The first human pregnancy from a frozen oocyte was achieved in Australia in 1985 by Dr. Christopher Chen (Lancet  1 (vol 8486): pp 884-886, 1986), just one year after the first baby had been born from a previously frozen embryo.  Since then we have come a long way and current reports suggest that preserved and then thawed oocytes can perform just as well as fresh oocytes.  In a recent study using oocytes from young donors, Peter Nagy from Reproductive Biology Associates in Atlanta showed that frozen oocytes actually outperformed fresh oocytes from the same donors in a side by side study.  134 out of 153 oocytes survived after warming and fertilization, embryo development and pregnancy rates were normal, leading to 16 pregnancies among the 20 recipients in the study (Fertility and Sterility, 92: pp 520-6, 2009).  Pregnancies were also achieved from frozen embryos derived from the frozen oocytes in that study.

As a young embryologist in London in the late 1980’s I remember meeting Dr. Chen, and the wide skepticism that surrounded his work made an impression on me.  It hadn’t been possible to repeat his effort and his early success did not yield a method that could be reliably or consistently used to freeze oocytes.  He had used a procedure called “slow-freezing” which worked and continued to work reasonably well for embryos, but which has been unreliable for freezing oocytes.

The human oocyte is a huge cell at about 100 µm in diameter.  It is so big that under the right circumstances it can actually be seen with the naked eye, without the use of a microscope.  All human cells are smaller than this, with skin cells measuring about 30 µm and a sperm head (the smallest human cell) measuring just 5 µm long and 3 µm wide.  And this size has been one reason why oocytes have been so difficult to freeze successfully.  Cell freezing is dramatically influenced by the cell’s surface area to volume ratio, and by its water content, and since the oocyte is so big, these dimensions are very different from all other cell types.  One way to think about this is to compare the dimensions of the earth to those of the moon, which measures about 25% of the earth’s diameter.  The moon’s surface area however, is only about 7% that of the earth and its volume is just 2%.  Similarly, the dimensions of the human oocyte outstrip other cells when we look at these key measurements.  The table below shows the key surface area to volume ratios for a sperm and an oocyte.

Other than size and water content, there is one other significant hurdle to oocyte freezing that is not a factor when freezing other cells.  At ovulation, the oocyte is close to completing meiosis, a type of cell division that reduces DNA content by half in preparation for fertilization.  While the ejaculated sperm has fully completed meiosis and is in a state of nuclear stability, the oocyte in contrast pauses at a very sensitive stage, metaphase II, and as a result is very sensitive to physical (e.g. being handled too much), chemical (pH) and environmental (temperature) changes, which can cause irreversible damage if not carefully controlled.

Chen’s method, and the traditional method for embryo freezing involves slowly dehydrating cells and then cooling them very slowly over a period of approximately 90 minutes, until they reach a temperature of between -30 and -40⁰C.  The oocytes or embryos are then stored in liquid nitrogen at -196⁰C until they are needed, at which time they are thawed and rehydrated.  The removal of cell water is a critical part of the process as any water remaining inside the cell can freeze (turn to ice) that will rupture (kill) the cell.  Dehydration is achieved with the use of permeating agents (called cryoprotectants) such as ethylene glycol, glycerol or DMSO which enter cells and draw water out by simple osmosis.  Non permeating cryoprotectants such as sucrose, which will not cross a cell membrane by osmosis, are also used to further dehydrate the cell before freezing.  Slow freezing uses minimal concentrations of cryoprotectants and is a slow and delicate process that aims to prevent ice formation inside cells.

In the 1990’s and 2000’s as IVF became a widely available technology with ever improving success rates, several countries around the world moved to regulate the industry.  In Europe, Germany, Italy and Switzerland all established laws that limited the number of embryos that could be created and/or frozen in an IVF treatment cycle.  The law in Italy required that any oocyte that had been incubated with sperm had to be transferred to the mother, even if that oocyte did not fertilize.  Such laws limited the number of oocytes that could be inseminated, and since IVF usually generates multiple oocytes, research on improving oocyte freezing methodology became very important.  The Italians with their prohibitive laws lead this effort and several promising papers resulted that brought improvements in the slow freezing method for oocytes.  However, despite the considerable time and effort to develop a method for slow freezing oocytes, only about 300 babies were reported worldwide after about 2 decades of trying (Reprod Biomed Online, 18(6), pp 769-76, 2009).  The slow progress of this method together with disappointing efficiencies, suggested that slow freezing was unlikely to ever be a useful method for oocyte freezing.

In the quest to improve oocyte freezing methods, and in the face of disappointing results with slow freezing, scientists began to look for an alternative method that would improve outcomes.  In 1985, mouse embryos had been successfully preserved using a process called vitrification (Nature, 313, pp573-5) which prevents ice formation in cells by using higher concentrations of cryoprotectants and ultra-rapid cooling.  Vitrification is a technically more challenging procedure and depends a lot on the skills of the embryologist in handling oocytes or embryos through the process.  This handling skill (referred to as the “technical footprint”) is more critical since vitrification is an ultra-rapid procedure where the individual steps are timed to the second, and it requires higher concentrations of cryoprotectants that are extremely viscous and difficult to work with.  It also uses smaller vessels to contain the oocytes compared to the large ¼ or ½ cc straws, or 1 ml vials that were used with slow freezing.  The typical vitrification straw or container is just bigger than the oocyte itself, allowing for the maximum possible cooling rate when plunged into liquid nitrogen.  In a typical vitrification procedure, the final step requires that the oocyte be placed in a solution that is composed of about 30-35% cryoprotectant by volume, then aspirated in as small a volume as possible into a tiny container before plunging into liquid nitrogen and getting to -196⁰C in 1-2 seconds.   When we teach this procedure, practically all the emphasis is on the technical footprint.

By 2005, after years of experimenting on bovine and porcine oocytes (which are similar in dimensions to human oocytes) researchers led by Masa Kuwayama from Tokyo began publishing very exciting results from vitrified human oocytes (RBM Online, vol. 11, pp 300-8).  Survival of oocytes was over 90% after warming, and fertilization, embryo development and pregnancy rates were similar to those achieved with fresh oocytes.  Finally a reliable method was available and it could be learned and implemented by embryologists all over the world.  Vitrification worked so well that it was quickly adapted for embryos, and is now the standard procedure for preserving surplus embryos after IVF treatment.  The number of IVF clinics that still freeze embryos by the older slow-freeze methodology is constantly declining, and virtually all oocyte freezing is now done using vitrification.  By 2009, the number of babies born from vitrified oocytes had already passed the total number conceived over more than 20 years with slow freezing (Noyes st al., RBM Online, Vol 18 (6), pp 769-76), and the babies were normal when compared to naturally conceived children.

Here at PFC, we quickly abandoned slow freezing as soon as vitrification was seen to be safe and readily available.  We first vitrified embryos in March 2007 and have not regretted the move away from slow freezing which was completely phased out by June of that year.  We also began vitrifying oocytes in late 2007 and continue to use the procedure for patients facing cancer treatment and women wanting to preserve their fertility.  We constantly help other programs that are learning vitrification and have visitors from all over the world that come to watch us and learn the techniques.  In addition, we helped run hands-on workshops for embryologists domestically in Houston, Boston, Virginia and Austin, and overseas in Europe and South America in 2011.  Vitrification has finally allowed oocytes to be reliably preserved, and since it works so well, demand for the procedure is increasing steadily.

- Joe Conaghan, PhD, HCLD

A statistic that we follow closely at PFC is our cumulative pregnancy rate in a given year.  This is defined as a patient’s chance of taking home a baby after one IVF cycle, but it includes the fresh embryo transfer and any frozen embryo transfers resulting from that one cycle.  These rates are shown in the table and are broken down into maternal age groups.  The numbers are calculated by looking at how many patients delivered a baby from their fresh transfer (43% of patients under age 35) and then adding in pregnancies achieved from the frozen embryos for patients that did not get pregnant in the fresh cycle (totals 64% of patients in this group).  So in this age group, 2 out of every 3 patients had a baby from just one IVF cycle.  Similarly, for patients doing a single cycle with donor oocytes, 74% had a baby.

Cumulative pregnancy rates have special importance since PFC is a national leader in reducing the number of embryos transferred at one time while still maintaining exceptionally high overall pregnancy rates.  One healthy baby at a time is the goal of fertility treatment at PFC and for every patient, a singleton pregnancy is the safest and most likely way to have a healthy baby.  At PFC we work carefully with every patient to reduce their exposure to a multiple pregnancy and all its risks for mother and baby.  And a big part of our strategy involves freezing embryos successfully so that we can use embryos conservatively and efficiently to generate more singleton pregnancies, and fewer multiples. Multiple pregnancies are a complication of IVF treatment, and we strive to avoid them. 

Patients with the highest risk for multiple pregnancy are those where maternal age is <35, doing their 1^st or 2^nd IVF cycle or those patients using donor eggs.  We encourage these individuals to transfer just a single embryo during their IVF cycle and to freeze their surplus embryos for use later.  The frozen embryo program has been so successful here at PFC that it provides very high pregnancy rates for those patients that need to use their embryos from the freezer.  It also means that we don’t have to risk transferring many embryos in the fresh IVF cycle because we have the frozen embryos as a back-up. And most patients that are doing elective single embryo transfer qualify for one of PFC’s financial plans (e.g. the refund plan) that include the cost of frozen embryo transfer cycles in the original price.

We believe that using embryos conservatively is the safest treatment.  And we don’t see big differences in pregnancy rates between patients that transferred just one embryo vs. those that transferred 2.  In fact, patients that received donor eggs and transferred 1 or 2 embryos had the same delivery rates, but those transferring 2 had a 35% twin rate.  In our efforts to reduce this twin rate, we are now transferring 1 embryo 60% of the time in the donor egg program, and 40% of the time in patients aged less than 35.

We want our patients to have healthy babies and we are working to make this possible while still maintaining high success rates.  Our goal is one healthy baby at a time.

 - Joe Conaghan, Ph.D., HCLD & Embryologist Erin Fischer

Since March of 2007, PFC has been vitrifying embryos.  We have now completed over 600 warming cycles, utilizing those embryos.  Vitrification is proving to be a very reliable technology to preserve any unused embryos that remain after a fresh transfer. We continue to adjust our technique and thus increase the successful results of vitrification.  Last year, we introduced a modification to the procedure that allows us to remove the fluid from the cavity in a blastocyst before we begin vitrifying.  As with any freezing procedure, cell water must be substantially removed and replaced with cryoprotectants to avoid ice formation in the cells.  Five and 6 day old embryos, or blastocysts, can have a large fluid filled cavity that slows dehydration and passage of cryoprotectant into the cells.  Since vitrification is an ultra-rapid freezing procedure, any delays caused by the fluid in the cavity may affect the ability of the embryo to survive the procedure.  By making a small breach between two of the outer cells in the embryo, we are now allowing the cavity to collapse prior to beginning the vitrification procedure.  This artificial collapsing has further enhanced results.  We are seeing implantation rates with warmed embryos that are very similar to those achieved with fresh embryos.

Overall, from 636 warming cycles, we have achieved 284 clinical pregnancies (45%) in all age groups combined.  In younger patients (maternal age under 35), there were 103 successful clinical pregnancies from 190 transfers (54%) with an average of just 1.7 embryos transferred.  This pregnancy rate drops to 42% (41/97) in 36-37-year-old patients with an average transfer of 1.8 embryos.  In the 38-40 age group there were 31 pregnancies achieved successful from 79 transfers (39%). For patients over age 40, 8 of the 23 transfers were successful (35%).  In the donated oocytes group, 101 pregnancies resulted from 247 transfers (41% with an average of 1.7 embryos transferred).  For patients that had their embryos artificially collapsed, the results were better.  However, since this is a new technique, the number of cycles is small.

Overall, we are very pleased with the outcomes achieved with vitrified embryos.  We are optimistic that results will continue to improve.  The table above shows results for all cycles completed since the beginning of the vitrification program.  As our experience grows, so do our success rates.  Reviewing cycles of patients that had embryos warmed and transferred from just this year (Jan-Oct 2010), we see that the outcomes are exceptionally good, particularly  for patients whose embryos  were collapsed prior to vitrification.

At PFC we are continuing to vitrify all embryos by day 5 or 6 after oocyte retrieval if they are good or reasonable quality blastocysts.  We now routinely collapse any blastocyst with an expanding cavity.  These procedures have worked well.  Consequently, it has become necessary to reduce the number of embryos being transferred to avoid generating too many multiple pregnancies.  Our goal is to achieve a healthy singleton pregnancy in all patients; vitrification has allowed us to reduce the incidence of multiples by transferring just a single embryo most of the time.  For our 2009 fresh cycles, in patients under 35, 40% of the time we transferred just one embryo, and in patients using donor oocytes 60% of the transfers were a single embryo.  Vitrification has proved to be so successful that many patients have elected for a fresh single embryo transfer; virtually eliminating their risk of twins and knowing that their frozen embryos will be available should they be needed.

Authors: Paul and Shannon Morell
Published in 2010 by Howard Books, New York

In 2009 we read in the popular press that a woman in Michigan was mistakenly implanted with another woman’s embryos due to a mix up at an unnamed IVF Clinic. While this type of mistake is rare, the story was further sensationalized by the revelation that the pregnant woman, Carolyn Savage, intended to carry the pregnancy to term and then turn the baby over to its rightful parents, Shannon and Paul Morell. This was a heartwarming and wonderful outcome from an error that likely would have ended up in tragedy in any other situation.

I finished reading the book less than a week after it was published because I’m keenly interested in the events that led to an error like this. I believe that there is much to be learned from the mistakes of others and I encourage open discussion with the embryologists at PFC to see if it would be possible for us to make a similar mistake. Unfortunately however, the book shares few details of what exactly happened that caused one woman’s embryos to be placed in the uterus of another. We do know that both women used the same last name (Shannon Morell had been treated under her maiden name, Savage), and the error resulted from the thawing of frozen embryos, rather than from the use of fresh embryos. Additionally, we know that Carolyn Savage reported to the clinic for her frozen embryo transfer and somehow the embryos from Shannon Morell (Savage) were thawed and transferred. We do not know if the wrong orders were sent to the IVF lab (“thaw Shannon Savage’s embryos”), if the embryos were inappropriately labeled in the freezer, or if the embryologist simply was not paying attention and thawed the wrong embryos. Whatever the error, it seems likely that having the same last name somehow contributed to the problem.

Regardless of the error that led to thawing the wrong embryos, my opinion is that the major mistake happened when the embryologist went into a room with embryos from one patient and handed them over to another patient. That moment of transfer is the final checkpoint for error prevention. The embryologist is absolutely responsible for confirming that the patient in the room is the owner of the embryos that are to be transferred.

Therefore, even though the book does not share many details about the source of the error, in my opinion, the mistake was made by the embryology laboratory staff. A similar mistake happened in the UK in 2007. In that particular case, the pregnant woman terminated the pregnancy. The Morell’s were aware of this history and worried that the same fate awaited them. All of their frozen embryos had been thawed. Even though the Morells had 2 beautiful daughters, Ellie and Megan, they had planned on using every one of their embryos. Both couples were deeply religious and much is made of this in the book, from praying for a positive outcome to discussions on the embryo as a human being. The book tells a deeply human story and will likely be an emotional rollercoaster for any readers who have undergone fertility treatments.

The book is rich with information on how patients approach, cope with and understand fertility treatments. Shannon Morell appears to have been a typical patient, but with deep religious convictions and a belief that life begins at fertilization. As a sideline to the story concerning the mix-up, she also delves into the story of one of her other embryos that did not make it to transfer or freezing. She was upset that an embryo remaining after an embryo transfer in an earlier treatment cycle was not frozen for later use. Chances are that this embryo had either arrested or was not of sufficient quality to tolerate freezing and thawing. This issue could have been resolved earlier if she had spoken with the MD or embryologist at the time, but she was not aware until much later that the embryo had not been frozen. Similarly, in the pregnancy that is the focus of the book, six embryos were thawed but only three were transferred. Little if any information seems to have reached Shannon on the fate of the other three. It is very likely that these three embryos did not survive, as the freezing technology in use at that time was not as good as what we use today. Understandably with this lack of information, the Morell’s were left with a feeling that mistakes were happening. In sharing their story with the media, they reasoned that perhaps the publicity would force other fertility clinics to be more careful about how they handle embryos, and think twice about the ramifications of their mistakes.

Being that medical care, including fertility treatment, is provided by humans to other humans, it is inevitable that random mistakes will happen in fertility clinics, medical offices, hospitals and every other workplace. However, the more we talk about them, study them and increase awareness, the less likely they are to be repeated. At PFC, we study all these cases in great detail, to see what we can learn, to find out if we are vulnerable to similar errors, and to modify our processes to ensure that we cannot make the same mistake. Unfortunately, with many of the errors that have occurred in IVF clinics, the staff appear to wait until the patient has had a pregnancy test to determine whether the embryo(s) have implanted, before disclosing the error. To me, this delay appears a further insult to the patients involved, since it may remove some of their options for remedy (such as taking the morning after pill to prevent the embryo from implanting). The gamble the clinic is taking is that the patient will not become pregnant and then perhaps the error will not seem so bad, or worse still, not be disclosed at all. The proper path is to fully disclose any mistake immediately after it happens, so that all parties can make fully informed decisions and have as many choices as possible. Mistakes, like secrets, only get worse with time, not better. And they never go away. Waiting makes mistakes worse, suggests deceit, and potentially ruins the lives of one or more families.

At PFC we have never had an embryo mix-up incident, and we have never inseminated a patient’s oocytes with the wrong sperm. In our laboratory, we have procedures in place that require a double witness of these and other critical steps in the IVF process. We are also very diligent about personally checking each patient’s identity when sperm or eggs come into the laboratory, and when embryos leave. Additionally, we review all our processes on a regular basis and look for new ways to improve. Interestingly, both the College of American Pathologists (our accrediting agency) and more recently the FDA, now specifically assess our procedures and processes for identifying and tracking gametes and embryos and our ID checks on patients. In fact, the FDA conducted an unannounced inspection here at PFC this summer specifically to look at this area of our practice, and we passed with flying colors. PFC will continue to demand improvements in our protocols and procedures, so that we will continue to avoid errors and continue to provide the same quality of care that we have given for the last 10 years.

By the end of the year we will have started a new and very exciting research project in our lab. We have partnered with a company called Incept Biosystems (www.inceptbio.com) to do a clinical trial of a new embryo culture system called microfluidics.

The traditional culture dish with medium droplets under oil as described by Brinster, R.L., 1963, Exp. Cell Res., Vol. 32

This involves culturing embryos in very small volumes of culture media inside a chip specifically designed for this purpose. Tiny pumps regulate the flow of culture medium in and out of the chip without causing the embryos to move around.

The traditional vessel for embryo culture is the petri dish, where small droplets of culture medium are overlain with a highly purified mineral oil. The culture medium, regulated in much the same way as pharmaceuticals, is one of the most highly tested and expensive components of the IVF laboratory operation. We typically make droplets of medium that are in the 50-200µl size range, and the oocytes or embryos are placed in the droplets for 24-48 hours at a time. This is a static culture system where nutrients are depleted by the developing embryos and waste products (e.g. ammonia from amino acid breakdown) accumulate over time. The droplets are large enough to make sure that the supply of nutrients is more than adequate and that waste is diluted to the point of not harming the embryo in any way. The embryos are changed into fresh medium at least every 48 hours.

This system for embryo culture has been in use since human IVF began in the late 1970′s and early 1980′s. It was actually developed in the early 1960′s by a pioneer of mouse embryo culture, Dr Ralph Brinster, at the University of Pennsylvania. Some early human embryologists cultured embryos in small test tubes without the mineral oil, but nowadays, despite the age of this technique, it is very unusual to find a facility that does not use the droplets under oil method. After 45 years, perhaps it is time for a change?

A microfluidic system for embryo culture has been in development for over 5 years at the University of Michigan in Ann Arbor. Professor Gary Smith combined the talents of his graduate students in physiology with those of engineering students and came up with a device that has had outstanding results with growing mouse embryos. Professor Smith is no stranger to IVF, as he was the director of the University’s IVF Laboratory for many years and he was instrumental in designing and testing the vitrification system that we now use to preserve oocytes and embryos. He solicited venture capital to start Incept Biosystems with the intent to bring microfluidics into human IVF labs. Incept Biosystems were onsite at PFC during the last week of October to train our embryologists on the use of the system. We did several trials with mouse embryos to achieve proficiency with the system and then we will actively recruit patients to enroll in a clinical trial using the system.

The clinical trials are being run at 3 centers in the US. In addition to PFC, patients will participate at the Fertility Center of San Antonio and at Southeastern Fertility Center in Charleston, South Carolina.

A schematic of a microfluidic embryo culture device with fresh medium in blue and spent medium in red. The embryo is contained at the base of the chamber, where the blue medium ends.

Patients that are asked to participate will have to consent to the study, where their embryos will be divided into 2 groups for culture in the microfluidic device and in the traditional petri dish. The culture media will be the same for all the embryos, but half will be in a replenishing media current (microfluidics) and half will be in our traditional static culture.

Microfluidics has had impressive results with mouse embryos where it significantly increased rates of development and implantation over those for embryos grown in static culture. Cell numbers for the microfluidic embryos were almost twice as high as for traditional culture (110 vs. 65), and pregnancy rates from transferred embryos were increased by 22%. Incept Biosystems have tested the new technology extensively and have been able to obtain surplus IVF embryos donated for research for human trials. There are some nice videos on their website that showcase the equipment and procedure, and detail the mouse embryo results. Professor Smith presented the results and won the prize paper at the 2008 American Society for Reproductive Medicine (ASRM) meeting (Smith et al., 2008, Fertility and Sterility, Vol 90, pages S1-S2), and these results will soon be published in a peer reviewed journal.

We will be asking for participants to join the study, beginning in November and continuing for 2-3 months. This is a short study requiring enrollment of only 20 patients, but a larger study is planned for next year subject to favorable outcomes here. If you are interested in the study and would like more information, please ask your physician at your next visit.

This summer, we are introducing a new procedure in our laboratory that will allow us to do genetic testing on embryos that have reached the blastocyst stage of development. Traditionally, embryos are biopsied when they are just 3 days old at which time they should have reached the 8-cell stage (see figure 1). The biopsied cell is sent to the genetics laboratory for testing while the remainder of the embryo continues to grow in our laboratory. The genetic testing results are received 48 hours later, when we hope that the embryo will have reached the blastocyst stage (see figure 2). Blastocysts that have passed genetic screening can be transferred or frozen for later use.

Performing the biopsy when the embryo has become a blastocyst is more technically challenging, and it allows less time for the genetics lab to do their testing. However, in a blastocyst, we are specifically able to biopsy from the part of the embryo that will become the placenta, and we can get more than 1 cell, which allows for greater accuracy in the genetic testing. Depending on how quickly the test is run, the embryo may have to be frozen while we wait for the results.

While freezing is inconvenient, it does allow time for more complex genetic testing, and for multiple tests if necessary. And, with the success of vitrification for preserving embryos (see Fertility Flash Vol. 7, Issue 3), we are confident that the frozen embryos will survive and implant at high rates when thawed.

In the next few years, we expect that the traditional methods for biopsy and genetic testing will disappear and that blastocyst biopsy will be the standard procedure. As genetic testing evolves, it will not be possible to rely on just a single cell from an embryo to get dependable results. We already know that there is genetic variability among cells in an individual embryo, a phenomenon known as mosaicism, and our new procedure will overcome this problem.

In the coming months, we will announce an exciting new partnership with a Bay Area genetic testing lab, and we will keep readers informed on our progress with genetic testing in embryos. This is an exciting field that continues to evolve.

In the press today we see that the “world’s oldest new mom dies” at age 69 (see our earlier blog post clarifying that PFC did not treat this patient), three years after giving birth to twins conceived through IVF. Maria del Carmen Bousada apparently lied about her age to the Los Angeles Physician who helped her become pregnant, creating a firestorm of criticism in the press.

The case demonstrates one of the most basic dilemmas that we face in helping women become pregnant: at what age is a woman too old to become a mother?

Most of us might agree that a 25 year old woman is young enough to receive help, but that a 70 year old is too old. However, drawing the cut-off line at some point between these extremes is not easy. With the help of in vitro fertilization and donated oocytes, women like Maria can become pregnant at an age where nature would naturally prevent the possibility of conceiving. Typically, women run out of oocytes in their early 50’s and without oocytes and the granulosa cells that surround them, they lose their ability to make estrogen. This natural process, called menopause, can happen earlier or later for a given individual, but the ability to get pregnant and deliver a healthy baby declines rapidly for women in their late 30’s and on into their 40’s. The age of the woman is a determining factor of her since a 40 year old woman is trying to get pregnant with a 40 year old oocyte, and these older oocytes don’t perform well. For example, the older oocyte is not good at keeping track of its own DNA, as evidenced by the increasing incidence of genetic defects such as Down syndrome in older mothers. And as if this wasn’t bad enough, the rate at which oocytes are lost from the ovaries (also know as a woman’s biological clock) doubles at about age 38. If this doubling didn’t happen, we think that women wouldn’t reach menopause until their early 70’s. It is thought that the speeding up of the biological clock in the late 30’s is nature’s way of clearing out the remaining oocytes, so that women lose their ability to become pregnant but are then around to raise the children that they already have.

Based on nature’s model, we might consider limiting IVF treatment to women that are in their early forties or younger. But with donated oocytes, this limit can be pushed and there are no legal age limits for pregnancy. So, who gets to decide when it’s too late to become pregnant? As far as following “nature’s model”, is age different than other factors that lead to infertility? Do we make rules? And do the rules apply to men too, where nature doesn’t have limits?

Note: Pacific Fertility Center does have both lower and upper age limits in place.

In January, Dr. Carolyn Givens and I attended a meeting in Hawaii organized by the American Board of Bioanalysts (ABB). This organization board certifies and licenses embryologists, andrologists, and a number of other laboratory specialists in the United States. Our meeting was under the direction of the College of Reproductive Biology, a special interest group within the ABB and for which I am the immediate past Chair.

The meeting was small and intimate, a situation always welcomed among reproductive biology professionals. The location allowed for good interaction with embryologists from Japan who have always been a great source of ideas and innovation within our specialty.

In fact, the highlight of the meeting was a series of videos shown by Dr. Yasuyuki Mio from the Mio Fertility Clinic in Yonago, Japan. He was able to take time-lapse cinematography of human embryos in culture, and as a result reported some novel observations on how oocytes fertilize and how embryos develop. The actual moment of sperm entry into the oocyte was recorded and it was possible to see that human oocytes form a fertilization cone (a membrane that helps bring the sperm into the oocyte), shortly after sperm entry. The events that follow (2nd polar body extrusion, which is the egg extruding a set of chromosomes, and pronuclear formation, alignment of the nuclei from the egg and sperm) occurred as expected, but for the first time the male and the female nuclei could be distinguished from each other.

After fertilization, the embryos were seen to change dramatically as they developed. In particular, they appeared more disorganized and untidy immediately after a cell division event and more symmetrical and organized several hours later. This discovery has implications for those embryos that sometimes may appear poorly. It suggests that they may look better later in the day when they are clear of the cell division process. Another important observation regarding blastocysts, is that those that develop 2 inner cell masses (ICM: the precursor cells of the fetus) do so in a predictable way. At PFC, we avoid using embryos with two ICMs whenever possible, as they are likely to lead to the formation of identical twins. A normal embryo should have only a single ICM. Currently, it is possible that one of the ICMs may be small enough to avoid detection. The observation was made that the fine cellular bridges within the embryo cavity appear to correlate to the presence of an extra ICM.

Another notable presentation was that of Dr. Tetsunori Mukaida, of Hiroshima HART Clinic, on sperm morphology. He demonstrated that observing sperm under ultra-high magnification can show structural defects that are not always visible when using standard microscopes. While magnifying sperm thousands of times has its difficulties, Dr. Mukaida reported that sperm with subtle physical defects have a much lower chance of making an embryo that can become a baby. Sperm that are close to perfect in size, shape and structure are difficult to find in any sperm sample and it can take hours just to find a few ideal sperm. However, the extra effort may be worthwhile, especially in patients that have had a previous IVF cycle where the embryos did not develop well or implant after transfer. PFC is currently looking into this technology and we will report more details in a future issue of Fertility Flash.

Attending meetings like this and keeping up with the latest developments in our field is an important part of the culture at PFC. We share the load of traveling to educational events and are always excited to bring home ideas and thoughts to share with our colleagues. PFC is committed to implementing the latest technology and innovations to maximize pregnancy rates for our patients. We will continue to stay updated with all of the research and development in our specialty.

Both Dr. Givens and Dr. Conaghan contributed to this article.

	Joe Conaghan, Ph.D., HCLD is PFC’s laboratory director. Dr. Conaghan is internationally recognized for his work on improving embryo culture conditions. His interests include developing programs for the treatment of severe male factor infertility; diagnosis of genetic disease in embryos; and improved embryo culture. See more posts
	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. See more posts

In October of 2008, after many months of planning, PFC began construction on our new lab. The design called for an environmentally friendly facility that was bright, open, and efficient for our 8 embryologists and the many thousands of embryos that we care for each year. The size of the lab was doubled to allow for the addition of more embryo incubators and ensure room for future growth.

Traditionally the embryology lab is an area that is not accessible to patients or visitors, but our new design utilizes glass walls in key areas and makes our activities more transparent to the outside world. While the lab remains a secure area with limited access, the activities inside can be observed from the outside by anyone passing through our facility. This openness is important to all of us at PFC; we want to remove any mystery associated with the IVF lab, and allow patients free “visual” access. Large TV monitors are installed above several of the microscopes to further open up the world of IVF. We are proud of the work we do at PFC and we want to share.

While the glass walls are largely a cosmetic change, almost every other part of the new lab was designed with the health of our embryos in mind. The installation of a specialized and custom-designed air filtering unit consumed over 20% of the budget for the project. Our goal is to have highly purified air circulating in the lab. The new air handler achieves this goal with the use of a series of filters that remove all particles and chemicals from the air. The lab is further protected from the outside by two separate air lock doors that use positive pressure from the inside to the outside to keep unclean air out.

All supplies and consumables for the lab are handled by the embryologists only, who also take responsibility for all cleaning and other custodial functions. We empty our own trash and wash our own floors. These precautions are in place not only to keep custodians and other building staff out, but also to control the chemicals and cleaners that might con- taminate our clean environment. All com- pressed gases (which feed our incubators) and liquid nitrogen (for our frozen embryo tanks) is piped in from outside the lab, so that it is not necessary for delivery people to enter the lab.

Our incubators in which the embryos develop are fundamentally the most important pieces of equipment in the lab. These incubators are monitored, serviced, and maintained by the embryologists, who have specific training in the use of all of our equipment. Quality control checks are exhaustive and performed daily to make sure that all equipment is functioning exactly as specified. A change of even a half degree in temperature could cause problems in an incubator, so monitoring is continuous and detailed. The gases that are piped into the incubators (carbon dioxide and nitrogen) are filtered as they enter the lab to make sure that they are pure.

The laboratory is supplied with emergency power from a large dedicated back-up generator located at the side of our building. Should there be a major power outage this generator produces power for at least 36 hours before it needs additional diesel. Our generator has proved itself many times over during the years of rolling black outs and other power failures.

We continue to upgrade our facility and maintain the standards of excellence that makes PFC the choice of patients. At the time of writing, we are working toward installing a second back-up system in the event that there is a power outage and our back-up generator fails. We are also always examining new equipment that will allow us to offer new technologies to patients. As we move forward, we will report our latest developments at PFC in the Fertility Flash. In the meantime, come by and visit with the embryologists through the glass. We won’t wave at you, but we’re happy to see you checking up on us. We want you to know that your embryos are in good hands.

To schedule a tour contact one of our New Patient Coordinators at 888-834-3095.

Participants in a workgroup.	Practicing micromanipulation

On November 8 th and 9 th, 2008, PFC held a training course for embryologists interested in learning the techniques associated with Preimplantation Genetic Diagnosis (PGD).

The course was organized in conjunction with the Genetics and IVF Institute (GIVF) of Fairfax, Virginia and was originally scheduled to run on the Sunday only. However, due to the overwhelmingly positive response to attend the course, PFC decided to offer the course on Saturday as well. Forty seven individuals heard excellent lectures over the 2 day period.

Dr Dagan Wells from Oxford, UK and Dr Alan Thornhill from London, UK, gave talks on current and future technologies for genetic testing on embryos. Participants were then divided into 4 working groups that spent the rest of the day rotating between activities. The activities included embryo biopsy training, cell fixation training, media and solution making and PGD troubleshooting. Lauri Black, MS, CGC, a Certified Genetic Counselor at California Pacific Medical Center and Mary Sands of GIVF gave talks on genetic counseling. The course also allowed embryologists from all over the world to view the new state of the art laboratory at PFC.

Joe Conaghan, PhD, HCLD