Since Pacific Fertility Center came into existence in November of 1999, we have been offering genetic pre-screening of IVF embryos for couples with recurrent miscar- riage, repeated IVF implantation failure and sex selection for family balancing. For most of the last decade, a technology known as Fluorescent In-Situ Hybridization, or FISH has been used to screen embryos. FISH is employed to probe a cell removed from a Day 3 embryo to determine the chromosomal makeup for anywhere from three to twelve of the cell’s 23 pairs of chromosomes. With time, we, as well as everyone else in the reproductive genetic world, came to realize the serious limitations of this technology.

In the last 2-3 years, as the Human Genome Project has been completed and as more DNA-related biotechnologies have emerged to evaluate human genes, these methods are being utilized to analyze human embryos. The technology now available—the ability to analyze large numbers of genetic locations on each human chromosome, and quantify that genetic material, with the previously well-established techniques to amplify a single cell’s genetic material up to hundreds of thousands of copies—has allowed PGS to take a quantum leap forward. It is now possible to more accurately analyze all 23 chromosome pairs from a single embryo; not only to determine if the correct number of copies of each chromosome is present, but also to look at single gene mutations.

At the end of 2009, Pacific Fertility Center began working with a new biotech company called Gene Security Network, located in Redwood City (genesecurity.net). This company uses gene microarray technology to analyze amplified DNA from a single cell.

It then uses microchips to analyze 30,000 genetic loci in a quantitative manner. In addition, their unique technology allows us to compare the analysis of the embryos’ cells to the parent’s chromosomes to ensure that all the genes are being properly analyzed. It does appear that the error problems that plagued FISH technology have been overcome with this new, more sophisticated, method.

In October of 2009, Dr. Conaghan and I were invited to tour the GSN laboratory and see the technology in action. We met with David Johnson, the lead scientist at GSN, who explained the cell process; from the amplification of the DNA, to arranging the chromosomes on chips, to DNA analysis, to synthesizing the data generated with the parental genetic data to come up with a full analysis of that cell’s genome. In order to process the cells between the day of embryo biopsy (Day 3) and receive the results on the day of embryo transfer (Day 5), their technicians work around the clock in shifts. GSN has a very cold, clean room to replicate the single cells into multiple copies. They cannot allow any outside contamination, not even from a single cell. They videotape the cell duplicating process so if any errors subsequently arise, they have a video record of what the laboratory technician did. We found this to be very impressive. We also saw how the chips were coated with DNA and analyzed. We were shown the sophisticated software that generates the final report detailing the genetic makeup of each embryo from the cells in which they originated. All in all, the tour gave us great confidence in the quality control and scientific integrity at GSN.

Even with this 21st century technology, we continue to biopsy Day 3 embryos because it provides us with a 48 hours window to send the cells to the lab and complete the analysis in time for transfer. However, we have not yet found a way around the problem of mosa- icism. GSN and microarray technology appears to have largely solved the resolution error problem but it can only tell us what is in the chromosomal make-up of the single cell. It cannot tell us whether or not that cell represents what is truly going on with the rest of the embryo. We are currently looking at the possibility of biopsying Day 5 embryos. The set back would result in having to freeze these embryos due to the time constraint in analyzing the genetic material in time for fresh transfer. With all of the innovation occurring daily in the genetics field, we hope that this puzzle will be resolved.

— Carolyn Givens, M.D.

Previous Fertility Flash articles about PGS:
2 Methods of Gaining Info Prior to Implantation
PGD & PGS: Why Genetic Counseling is a Prerequisite
The Benefits and Pitfalls of PGS

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 past summer, I had the opportunity to travel to Amsterdam, Holland for the annual meeting of the European Society for Human Reproduction and Embryology (ESHRE). Though largely attended by Europeans, this scientific meeting draws physicians, embryologists and scientists from around the world to discuss their research, attend courses and lectures, and discuss the latest topics in our field. Although I don’t think this year’s meeting was as quite as good as last year’s ESHRE in Barcelona, there were still some good learning opportunities. Here are some of the highlights of the meeting:

“From Gamete to Heartbeat: The Missing Link”

This was a post-graduate course offered in conjunction with the meeting. The course covered sperm and egg evaluation,

expression of genes in the early embryo and in the endometrium (uterine lining) and some of the latest research into basic embryo implantation mechanisms.

One of the interesting talks was on gene expression in the early embryo. We have come to believe that the differences in pregnancy rates between younger and older women is mainly due to an increase in the number of abnormal chromosomes in embryos from women as they age (such as increased risk for Down Syndrome). However, this only explains part of the differences in successful pregnancy in younger compared to older mothers. New research into expression of proteins from embryonic genes is showing that in both chromosomally normal and abnormal embryos, there are differences in the number and types of genes encoding proteins in younger and older women. This suggests that it is not just changes in the number of chromosomes but subtler differences in the way individual genes are being expressed that affect the developmental competence of their embryos. Determining which genes and proteins are involved, and what the mechanisms are for regulating the expression of these genes in early embryos, will be an area of focused research in the coming years.

“Hyaluronic Acid (HA) favors selection of spermatozoa with intact DNA and normal nucleus, resulting in improvement of embryo quality” (Bologna, Italy)

This presentation (Parmegiani, et al.) looked at the percentage of sperm showing DNA fragmentation based on several methods of sperm preparation for IVF-ICSI (in vitro fertilization with intracellular sperm injection). They compared sperm in the fresh specimen 30 minutes after ejaculation, sperm that had been processed with a standard “swim-up” technique, and sperm that were placed in PVP (polyvinyl propylene), a substance used to slow sperm down so they can be picked up from a culture dish just prior to injection into the eggs. Lastly, they looked at sperm that had been placed into dishes that contain a ring of hyaluronic acid at the bottom of the dish, a substance to which some sperm will automatically bind. They looked at the percentage of sperm showing total or partial fragmentation of the DNA with each of these steps in the sperm preparation process. In the freshly ejaculated sperm, the DNA fragmentation was 16.5% of tested sperm. In the “swim-up” sperm prep, 11% were fragmented and in the PVP-exposed sperm, it was also 11%. Sperm that had bound to hyaluronic acid showed the least amount of fragmentation, at 5.3%.

These findings suggest that using HA binding to select sperm for sperm injection may result in fewer abnormalities in embryos, and possibly higher pregnancy rates. PFC is currently investigating HA binding on our own to see if it is something we would wish to routinely incorporate into IVF. The downside (like everything else!) is that HA plates are expensive.

Stress and Fertility – an enlightening symposium

Jacky Boivin, PhD., a researcher from Cardiff University in Wales, presented some very interesting data about the stresses of infertility treatment. She discussed a new study from Alice Domar’s group in Boston that surveyed why women/couples discontinued IVF treatment before achieving pregnancy (Fertility and Sterility, in press 2009). In this study, 132 women who had insurance coverage for IVF were surveyed. The two main reasons given for dropping out of treatment were the toll that infertility took on the couples’ relationship and being too anxious or depressed to continue. Among the less common reasons for dropping out were medication-related issues (such as difficulty with injections) and feeling the need for a female doctor. Dr. Boivin also discussed results from her own study that was published in the journal Human Reproduction in 2008. In that study, she developed a copingstratagem for women awaiting results of their treatment (i.e. the time between embryo transfer and first beta hCG). It is known that this is a most anxious time for women and the stress of waiting can become overwhelming. She utilized something called the “positive reappraisal coping intervention” card, or “PRCI” card. This is a small printed card that a patient can carry around in his or her pocket and it is meant to be read 2 times per day, every day during the 9-11 days between embryo transfer and first pregnancy test. The card has several little sayings such as: “During this experience I will try …to do something that makes me feel positive” and “During this experience I feel that….I’m energized or I’m creative.” This is a way of programming thoughts towards the positive and away from the negative. She and her colleagues were able to show that patient felt less stressed and felt that the PRCI was helpful during this period.

Currently, at PFC, we have begun a task force to look into ways to better incorporate counseling and tools for stress management for our patients. Please also see this recent Patient Odyssey. Support groups are a wonderful way to diffuse stress and feel more positive.

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

As most people know, the medication we most commonly use for fertility treatment, Follistim, is pure human FSH, manufactured using recombinant DNA technology. The company that makes Follistim, Schering Plough, is working towards FDA approval of a modified version of Follistim, called Corifollitropin, that will make the drug very long-acting.

For those interested in the details; Corifollitropin is the recombinant FSH molecule + 22 C-terminal peptides from betahCG. It does not bind to the LH receptor. This modification lengthens the half-life of Follistim from 22-34 hours to 60-74 hours for Corifollitropin. The recommended regimen will be one dose per week, starting at baseline, then switch to daily recombinant FSH after that. After injection, peak levels are reached in 2 days then they slowly level. It may be possible to only take one injection per week!

A symposium at ESHRE presented information from the ENGAGE trial with data from 14 European and 5 Asian IVF centers, using women with body mass indices (BMIs) between 18 and 32 (generally less than 60 kg -132 lb). The patients were randomized to receive either Corifollitropin or conventional daily recombinant FSH for oocyte recruitment. The number of days of stimulation was the same in both groups (9). The number of eggs retrieved was significantly higher in the Corifollitropin group (13.3) vs. the FSH group (10.6). The rates of ovarian hyperstimulation syndrome were the same in both groups (about 3%). The pregnancy rates were 25% in the Corifollitropin group and 34% in the FSH group, a difference that did not quite reach statistical significance.

Data were also presented on a second study of Corifollitropin from the U.S. and Europe, comparing two doses of the drug. In the study, 100 mcg/dose was given to women less than or equal to 60 kg and women greater than 60 kg were dosed at 150 mcg. Over 1500 patients were included in this large trial. In this study, the average number of eggs recovered was 13.7 for the Corifollitropin group and 12.5 for the Follistim group. The mature egg and fertilization rates were the same. The percentage of good quality embryos was the same.

The clinical pregnancy rate in the Cori group was 38.9% and was 38.1% in the Follistim group. These rates were statistically the same. We expect that Corifollitropin will likely be available in the U.S. in 2010 or 2011.

Many populations of the world have specific genetic conditions that are prevalent within their ethnic group. Consequently, numerous medical organizations have recommended that genetic screening for these conditions should be offered when women are either planning for, or are currently pregnant. We are all carriers of genetic conditions: generally this is of little concern, as it is highly unlikely that we would have children with a partner who is a carrier for the same condition.

The genetic conditions listed in the table below are recessive. A recessive gene mutation is “carried” by someone who is unaffected by the disease, and thus unaware of their carrier status. Men and women have equal potential to be carriers for recessive conditions. Even if someone is a carrier, we would not expect to see a family history of the disease. If there is a family history, the likelihood of being a carrier of that condition is generally greater than in the general population. Being a carrier for a genetic condition typically has no impact on the carrier’s health and development. However, if a carrier has a child with another carrier of the same genetic disease, the chance that the child will be affected with the disease is 1 in 4 (25%).

If only one partner is a carrier, and the other tests negative, then the risk of an affected child is low, but not zero (a result of the limited ability to test for all defects that would make one a carrier; see table). These genetic screening tests are typically performed on a blood sample.

Below is a table listing the minimum number of tests for various ethnic groups recommended by the physicians at Pacific Fertility Center prior to starting assisted reproduction treatment. Additional genetic tests may be considered after a discussion with your physician.

If you know that both you and your partner are carriers of the same genetic defect, you may be able to have embryos created in an IVF cycle and tested for their status. Preimplantation genetic diagnosis (PGD) is a technology that allows embryos to be tested for specific disease causing mutations. PGD can identify unaffected embryos for transfer back to the uterus or freezing.

—Guest Contributor – Certified Genetic Counselor Lauri Black, M.S., C.G.C

*See lab specific accuracies on test result

It is always frustrating, both for patient and physician, when we perform a full fertility evaluation and conclude that that patient’s diagnosis is “unexplained infertility.” But unexplained infertility does not mean “no” infertility. It means that we recognize there is some abnormality or inefficiency in the process of getting pregnant, but that we do not have a good test to assess what is the actual problem.

There are many critical steps in the process of getting pregnant which we can not directly assess; such as how efficiently the eggs are captured by the fallopian tubes, how efficiently the tubes push the eggs along to the uterus, how well the sperm are fertilizing the eggs, how well the fertilized eggs are developing to embryos, and how efficiently the embryos find their way to the uterus. All these steps are critical—yet we have no direct test. If one were to proceed to an IVF cycle, we can then make some assessment of these steps, but not prior.

So, if the evaluation shows normal egg quality (FSH, Estradiol, ultrasound), dye study (HSG) shows open tubes, sperm test shows normal count and motility, and there is no evidence of endometriosis—this typically defines unexplained infertility.

One of the first studies addressing the efficacy of treatment for unexplained infertility was published in 1998 (1), and indicated that patients who underwent no treatment had a 1-4% pregnancy rate per month; insemination alone, 3.8% pregnancy rate/cycle; clomid alone, 5.6% pregnancy rate/cycle; Clomid plus insemination, 8.3% pregnancy rate/cycle; gonadotropins alone, 7.7% pregnancy rate/cycle; and gonadotropins plus insemination 17.1% pregnancy rate/cycle. This study was retrospective, and did include patients with mild endometriosis, so while providing good insight, more studies were needed to better define treatment efficiency for these patients. However, it was important to see that insemination alone did not provide an improvement, and best improvement was noted with combination therapy.

Next, Guzick and colleagues published a large randomized study addressing superovulation with gonadotropins and insemination, for patients with unexplained infertility (though again mild endometriosis was included) (2). The control group was patients who had intracervical inseminations (to assure sperm exposure). Pregnancy rates for this group were 10% per cycle. Those who underwent intrauterine insemination had an 18% pregnancy rate. Those who underwent gonadotropin stimulation plus intracervical insemination have a 19% pregnancy rate, verses 33% for those with gonadotropin plus intrauterine insemination. Therefore, couples who undergo superovulation and intrauterine insemination have a 3 times higher chance of achieving a pregnancy, than the control group.

The question of which type of ovulation induction agent to use, oral verses injectable, has been addressed in a meta-analysis published in 2002 (3). This review of 5 randomized controlled trials shows that pregnancy rates were higher in injectable cycles (gonadotropins), though live-birth rates were not different between oral and injectable cycles. Their conclusion was that oral agents may therefore be more suitable for ovulation induction, since the multiple rates were lower, and cost of the cycle less.

Last year, a prospective trial looking at using oral Clomid verses Letrozole with inseminations in patients with unexplained infertility was published (4). This study showed that the total number of follicles was greater for the Clomid users (3.1 vs 1.6), but the pregnancy rates were the same for each group (Letrozole 19%/cycle verses Clomid 18.3%/cycle). Therefore, both agents are equally effective, and the multiple rate may be less with Letrozole.

Last year, another randomized control trial evaluated expectant management (no treatment), verses Clomid alone or intrauterine insemination alone in patients with unexplained infertility ( though included mild endometriosis patients) (5). This study again confirmed that Clomid alone (14% live-birth rate) or insemination alone (23% live-birth rate) was not statistically different than expectant management (17% live-birth rate). They evaluated patient satisfaction with the treatment process, and patients who were randomized to the “expectant management” arm were much less satisfied than those who were doing Clomid or insemination therapy, despite no improvement for those patients’ live-birth rates.

So, some take home points are as follows:

• Unexplained infertility does not mean “no” infertility
• Empirical clomiphene and/or unstimulated intrauterine inseminations are unlikely to offer an improvement over expectant management (no treatment)
• Best options are to consider Clomid or gonadotropins with intrauterine inseminations (depending on patient age, etc..)
• If this fails, then IVF is best option
• Depending on age and other evaluation, IVF may be best first option

Footnotes:

1. Guzick DS, et al. Efficacy of treatment for unexplained infertility. Fert Ster 1998; 70:2, 207-213.
2. Guzick DS, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. N Engl J Med 1999;340;177-83.
3. Athaullah N, et al. Oral vs injectable ovulation induction agents for unexplained subfertility. Cochrane Database Syst Rev 2002;3:CD00352
4. Badawy A, et al. Clomiphene citrate or aromatase inhibitors for superovulation in women with unexplained infertility undergoing intrauterine insemination: a prospective randomized trial. Fert Ster Aug 2008
5. Bhattacharya S, et al. Clomiphene Citrate or unstimulated intrauterine insemination compared with expectant management fro unexplained infertility: pragmatic randomized controlled trial. BMJ 2008;337:a716

A human embryo

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

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

Frozen embryos are stored in liquid nitrogen

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

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

The article in January’s issue of Fertility Flash, Conception at 40 and Beyond – Does IVF Help? contained some errors in the table. The following is a reprint of the article with corrections.

We all know that fertility declines with female age but what is not certain is how much does in vitro fertilization improve one’s chances of conception if a woman/couple is having problems conceiving on their own?

The table below is one I often use when counseling patients 40 and over about their chances of conception with in vitro fertilization.

This table represents pregnancy outcomes with PFC patients from January 2003 to March 2008, so most of the viable pregnancies tabulated here have been delivered.

One thing to note is that over half of the patients that get a positive beta-hCG result do not end up delivering a baby. This is consistent with the observation that most embryos from women 40 and over have abnormal numbers of chromosomes.

Another thing to note is that pregnancies after age 43 are exceedingly rare, even with IVF. We encourage most women over age 43 to strongly consider ovum donation.

World-wide, over half the babies born from assisted reproduction to women over age 40 are born from ovum donation, not from their own eggs.

Pacific Fertility Center is pleased to share our delivered pregnancy rates for 2007 and our preliminary clinical pregnancy rates for 2008. These outstanding pregnancy rates are made possible thanks to our team of board certified Reproductive Endocrinology and Infertility specialists, as well as, our highly trained embryologists.

Clinical pregnancy reflects the finding of a pregnancy sac in the uterus following transfer. Delivered pregnancy rate will be lower after accounting for miscarriage and pregnancy loss, particularly in older age groups.

Pacific Fertility Center Preliminary Clinical Pregnancy Rates for 2008

Delivered Pregnancy Rates 2007 (as reported to SART and CDC)

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

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

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

Control group: Fresh IVF pregnancies

Only 14% were twins

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

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

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

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

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

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

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

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

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

Results: Avg. # embryos transferred = 2.3

Clinical pregnancy rate = 27.8%

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

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

They experienced no congenital malformations at birth.

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

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

His arguments to support this opinion:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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