: The cells are elongated and pressed against one another. The inner and outer cells are clearly visible, as is the cavity.

Two Vitrified Embryos at Blastocyst Stage After Warming: Though their appearances differ, both embryos implanted and created viable pregnancies.

This embryo looks perfect, as if it was never frozen. The outer and inner cells are clearly visible, as is the cavity.

This embryo has rounded, more dissociated cells resulting from shrinkage during incubation in cryoprotectant, (as cells shrink they pull away from each other). The cavity is small, but visible.

The first human pregnancy from an embryo that had been frozen and thawed was achieved in Australia in 1984, 6 years after the birth of the first IVF baby in the UK. The method used to preserve that embryo is called “slow freezing” and it is still the preferred method for preserving embryos throughout the world today. Slow freezing is a reliable and established technique that has served the IVF community well for over 20 years. The procedure has been refined throughout those years and it works, with slight modifications, for freezing all embryo stages and for sperm. However, despite many years of trying, slow freezing has never worked very successfully with oocytes. Frustrated by years of failures, scientists turned to an alternative procedure called vitrification in their quest to preserve oocytes. This approach is relatively new, but appears as through it will be preferentially used for oocyte preservation as we go forward. Vitrification kits are just beginning to get FDA clearance following scientific trials, and embryologists are being trained in the use of the new technology.

The main concern during the freezing of any cell is the removal of water without actually killing the cell. Since water expands in volume as it freezes, ice formation inside a cell would cause the cell to rupture and die. Therefore, cell water is traditionally replaced with a cryoprotectant (antifreeze) prior to cooling of the cell. This is achieved by sequentially incubating the cell in increasing concentrations of cryoprotectant. The cryoprotectant draws water out of the cell and itself enters the cell, all by osmosis. Once most of the water has been removed, the cell is cooled at the very slow rate of -0.3° C/minute until it has been cooled to below -30° C and is therefore fully frozen. Thereafter, storage of frozen cells is in liquid nitrogen (-196° C), which is a simple and practical storage medium.

Vitrification still requires the use of cryoprotectants and the cell is also ultimately stored in liquid nitrogen, but the journey from the incubator (at 37° C) to the nitrogen (-196° C) is much faster. The word “vitrum” in Medieval Latin means “glass” and the process turns the cell contents to a glass like substance instead of ice. Since no ice forms, the risk of rupturing the cell is eliminated. For glass to form instead of ice, the rate of cooling must be thousands of degrees per minute instead of the 0.3 degrees/minute that we use in slow freezing. Therefore, the process is sometimes referred to as ultra-rapid freezing, although the word “freezing” is really inappropriate here since the cell is not really frozen (i.e. no ice is created).

One of the big stumbling blocks during oocyte freezing was the sheer size of the cell (the oocyte is the largest human cell by some margin) and therefore its high water content. Just getting the cell to survive, (an oocyte has only one cell), was a huge stumbling block. Studies where 50-60% of the oocytes survived were considered groundbreaking, and still today there are few studies that have done better. Vitrification as a technique had been largely ignored by the IVF community as it was technically more challenging and used much higher concentrations of cryoprotectants. Cryoprotectants were thought to be toxic to cells. Today we know that they are safe and effective and do not contribute to cell death. It is possible that cryoprotectants may have deleterious effects on cells if they are metabolized, but virtually all freezing protocols utilize them at room temperature or below, where cell metabolism is significantly slowed or stopped. So, with success rates using traditional slow freezing failing to improve, vitrification has been given serious consideration as an alternative. In the few years since its introduction, vitrification has shown promising and excellent results in clinical studies (see Oktay et al., Fertility and Sterility, 2006, Vol 86(1), pages 70-80 a comparative review of slow freezing and vitrification results with human oocytes).

Making the transition from slow freezing to vitrification has been a challenge for the IVF community. As already stated, it is a technically challenging procedure, and training of embryologists in the technique has been slow. With slow freezing, embryos are placed in relatively weak solutions of cryoprotectant for as long as 15 minutes at a time. Then, they are usually moved on through slightly stronger solutions before being placed in large straws or vials which are then loaded into a computer controlled freezer for the long journey to -30° C. The embryologist can spend about 30 minutes with a set of embryos from the time that they come out of the incubator until they go into the controlled rate freezer. After 2 or more hours, the embryos can be placed in liquid nitrogen and the process is complete.

During a vitrification procedure, where typically only one oocyte or embryo can be worked on at a time, the transition from incubator to nitrogen takes only a few minutes. The embryo is stepped through solutions containing high and then higher concentrations of cryoprotectants where it shrivels and swirls in the extremely viscous medium. In the final stage, which is measured in seconds, the embryo is placed in an extremely concentrated cryoprotectant solution and then quickly loaded up into a tiny straw that is barely larger than the embryo itself. The straw is then sealed at both ends and plunged immediately into liquid nitrogen. The straw is so fine that it freezes in an instant, an important part of the vitrification process. The loading of the straw occurs at room temperature (25º C in the IVF lab) and it is cooled to -196º C in one or two seconds, giving a cooling rate of 6000-13000º C/min. The faster the straw can be cooled, the more successful the procedure. Performing this final step too slowly or too quickly can be the difference between success and failure and therefore requires extensive training.

At Pacific Fertility Center, we have been working on vitrification for over 2 years. Our initial interest was in oocyte freezing, but we were also interested in extending the technique to be used with embryos, and in particular to blastocyst stage embryos where slow freezing has not always worked well. Slow freezing has served us well over the years for embryos being frozen 1, 2 or 3 days after an oocyte retrieval, but blastocysts (5 or 6 day old embryos) did less well. With an industry wide transition to blastocyst stage embryo transfers, we looked at vitrification as an alternative method of preservation for these precious embryos.

A blastocyst is an embryo that has developed to the stage where it is ready to implant in the uterus. Instead of having a small number of loosely associated cells characteristic of earlier embryonic stages, it has 2 defined cell populations and a fluid filled cavity (or cyst). The cells that surround the cavity will form the placenta, and the cells within the cavity will develop into the embryo proper, or fetus and some of the extraembryonic membranes, such as the yolk sac. It is these interior cells that cause trouble during freezing since they are on the inside and difficult to expose to cryoprotectant. Slow freezing relies on cryoprotectant traveling through the outer placental cells, then the cavity, and finally into the fetal cells while water travels in the opposite direction. Fully dehydrating these fetal cells has always been a challenge and an embryo where these cells do not survive freezing and thawing will not result in a viable pregnancy. And with slow freezing, embryos tend to collapse in on themselves during dehydration, making it difficult to assess survival after thawing.

After investing heavily in vitrification training and implementing a successful oocyte vitrification program, PFC began working on blastocyst vitrification in January of 2007. By March we had a program established and were delighted by how easily blastocysts seemed to tolerate the procedure. Often, blastocysts looked no different after vitrification when compared to how they looked before the procedure. This result was in stark contrast to slow freezing where blastocysts always look shriveled and deflated after coming out of the freezer. By July 2007, we had switched completely to vitrification and currently we are enjoying the successes that it is bringing to our patients and us.

Our vitrification team consists of 3 embryologists: Mariluz Branch, the team leader, with Erin Fischer and Liz Holmes. Because of the technical challenges involved, we have to be cautious with involving other embryologists. So one of the three team members must be on duty every day (our lab is open 7 days a week). I am grateful to this team for their flexibility in accommodating our needs. By the end of the year we expect to have 2 more embryologists on the team, and then the final 3 in 2008.

Vitrification has been an exciting and challenging technique which we have embraced and conquered in 2007. We look forward to the gradual elimination of slow freezing and the successes that vitrification will bring us in the future.

Joe Conaghan, PhD, HCLD

Many IVF programs routinely schedule frozen embryo transfers (FET) to occur on specific days by putting their patients on estrogen and progesterone to prepare the uterine lining for implantation. This allows for a flexible schedule for the clinic and the patient, i.e. it allows the clinics to group FETs together and avoid weekend transfer procedures. However, the patient must remain on both estrogen and progesterone to support the pregnancy for up to 12 weeks.

More and more, clinics are starting to schedule FETs in natural cycles, timed to natural ovulation with minimal medications. This does mean that a transfer can occur any day of the week. Due to tradition and convenience, some clinics remain hesitant to switch to natural cycle FETs. Part of the problem is that there have been very few studies showing what the success rates were in natural vs. programmed FET cycles. The few studies that have been published have reported on a fairly limited number of cycles.

Pacific Fertility Center has always been a proponent of natural cycle FETs. Because we do about 400 FETs each year, we have been able to gather a large number of cycles to evaluate. Most of our patients we evaluated for this study were in natural cycles but some patients had to do programmed cycles because they did not ovulate regularly or because they had to travel some distance to come to PFC for their FET and needed to have the more precise scheduling that a programmed cycle affords.

In our study, we looked at 1,378 frozen embryo transfers done between 2000-2005. Of these, 934 were done in patients using embryos from their own eggs and 444 were done in patients using embryos from donor eggs. The bottom line is that there were no differences in delivered pregnancy rates within both groups of patients (own eggs and donor eggs) between those patients having a transfer timed to natural ovulation or those patients with estrogen-progesterone uterine preparation.

Because we feel that a natural cycle is less costly, requires no blood tests and (usually) fewer ultrasounds and injections, patients find this a desirable alternative to the more common, programmed FET. In addition to these patient-friendly reasons for choosing natural cycle FETs, we now feel PFC has solid data to justify this approach.

Preliminary results of this study were presented at an oral presentation at the Pacific Coast Reproductive Society meeting in Palm Springs this past April (see sidebar).

This study has just been submitted to Fertility and Sterility, the major reproductive endocrinology journal of the American Society for Reproductive Medicine. We expect full publication after the peer review process is completed.

Carolyn Givens, MD

“Outcomes of Natural Cycles vs. Programmed Cycles for 1378 Frozen Embryo Transfers” Carolyn R. Givens, M.D.a, Leslie C. Markun,b Isabelle P. Ryan, M.D.,a Philip E. Chenette, M.D.,a Carl M. Herbert, M.D.,a and Eldon D. Schriock, M.D.a Submitted July 2007 to Fertility and Sterility.

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