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

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

We typically freeze embryos one, three or five days after an egg retrieval procedure. The day we freeze embryos depends on the individual circumstances of a particular patient, how well embryos are developing in the laboratory, how many embryos we have, and when a patient is having their embryo transfer. Most patients have a transfer on the third day after retrieval and we freeze surplus healthy embryos the same day. At this time, we can see how well the embryos are developing and choose the best embryos for transfer and freezing. Embryos tolerate freezing relatively well on day 3 and about one third of patients become pregnant after a transfer using thawed embryos.

It is not common to freeze embryos on day 1 after retrieval since at this time we have very limited information on their development, but this is the stage at which embryos best tolerate freezing and thawing. In 2005, 92% of embryos thawed at this stage survived, compared to 64% of embryos surviving after freezing on day 3. However, the lack of embryo development information on day 1 means that we are probably freezing many embryos that have little or no chance of establishing a pregnancy. We therefore prefer to let the embryos grow for at least another 2 days to make sure that we only end up with good quality embryos in our freezer.

In the last 2 years we have been doing more and more transfers on day 5-post retrieval (blastocyst transfer). Delaying the transfer an additional 2 days allows us to get a much better picture of which embryos in a cohort are really strong and healthy. By day 5, the embryo should have reached the blastocyst stage, which is characterized by the presence of a fluid filled cavity or cyst in the embryo. Embryos that reach the blastocyst stage by day 5 have a higher chance of implanting after transfer when compared to embryos transferred on day 3. However, not all embryos that look healthy and strong on day 3 will make a blastocyst. We estimate that it takes 3-4 nice day 3 embryos to achieve a nice blastocyst on day 5. Therefore, blastocyst transfers are usually undertaken only by patients with many nice embryos on day 3. Also, patients at high risk for a multiple pregnancy and/or those wishing to transfer only one embryo often decide to do their transfer on day 5.

After a day 5 transfer, surplus blastocysts can be frozen for later use. They can be frozen on day 5, or if they are developing a little more slowly, on day 6. Blastocysts have many more cells (up to 200 cells) than day 3 embryos (up to 12 cells) and we employ a different method for freezing them. All freezing techniques involve dehydrating the embryo, so the fluid filled cavity in a blastocyst will collapse during freezing. When thawed and placed inside the incubator in the laboratory, the cavity will begin to re-expand and the blastocyst should be fully inflated about 4 hours later. Blastocysts that show little or no signs of re-expansion are unlikely to implant after transfer.

The technology that allows us to grow embryos to day 5 or 6, continues to improve, and in line with this, we are offering blastocyst transfer and freezing to more patients. In particular, our ability to culture embryos in a reduced oxygen environment reduces stress on the embryos and therefore provides healthier embryos for transfer and freezing. In 2005, slightly less than 10% of our fresh and frozen embryo transfers were performed on day 5, but this number is likely to increase dramatically in 2006. Individuals using donor oocytes comprised almost half of the day 5 transfers, since we tend to have many embryos to choose from in these cases. Blastocyst transfer will not be an option for everybody, and not everyone will have enough blastocysts to transfer and freeze. We are freezing blastocysts almost every day now and transfers with thawed blastocysts are becoming a regular part of our laboratory routine. If you think that you might be a candidate for a blastocyst transfer, please talk to your physician for more information.

– Joe Conaghan, PhD, HCLD

The field of assisted reproduction is continually changing and this is a good thing because, for the most part, these changes have been for the better. Better medications, improved treatment strategies and a better understanding of laboratory techniques are resulting in constantly improving embryo implantation rates. This will allow us to transfer fewer embryos, reducing the risks for twins or triplets, which will result in better obstetrical outcomes for our patients. Our goal in assisted reproduction is to do all we can to ensure that chromosomally normal embryos have the opportunity to result in a healthy pregnancy.

More and more, Pacific Fertility Center is utilizing the day-5 embryo transfer procedure. The benefits of this are to 1) improve implantation and pregnancy rates and 2) lower the number of embryos transferred. Allowing embryos to remain in the laboratory for 5 days after egg retrieval (as opposed to the more standard 3 days) gives us an opportunity to choose those embryos most likely to carry normal chromosomes and those most likely to keep developing in the womb after embryo transfer. PFC utilizes low oxygen incubators almost exclusively. As such, we are seeing an improvement in the percentage of embryos that are developing well in the lab environment, as evidenced by their continued progression from a cleavage stage embryo to a blastocyst stage embryo.

Although Day-5 embryo transfer has been around for a while, we have been hesitant to use it exclusively for our patients. Clinical data suggests that unless a woman has a good number of nice looking embryos on day 3, the risks of having no well developed embryos on day 5 is fairly high. Therefore, the patient choosing to attempt a day-5 transfer might end up with virtually no embryos to transfer. One argument asserts that this is what would probably occur within the uterine lining anyway. However, there are studies suggesting that pregnancy rates in women with less than three or four nice 8-cell embryos on day 3 will have a higher chance of pregnancy with a day-3 transfer as compared to women with less than three or four nice 8-cell embryos who have a day-5 transfer. We are noticing that, with the use of our new low oxygen incubators, we are getting better developed embryos on day 3. These embryos, in turn, have a greater chance of being a nice embryo on day 5.

We are also seeing a small but growing number of couples interested in the transfer of only one embryo because they wish to avoid the risks of having a twin pregnancy. Although the over-whelming majority of babies born as a twin do well, there is a measurable increase in the incidence of perinatal death and cerebral palsy in twins as compared to babies born as a singleton. Also, we have many patients returning to us for baby #2 or #3 and they would like to avoid a multiple gestation. This has been particularly true for our patients using donor egg-derived embryos. Last year, we saw a 50% pregnancy rate in women electively transferring one embryo in the donor egg program. We will definitely support any patient that wishes to transfer only one embryo at a time, and we will likely encourage day-5 transfers to better select the one embryo most likely to implant.

One potential downside of attempting day-5 transfer is the question of whether or not we will increase the number of pregnancies in the fresh IVF cycle at the expense of additional attempts with frozen embryos. This is because embryos frozen at a cleavage stage [day 3] have historically done better with freezing and thawing as compared to day-5 embryos.

Pacific Fertility Center has always had a strong freezing program with excellent success rates with frozen embryo transfers, mostly at the day-3 stage of freezing and thawing. Many patients have asked us why we do not freeze some embryos at day 3 and culture some to day 5. One reason is that when we freeze some embryos at day 3, we are taking them out of contention for fresh embryo transfer and therefore, we may be losing some of the selective advantage of doing a day-5 transfer. With patients that have a very large number of embryos on day 3 that look good (say 15 or more), this may still be a viable strategy. Most patients don’t have many good quality cleavage stage embryos, however. So when we are planning to attempt a day-5 transfer, we will usually plan to culture all embryos to day 5 and select the best one or two for transfer and freeze the remainder as a day-5 embryo. As a result, we are now seeing a larger number of our patients returning for frozen embryo transfer with their day-5 frozen embryos. Our laboratory director, Dr. Joe Conaghan is currently reviewing the data on implantation rates from frozen-thawed day-5 embryos from 2005.

Listed below are some of the situations for which we are more likely to recommend a day 5 transfer. These include:

1. Any woman undergoing transfer with donor egg-derived embryos (anonymous donors)
2. Any woman less than 40 with a large number of eggs/embryos/good quality day 3 embryos
3. Any woman that has had poor success with freezing and thawing on day 3 in prior ART cycles
4. Any patient considering transferring only one embryo
5. Any patient with a history of multiple ectopic pregnancies (one recent abstract from the ASRM
meeting suggested a decrease in the ectopic rate after IVF with a day 5 vs. day 3 embryo transfer).
6. Any woman with unexplained IVF implantation failures with day-3 transfer.

– Carolyn Givens, MD

Related Posts:

Low O2 Incubators
Stages of Embryo Development
From Egg to Blastocyst
Day 3 vs. Day 5 Transfer – Photos

My infertility journey started when I was only 17. I was diagnosed with endometriosis and underwent my first laparoscopy. I had temporarily relief and then my symptoms returned. I tried various alternative treatments but they too offered only temporarily relief. This was not the life that I wanted to have as a young adult who wanted to have children more than anything in the world.

Surgery after surgery, specialist after specialist, my quality of life was slowly going down the drain. Initially, I told doctors that I didn’t want to have a hysterectomy but later, something had changed. I was eight surgeries into my journey and I asked my doctor if I could have just my uterus removed so I could still try and have a biological child. He said yes and I was quite relieved. After the surgery, I felt better for a while, but the pain still continued. I had to evaluate my life and decide what was important to me. I knew I wanted to live, but the pain had me in and out of the hospital and often times feeling suicidal. I had no other choice but to have my ovaries removed.

Luckily, I thought about freezing embryos and called Pacific Fertility Center. I met with Dr. Isabelle Ryan and she changed my life. My boyfriend and I knew we wanted to get married and I was on a limited time line until I had my ovaries removed. We only had one chance to do this and we were determined to do it right. We underwent one cycle of In Vitro Fertilization and froze all of our embryos. We froze our embryos at a 2PN stage* per Dr. Conaghan and Dr. Ryan’s request. This would help our chances of having them thaw better but we don’t know how they will turn out. We were willing to take that chance.

Two weeks later, I had my ovaries removed and then felt I was ready to move on with my life. My boyfriend and I got engaged and together dealt with the loss of having me carry our child. In our counseling session with Peggy Orlin, MFT at Pacific Fertility Center, we talked about what if a gestational cycle didn’t work. We knew that we would be parents no matter what and if it wasn’t our biological child we could be ok with that.

From time to time, I still grieve the loss of being pregnant, but know that I did everything that I could. Since then we have gotten married and have been offered the opportunity of a lifetime. A dear friend has said that she would like to carry our child. She has restored our faith in humanity. What an offer!

As we are working out the details, we are thankful for her commitment to us and our journey. We will transfer some of our embryos into our gestational carrier and hope for the best. Dr. Ryan and all of the staff at Pacific Fertility Center have been so supportive of us that we can’t wait to come back when we are ready to do our transfer.
Anonymous, San Francisco

* A note from Laboratory Director Joe Conaghan, PhD:
Embryos can be frozen at different stages of development, usually 1, 3 or 5 days after oocyte retrieval. In general, the earlier they are frozen, the better they tolerate the freezing process. Embryos frozen on day 1, or at the 2 pro-nuclei stage, survive freezing and thawing at a rate over 95%.

Q.
We have frozen embryos at PFC that we would like to donate to research. Now that California has passed the Stem Cell Research Initiative, how would this affect the donation process?

A.
The status of your frozen embryos at PFC will not change as a result of this new initiative. Currently, all patients with frozen embryos in storage at PFC are contacted by us on an annual basis to reconfirm their wishes for their embryos for the upcoming year.

All patients are given 5 choices:
1. Return to PFC for a frozen embryo transfer
2. Continue storage (an annual storage fee payment is remitted)
3. Thaw and discard all remaining embryos
4. Donate embryos to another party (known or anonymous donation)
5. Donate embryos for medical research

If option #5 is chosen, we will transport the embryos in a liquid nitrogen carrier tank to one of several scientists (primarily at UCSF) for use in their research. Our patient’s privacy is maintained because we only give UCSF an identifying number with each set of embryos.

Currently, we are working with several researchers who are attempting to develop embryonic stem cells under non-federally funded research grants. The new state initiative may provide these scientists and others with more funding to continue and expand their studies. With the added state funding, their research could contribute to the body of knowledge about how undifferentiated human cells become specific tissues, hopefully leading to the development of specific tissues to treat diseases and conditions such as Parkinson’s, Alzheimer’s, diabetes and spinal cord injury.

Q. How can I be sure that PFC will not accidentally confuse my eggs and my husband’s sperm and our embryos with someone else’s?

A. PFC recognizes that even with the best intentions, human error can occur. We’ve therefore designed our strict SurTransfer^SM laboratory security system of color-coding and clearly labeling all specimens and verbally identifying all patients. We have also devoted considerable time and effort into assembling one of the most highly trained teams in the country. Each of our Embryologists is Board Certified and Licensed, even though the State of California does not currently require licensure for Embryologists.
When a patient is scheduled for a procedure, a written procedure requisition is sent by the Physician to the laboratory staff, giving them at least 24hour notice and clear instructions on what is to be done. Each patient is assigned a specific color for their test tubes and Petri dishes; no two patients having procedures on the same day will be assigned the same color. Each of the patient’s specimens is carefully labeled with clear and unique identifying information that includes the patient’s name and date of birth.

During their stay in the lab, eggs, sperm and embryos are kept in incubators. We avoid assigning two cases to a single incubator on the same day. Each incubator has an exterior door and an interior door. Both doors are clearly labeled with name and color code. This labeling protocol allows the embryologist to verify the name twice before ever handling the specimen.

We have two embryologists performing all critical procedures to ensure accuracy; generally one handles the material while the other observes and verifies. We are not required to assign two people to procedures, but redundancy eliminates the possibility of an error.

Both embryologists sign off after checking the paperwork, labeling the specimen and performing the procedure.
Accepting sperm samples: When a man delivers his sample, we require it to be labeled with his unique information, including name, birth date and signature. We ask to see identification. The embryologist receiving the sample will sign that s/he received it and note the time and date of receipt. If s/he passes the sample to another member of staff, that individual will sign for it, thus continuing a chain of responsible custody.

Egg retrieval: A patient undergoing egg retrieval is asked in the retrieval room to identify herself before receiving sedating drugs. The embryologist will not rely on the physician, nor state the patient’s name and ask for a “yes or no” answer, but will instead ask her to state her full name. This avoids any possible miscommunication. As the procedure gets underway, two embryologists will take responsibility for accepting the collected eggs.

Inseminating eggs: This is arguably the most important part of the IVF procedure. While it is a relatively simple procedure to perform, we are sensitive to its significance. Without any exceptions, two embryologists perform the insemination. Even if there is only one egg to inseminate, or even if there is only one insemination on a given day, two people do it.

Embryo transfer: Similar to the retrieval procedure, one embryologist will ask the patient her name and a second embryologist will witness and verify that the correct embryos are loaded into the transfer catheter. As a final check, the embryologist will hand the catheter to the physician and state the patient’s full name and the number of embryos.

Freezing and thawing of sperm or embryos:

Frozen specimens are extensively labeled and catalogued. Thawing can only be directed by a physician, and as a rule an embryologist never handles or thaws a specimen without a witness. Once a specimen is thawed, there’s no going back.