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.

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.

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.  Joe Conaghan, Ph.D., HCLD