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

ASRM 2011 Updates

In addition to the magical wonders of Disney, Orlando welcomed reproductive endocrinologists from around the world this October to attend the annual meeting of The American Society for Reproductive Medicine (ASRM).  Several members of Pacific Fertility Center were among the participants. 

Preimplantation Genetic Screening (PGS)

PGS was again a hot topic of discussion.  Multiple presentations showcased the recent technological advances in this field.  The ability to perform comprehensive chromosome analysis using microarray technology instead of the first generation method of FISH (fluorescent in situ hydridization), which could only test a selected number of chromosomes at a time, has increased the accuracy and the detection rate of embryonic aneuploidy (abnormal number of chromosomes).  Laboratory advances such as biopsy of the trophectoderm (the outer cell layer of a day 5 embryo) and vitrification (a method of rapid cooling of embryos that minimizes ice crystal formation) have further improved success.  As the result of the above-mentioned technical breakthroughs, we have seen a measurable increase in the pregnancy rate and a decrease in the miscarriage rate from IVF using PGS.  Additionally, two respected groups independently presented data supporting the use of PGS as a successful embryo selection tool to promote elective single embryo transfer (the process of transferring one embryo at a time into the uterus to reduce the risks of multiple gestation).  The pregnancy rates from a single PGS-selected euploid embryo were 58% and 60.7% compared to 42% and 40.7%, respectively, from a morphologically comparable but non-PGS-selected embryo.  Moreover, the miscarriage rates decreased to 6% and 6.3% from 12% and 12.5%, respectively.  The risk of multiple gestation was essentially eliminated (1-2% monozygotic twining).

We were excited to note the parallels between the data presented and our own work at PFC.  Several years ago, we made the commitment towards decreasing our multiple pregnancy rates by adopting a policy of encouraging elective single embryo transfer in qualified patients.  We have found that 24-chromosome aneuploidy screening (via informatics-based single nucleotide polymorphism microarray technology by Gene Security Network) of trophectoderm biopsy has significantly enhanced our ability to select the embryo with the best implantation potential.  Our improved vitrification program has also allowed us to reassure our patients that their unused embryos can be safely stored for future use, thus removing the pressure to transfer more embryos at one setting.  We are very proud of our success so far in achieving our goal as we are currently the number one ranked program in the nation of the fewest number of embryos transferred in donor cycles (1.4 embryos per fresh cycle) while maintaining a high pregnancy rate of 57% (of all programs with more than 20 donor cycles per year, 2009 SART).  For more details on our experience with single embryo transfer and its pregnancy rates, please read “What are my chances of having a baby from a single IVF cycle” by our embryologist, Erin Fischer, and laboratory director, Dr. Joe Conaghan, in this issue of Fertility Flash.

Fertility Preservation

Another interesting topic that deserves attention is fertility preservation using oocyte cryopreservation. Two centers with extensive experience in this area shared their outcome data from both methods of cryopreservation, slow freeze and vitrification.  A center in Atlanta vitrified over 2000 oocytes from donors with an average age of 26 years.  Of the 1772 oocytes rewarmed, 88% survived, 75% fertilized, and 51% resulted in viable cleavage stage (day 3) embryos.  Live birth rate per cryopreserved oocyte was 11%.  The other presentation by a group in New York reported their experience of rewarming 536 cryopreserved oocytes using both slow freeze and vitrification from non-donors with an average age of 32 years.  The overall live birth rate per rewarmed oocyte was 5.5%.  Study is ongoing to compare the efficacies of slow freeze and vitrification.     

PFC’s own data with vitrification of oocytes is comparable to, if not better than, the results presented at our national meeting by various groups across the US.  A 5-10% live birth rate per oocyte in women under the age of 35 years translates to a respectable chance of having a baby in the future from one to two treatment cycles in the present (10-20 oocytes can be expected to be cryopreserved per cycle).   As we further perfect our own techniques of vitrification, we will be increasingly more confident in our ability to offer young women with a viable option for future family planning in addition to embryo freezing and donor gametes.  Future research is needed to achieve the same type of success rates in older women.   

Participating at ASRM is always an educational experience.  We enjoyed sharing our own clinical and research endeavors with our colleagues across the US and all over the world.  Our position as the nation’s leader in many of the most cutting-edge technologies in our field is a validation of our commitment to excellence and to provide our patients with the highest quality care available.

Embryo freezing is a routine part of the IVF process.  Approximately 60% of patients have embryos in frozen storage after their cycle is complete.  These embryos can be used at any time; but it is common that some embryos remain after couples have completed their families.  This situation leaves patients facing a very difficult decision regarding the final disposition of any embryos still frozen.  Quite often patients are not prepared to make such a decision, nor are they aware of their disposition options. Patients were so focused on simply getting pregnant, they had not considered what to do with any remaining embryos after the cycle was complete. This article provides a brief explanation about the three disposition options available at PFC for surplus frozen embryos: disposal, research (and then disposal), or donation to another couple for use in achieving pregnancy.

Disposal of your embryos means they are removed from the storage tank and placed in a biohazard waste disposal container. Once the embryos are removed from the liquid nitrogen storage tank, they lose all viability in a matter of seconds. The embryos are not used for research purposes, not donated to any individual or company, and are not cultured beyond the stage of development at which they were frozen.  They are disposed of as medical waste.

Donating your embryos for use in research requires that the embryos be shipped to a company called Reprogenetics, LLC, based in New Jersey (www.reprogenetics.com). At Reprogenetics, the embryos are studied to understand normal and abnormal development.  Donating embryos specifically for stem cell research is also possible.  Reprogenetics offers a stem cell research option, however,  some additional paperwork must be completed directly with Reprogenetics  Whether donating to Reprogenetics for stem cell research or basic research, a PFC Research Disposition form must be competed.

Donating your embryos for use by another couple can be broken down into three sub-categories: known donation, open donation and anonymous donation. Known donation, also called directed donation, is the donation of your embryos to a person or couple that you know personally, perhaps a good friend or family member.

Anonymous donation of your embryos means that you donate your embryos to an organization, and the organization places your embryos with a family that you do not know and will not meet. The identity of both the donors and the recipients is not disclosed to either party. Through the PFC Embryo Placement Program, only anonymous embryo donations are accepted. Any stipulations about to whom or to what type of family situation the embryos are donated cannot be accommodated (i.e.: that the embryos be donated to a two-parent household, or a household of a certain income level, or living in a certain geographic area). The placement of anonymously donated embryos operates on a first-come, first-serve basis. At the moment, we have a very long list of patients wishing to receive donor embryos. Currently there is nearly a two year wait).

Open donation is the donation of your embryos to a party that you do NOT know, but wish to meet, and/or possibly remain in contact with, after the embryos are donated. Open donations require further legal expertise and overall guidance and handling beyond PFC’s current abilities. For these reasons, PFC is unable to offer open donations to our patients. For those interested in an open donation, or for those requesting certain criteria be met by the recipients, patients are encouraged to research third party agencies that facilitate embryo donations, both anonymous and open. One such program is the Snowflakes Frozen Embryo Adoption and Donation Program (www.snowflakes.org), operated by Nightlight Christian Adoptions. Snowflakes facilities both open & anonymous donation of embryos and can accommodate most requests from the donors and the recipients. Another possibility is Miracles Waiting (www.miracleswaiting.com), an online do-it-yourself matching program for donors and recipients. More general information about embryo donation and adoption can be found at the National Embryo Donation Center (NEDC): www.embryodonation.org.

At PFC, all embryo dispositions are handled by our tissue bank manager Alexis VonAustin.  Her contact number is 415-249-3636. She can assist you with information, paperwork, and if necessary, with the shipping of embryos to the agencies listed above.

- Alexis VonAustin and Joe Conaghan, Pd.D., HCLD.

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.

Last November, PFC embryologist Erin Fischer had the opportunity to travel to Cairo, Egypt for the 16th Annual Meeting of the Middle East Fertility Society (MEFS). At the meeting, she presented an abstract on our vitrification results at PFC and also assisted with a vitrification workshop. She met many remarkable embryologists from all over the Middle East while teaching the embryo vitrification and sharing the successes that PFC has had with vitrification.She also had the chance to visit three IVF clinics in Cairo during her stay. “Seeing other IVF labs was the highlight of my trip, I was impressed by the large number of cycles that these teams performed daily,” comments Erin. She adds,“Traveling to Egypt was an amazing experience. I was proud to represent PFC and appreciate our state of the art facility.“

Every year thousands of families are created with the assistance of in-vitro fertilization. Many of those newborns are twins. While some may see this as a double blessing, it is important to understand that there are many potential risks associated with multiple gestation. Statistics show that a higher percentage of twins are born prematurely compared to singleton pregnancies. Premature birth can cause complications resulting in physical impairment, learning disabilities, and even death. In addition to the increased risk to the children born from a multiple pregnancy, there is also an increased risk for the pregnant woman of complications associated with carrying multiples.

Pacific Fertility Center (PFC) has been taking steps to minimize the risk of multiple gestation for several years. “We have worked actively to increase pregnancy rates and decrease the number of multiples,” comments Carolyn Givens, M.D. “Balancing high pregnancy rates with low pregnancy risk improves pregnancy outcomes. Our goal is to achieve this balance and reduce the risk of multiple gestation.”

PFC recently completed the analysis of our Elective Single Embryo Transfer (eSET) program for 2009. The twin rate was significantly lower, and, triplets were eliminated entirely. 79 patients underwent an embryo transfer procedure where they elected to transfer only one embryo created from their own eggs; these 79 transfers resulted in 38 pregnancies, two of which were identical twin pregnancies (the embryo split from one into two) and NO triplets. Compare this statistic to patients choosing to transfer two embryos: 159 patients, with embryos derived from their own eggs, transferred two embryos resulting in 80 pregnancies, of which 31% were non-identical twins and two triplet pregnancies (again from one of the embryos splitting).

Patients that choose eSET have excellent pregnancy rates with a single embryo. eSET embryos are grown for 5 days in the lab to the blastocyst stage, which allows for selection of the healthiest embryos for transfer. The transfer of fewer embryos provides for the healthiest outcomes; eSET produces high pregnancy rates while minimizing the risk of multiple pregnancy. “For many patients, there is no advantage to transferring more than one embryo. It is all about educating our patients. Given this information, these numbers and the potential risks of twin pregnancies, many will choose to transfer only one embryo,” says Carolyn Givens, M.D.

At PFC, careful consideration is given to the number of embryos transferred to each patient. Our goal is to create healthy singleton pregnancies. Utilizing advanced embryo culture techniques, the highest quality embryos can be selected for transfer. Special environmental conditions, advanced culture media, and the delicate handling of gametes and embryos is required; these efforts result in better embryos, with higher implantation and pregnancy rates.

In addition, PFC has developed an outstanding and robust program for freezing embryos not transferred in the fresh cycle. Using a technology called vitrification, we have been able to achieve pregnancy rates with frozen embryos that are very similar to those using fresh embryos. “The outstanding success of our freezing program has allowed us to be confident in transferring just one embryo at a time, which all but eliminates the risk of triplets or higher pregnancy,” says Dr. Joe Conaghan, PFC Lab Director. He adds, “We have been so successful with embryo freezing over the last 3 years that our embryologists are in high demand to provide training across the country and around the world. Our goal is to help our patients overcome infertility and build their family; one healthy baby at a time.”

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.

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

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

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

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

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

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

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

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

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

Question: I am an educator for a human sexuality class. A student asked me an interesting question that I was unsure how to answer. Given that we know sperm can survive about 72 hours in a woman’s body, how is it possible to keep sperm viable by freezing them?

Answer: Sperm can survive for a long time under the right circumstances. In a woman’s body we think that 72 hours is approximately correct, but the data supporting this estimate is not conclusive. In the lab, sperm can live 5 days or more provided they are removed from the seminal fluid and placed in a more nurturing environment. Seminal fluid contains many enzymes that first clot and then liquefy. This change in the fluid allows the ejaculated sperm to stay in the vagina initially, but then swim out as the seminal fluid becomes more liquid. These enzymes quickly destroy any sperm that can’t swim out of the semen within a few hours.

It takes approximately 72 days for sperm to mature in the body. During the last 14 days of this process, the sperm are very much alive and swimming. They are alive a long time prior to leaving a man’s body.

During freezing, sperm are cooled to a very low sub zero temperature (minus 196 degrees Centigrade). At that temperature, all biological activity is effectively stopped. The sperm cells are not metabolizing or depleting their energy reserves. They are truly in suspended animation. Bacteria or other microbes cannot attack or degrade the sperm in any way because they are also unable to function at such a low temperature. Everything is on hold.

Biologists believe that correctly frozen cells in long term storage can literally last forever, as long as the temperature is properly maintained. It is believed that constant exposure to normal levels of background radiation is the only thing that could cause loss of viability and this effect is difficult to measure. Studies done in the 1970’s, exposing frozen mouse embryos to the equivalent of 2,000 years of background radiation, showed no measurable mutagenic effects in offspring.

Cryobiology is a relatively new science, and human fertility treatments are newer still. Consequently, in humans there are no long term results with frozen sperm or embryos. There are a handful of reports showing babies born from embryos that had been frozen for 12-15 years. A couple in New York had a child in 2005 from sperm that had been stored for 28 years. Sperm frozen for domestic animal species have a longer record because samples frozen in the 1950’s are still viable.

The process used for freezing is very precise and works best when cells exist individually (such as sperm) or in very small groups (such as an embryo). Larger masses of cells, tissues or even whole bodies cannot be frozen and subsequently thawed alive. It is not currently possible to freeze and thaw a whole ovary or kidney.

To successfully freeze cells we must remove cell water (water expansion during freezing would burst the cell) and replace the water inside the cell with antifreeze. This is done by incubating the cells in a solution of antifreeze. The water and antifreeze swap places through the process of simple osmosis. In a complex tissue like an ovary, there is no way to get all the water out of all of the cells so easily, thus a whole ovary cannot be frozen. If the ovary is chopped up into tiny pieces however, more water can be extracted. Some success has been reported with freezing ovarian pieces in this way.

The following student experiment demonstrates the challenges of freezing. Place a whole peach into your freezer for 24 hours and then thaw it out and see what a mess you have. If however you slice the peach up and mix the slices with sugar for 15 minutes (the sugar will draw out water from the cells), you can freeze the peach quite successfully. If the technology is used correctly, you can keep your peach (or your sperm) for leaner times.

Joe Conaghan, PhD, HCLD