The American Society for Reproductive Medicine’s (ASRM) annual meeting was held in New Orleans. It is the largest meeting for reproductive medicine specialists and scientists in the world. From our practice, Dr.s Givens, Schriock and Conaghan attended, as well as embryologists Jean Popwell, PhD and Jennifer Andres. Also, PFC nurse Allison Chamberlaine and PFC’s Marriage and Family Therapist Peggy Orlin attended. In addition, the genetics counselor with whom we work closely, Lauri Black from California Pacific Medical Center, was an attendee and active participant.

PFC’s embryologists attending ASRM’s research poster session Jean Popwell, PhD (left) and Jennifer Andres (right).

Single-Embryo Transfer: Minimizing Risks & Maximizing Outcomes
Dr. Givens attended a post-graduate course entitled “Moving Toward Single-Embryo Transfer: Minimizing Risks and Maximizing Outcomes.” This two-day course dealt with a pressing issue in assisted reproduction: the high incidence of multiple gestations. With the ever-increasing success of in vitro fertilization and the significant improvement in embryo implantation rates, the incidence of twin and higher-order pregnancies has risen dramatically in this country. Many countries now regulate the maximum number of embryos that can be transferred into the uterus at one time. The course topics included a summary of optimal medication protocols, several lectures on pre-cycle evaluation and testing and embryo transfer techniques.

Oocyte Freezing, PGS & Blastocyst Embryo Transfers
On the laboratory side, there were several talks on evaluation of eggs and embryo selection techniques, embryo freezing technology, including a debate about the usefulness of pre-implantation genetic screening (chromosome analysis of embryos) embryo selection. The combination was a fascinating mixture of new ideas, refinements in current technology, as well as a welcome opportunity to network and discuss with others the latest developments in reproductive science. Topping the list of presentations in New Orleans were those concerning the continuing refinements in oocyte freezing technologies, the more selective use of preimplantation genetic testing and the ongoing scrutiny of blastocyst stage embryo transfers.

Slow-freeze vs. Vitrification
The traditional slow-freeze technology used so successfully with embryos for many years, has essentially stalled with oocyte freezing. It appears the slow-freeze technology has finally met its successor: a process called vitrification. Slow freezing has had very limited success with oocytes due to their large size, high water content and their extreme sensitivity to cryoprotective chemicals and to changes in temperature and pH.

Vitrification, a technology that cools cells so rapidly that ice does not form, has been such a success for oocyte freezing that many labs are now abandoning slow freezing altogether. Here at PFC, we have been developing protocols for oocyte vitrification throughout 2006 and are actively working on blastocyst vitrification. It was reaffirming to see that this technology has gained wide acceptance, and is showing excellent results.

Preimplantation Genetic Screening (PGS)
While vitrification is on the rise, it was interesting to see that another technology, Preimplantation Genetic Screening (PGS), was lacking in new improvements or viable alternatives. Embryos have been screened for extra or missing chromosomes for over 15 years now, but the technology has not advanced significantly over that time. It is still possible to count only 12 chromosomes in an embryo. Although the error rate per chromosome is very low, the accumulated error rate becomes significant as we count more chromosomes. PGS was “under the microscope” in several presentations in New Orleans and it appears PFC’s limited use of genetic screening is well justified. Specifically, PGS does not improve embryo selection and pregnancy rates in younger patients. Its use is limited in older patients because there are often too few embryos available to justify testing. The patients who benefit most from PGS are the younger patients who experience recurrent miscarriages. However, unless there is evidence that previous pregnancies were genetically abnormal, PGS may provide limited benefit to this group.

Blastocyst stage embryo transfers
While younger patients (those under 35) don’t benefit from PGS, they are the patient population most likely to benefit from blastocyst transfers. Culturing embryos for 5 days to the blastocyst stage, instead of the more traditional day 3 embryo transfer, is one of the main ways in which the laboratory staff can help in selecting the “best” embryo for single embryo transfer (SET) patients. Blastocyst culture techniques are well refined now and support the commitment within the community to transfer fewer embryos at one time. Furthermore, the promise of vitrification can reassure patients that their remaining embryos can be stored indefinitely when preserved at the blastocyst stage. Several presentations showed that blastocysts which were vitrified early, before their cavity (or cyst) had expanded too much, benefited most from the technology. In more advanced blastocysts, artificial reduction of the cavity gave superior results. It may not be long before vitrification is the procedure of choice for preserving all blastocysts.

2006 ASRM guidelines for numbers of embryos to transfer
The new 2006 ASRM guidelines for numbers of embryos to transfer were presented. See Tables 1 and 2 below.

The topic of whether or not federal or state legislation should regulate the maximum number of embryos to transfer was also discussed. Many people in the general public support such legislation but those of us in the field (and most patients) are opposed to the government regulating medical practice and arbitrarily setting limits on embryo transfer. In order to forestall such legislation, it is obvious that we must decrease the number of twin gestations (the number of triplet and higher-order gestations has already dramatically decreased in the last 5-7 years). At Pacific Fertility Center we have instituted a new emphasis on single embryo transfers and expect to significantly reduce the risk of multiples and achieve our goal of “optimal” pregnancy outcomes. (See From Us to You in this issue for a discussion of our 2006 statistics and please see Conception Health in this issue for a discussion of why it is important to try to achieve single baby conceptions.

– Carolyn Givens, MD and Joe Conaghan, PhD

For those of us with an interest in human reproduction, scarcely a day goes by without us hearing or seeing something related to oocyte freezing. The topic has generated a lot of hype and it is difficult to avoid the frequent magazine and newspaper articles, advertisements and TV features that generate excitement on the subject.

We have already discussed oocyte freezing in a previous newsletter article (Keeping Egg Freezing in Perspective; January 2005) and readers unfamiliar with the technology are encouraged to visit our website where they can read this in the newsletter archive. Having already discussed the methods for freezing, and their merits, we now address the achievements of oocyte cryopreservation on this, the 20-year anniversary of the first success.

There are two technologies used in oocyte freezing, and the primary aim of each is avoiding ice formation within the cell. The first is the slow freeze method (used so successfully with embryos) that dehydrates and cools the cells gradually, over three hours. The second is an ultra-rapid procedure that is performed so quickly that the cell contents turn to a glass-like substance. This latter method is called vitrification and it is gaining in popularity for oocyte and embryo freezing. And since no ice forms, the cells are technically not frozen, but “vitrified.”

In reviewing the scientific literature since the first success in 1986, the importance of oocyte freezing is apparent by the sheer volume of publications on the subject. For the purpose of this article, the many papers that report on the technique only have been excluded, and here we will only report on the pregnancy outcome data. However, even this is difficult since some patients may have become pregnant from the first few thawed oocytes, leaving us with no data on the many oocytes still frozen on their behalf. Also, even though there are reports that detail only one or two pregnancies, there are probably many other isolated successes around the world that have gone unreported in the scientific literature.

Most of the pregnancy outcome data has been pulled together in a single review paper by Dr. K. Oktay and colleagues at Weill Medical College in New York (Fertility & Sterility, 2006, Vol 86 (1), pages 70-80). The 47 papers reporting outcome data for slow freezing were analyzed and from these, only 26 provided sound usable data. The others were excluded either because sub-optimal procedures were used, the pregnancies had not yet delivered or the authors could not be reached to clarify the data. The 26 useful papers collectively documented the freezing of 4,564 oocytes from which 4,000 had been thawed in 397 patient cycles. Out of 95 pregnancies, 76 resulted in live births, and since some of these were multiple pregnancies, the total number of children born was 97. If we add in the excluded data, the number of pregnancies becomes 170, resulting in 106 live births and 11 ongoing pregnancies. Because of ambiguities in the excluded data, a final number of children is not stated. However, the data suggest that the number of children that are alive today as a result of 20 years of slow freezing of oocytes is approximately 200. Taking all the data into account, the clinical pregnancy rate per thawed oocyte was a mere 2.3%. The live birth rate in the 26 usable papers was 1.9% per oocyte thawed.

Unfortunately it is not possible to give rates per oocyte frozen since some papers are not complete, but more importantly because many oocytes are still in the freezer.

Vitrification, which is a technology that came late to oocyte preservation, is quickly gaining ground on the slow freezing method. By June of 2005 there were only 10 reported births following oocyte vitrification, but a year later the numbers reported by Oktay are 61 pregnancies from which 42 have delivered live infants and 7 are ongoing. With limited data, vitrification appears to be a more highly efficient preservation method than slow freezing. The latest numbers, based on admittedly limited data, shows that >90% of oocytes survive and about 90% of these fertilize. Overall, 50% of vitrified oocytes make blastocysts in culture which is as efficient as fresh oocytes. These numbers are reported by Masa Kuwayama at the Kato Ladies Clinic in Tokyo. Also, from 29 embryo transfers, 12 pregnancies have yielded 7 live infants with 3 not yet delivered at publication time (Kuwayama et al., 2005, Reprod Biomed Online, Vol 11 (3) pages 300-308). We can compare this data to the latest results with slow freezing where the experience of 20 years has been incorporated. Using sodium-depleted medium, in which oocytes are slow cooled and frozen, 59% of oocytes survived and 68% of these fertilized. Nine pregnancies were established in 28 thaw cycles from which 5 delivered and 1 was ongoing (Boldt et al., 2006, Reprod Biomed Online, Vol 13 (1) pages 96-100). For those women who want to rely on oocyte cryopreservation to postpone motherhood, these data should be sobering. While we don’t expect the technology to ever be 100% successful, it currently offers no guarantees.

Expecting too much from today’s procedures could leave many women very disappointed. Further, many of the pregnancies reported in these studies were achieved by preserving the oocytes from young women. Since oocyte quality declines as a woman ages, the success rates for older women are likely to be less than reported here. Women considering oocyte preservation will need careful counseling and a good understanding of the success rates before putting their eggs in this basket.

– Joe Conaghan, PhD

In the IVF laboratory we strive to provide the very best conditions under which we can maintain sperm, eggs and embryos. Sperm are resilient and could tolerate a little hardship, but eggs and embryos are extremely sensitive to their environment. Sperm are quite happy at room temperature, for example, but eggs suffer irreversible damage if allowed to cool.

A very important part of the environment is the air in the laboratory. Embryos are only indirectly in contact with the air since they are grown in a special culture fluid that is covered with a layer of mineral oil. But they do need some of the gasses from the air and these simply diffuse into the oil and then the culture medium. Carbon dioxide (CO 2) is present in only tiny quantities in air (less than 1% by volume), but since it is a product of respiration it is present in the body at higher levels. We therefore supply the incubators with CO 2 to create a more physiological environment. Oxygen (O 2) is necessary too, and because air has plenty (about 21% of its volume at sea level) we don’t need to supply more. In fact, we need to take some away.

Even though we all think of O 2 as a life giving force, there is a growing body of research showing that embryos from several species do better in a low O 2 environment. Human embryos can clearly do well without a reduced oxygen environment as evidenced by the many thousands of IVF babies that have been born to date. However, it is possible that they could do better. So far the research has shown that embryos do well under low O 2 when kept in the laboratory for 5 days and transferred when they reach the blastocyst stage.

At PFC, most embryos are transferred back to patients or frozen after 3 days in the laboratory.

So far, there is no evidence that a low O 2 environment is necessary for these embryos, but we are moving towards such a modified environment for all. Currently the laboratory has 13 incubators, of which only 2 have oxygen controlling capability. These are used for the 10% of cases that are having transfers at the blastocyst stage. However, 4 more of these incubators will arrive in the laboratory this October and another 4 by year’s end. Our goal is to replace all of the current incubators by early 2006.

Those of you who take a daily vitamin pill are probably aware that antioxidants are now a common dietary supplement. We even recommend antioxidants for men hoping to improve their sperm health. Examples are beta-carotene, vitamins C and E and selenium. Oxygen is an aggressive molecule that gives rise to superoxide radicals and numerous harmful intermediates that cause cellular damage (known as oxidation). These radicals are highly reactive and they oxidize other molecules in the cell. Consequently, organisms that live in oxygen rich environments have had to evolve enzymatic systems to prevent free radical damage. Eating a varied and healthy diet will of course provide individuals with the antioxidants they need, but still some 30% of Americans are thought to take antioxidant supplements. One example of oxygen damage in the body is the oxidation of low-density lipoprotein (LDL or “bad”) cholesterol that contributes to fatty buildups in arteries.

We supply antioxidants, such as pyruvic acid or taurine, in the culture medium in which we grow embryos, and these readily mop up products of free radical breakdown such as hydrogen peroxide. But reducing environmental oxygen is now an important target and by early 2006 we will be growing all embryos in a reduced oxygen environment.

A recent development in the laboratory at PFC is the acquisition of a laser for use in key procedures. The laser will be used to assist in the processes of Assisted Hatching (AH), Intracytoplasmic Sperm Injection (ICSI), and Pre-implantation Genetic Diagnosis/Screening (PGD/PGS).

All of these procedures require us to make a small opening in the outside shell of the egg called the Zona Pellucida (zona). Prior to laser technology this opening was made with an Acidified Solution, which would slowly dissolve away part of the zona until a small opening was achieved. Now with the laser, a beam of light creates a precise opening in the zona.

Laser use for PGD: The red “pilot light” marks target for the laser. The white circle marks a “safe zone”. The laser is usually fired 3 times for assisted hatching and 5 times for PGD embryo biopsy.

The zona pellucida is a non-living, but important part of the egg. It specifically allows only 1 sperm through to fertilize the egg, and then immediately hardens, preventing other sperm from getting in. After fertilization, the egg divides into 2 cells, and then these divide again into 4 cells. As the embryo continues through these rounds of cell division, the zona keeps all the cells together, since it encloses the embryo. After 5 or 6 days, the embryo has enough cells to begin forming a placenta and the embryo hatches from the zona and attempts to implant in the uterine lining.

Assisted hatching (AH) is a procedure that has been around for about 15 years and it is something that is often performed in the laboratory just prior to an embryo transfer procedure. It is a simple and precautionary procedure where we create a small hole in the zona just before transferring embryos to the uterus. Since the zona is not a living part of the embryo, making a hole does no harm, and in fact facilitates the embryo in hatching from the zona once it’s in the uterus. A normal embryo should be able to hatch all by itself, but in some patients we perform this procedure just to make sure a problem doesn’t arise when the embryo tries to escape from its shell. For AH, the laser will allow us to refine the procedure considerably. Firstly, we will be able to make a hole of an exact size, and secondly, the procedure will be performed more quickly and we will therefore further reduce the amount of time that an embryo is being handled. Traditionally, AH takes about 5 minutes per embryo, but with the laser this time will be reduced to less than a minute. For the process of embryo biopsy for PGD/PGS, an extremely precise opening is made in the zona to facilitate the removal of one cell. Again, the laser will speed the procedure up considerably and reduce the time that we’re working on each embryo.

By now you might be wondering if there are any harmful effects of using laser light on embryos. According to several studies and expert opinions, laser-assisted hatching is superior to chemical-assisted hatching as seen by improved development of “hatched” embryos to the blastocyst stage (the stage at which an embryo will implant in the lining of the uterus). Furthermore, laser-assisted biopsy of cells from embryos for PGD analysis does not appear to have a detrimental effect on the continued development of the embryos versus embryos not undergoing any biopsy procedures. This indicates that using a laser to do the biopsy procedure appears to be safe.

Current lasers have several built-in safety features. The laser system is equipped with a second non-laser beam of light, similar to a penlight, which allows the embryologist to observe where an opening of the zona would be created prior to firing the laser. Also, the temperature that the embryo is exposed to is controlled by the use of Isotherm rings. Isotherm rings help the embryologist prevent potential harmful thermal effects on cells adjacent to the zona due to heat from the laser beam. The rings indicate both the drill hole size and the safety region based on temperature. With this interactive feature, the user can predetermine the hole size and eliminate practically all risk of impacting cells within the embryo.

PFC’s new laser system has been tested for both accuracy and precision. In addition, the lab staff is undergoing training with Laser professionals on its use and maintenance. They will have unlimited practice time, ensuring the highest level of safety and technique when it comes time to use it on human embryos.

– Jean M. Popwell, PhD TS (AB, PFC Lab Embryologist)

Photos provided by 3DbabyVu

A first glimpse of a baby in the womb, especially for women who have faced an arduous route to pregnancy, is perhaps as euphoric a moment as the “You’re Pregnant!” announcement. It is only natural for parents to want a visual connection with the infant as early as possible.

Seizing on this yearning, a new crop of commercial ultrasound studios has mushroomed all over the country, offering parents a chance to have a first look via an elaborate 3D and even 4D video ultrasound. At least three such businesses are in the Bay Area. Yet new parents contemplating a nonmedical 3D ultrasound simply for novelty or posterity should be fully aware of this technology in a rapidly evolving marketplace.

The safety of common medical ultrasounds is undisputed. For over 35 years, ob-gyns have used 2D ultrasound technology as standard practice to medically diagnose the health of a weeks-old fetus, enjoying an early glimpse of its emerging shape, major organ development, tissue and blood flow and when desired, the gender. The ultrasound repertoire is so common; over 80 million procedures are now performed in the US each year, reports one clinic.

Nevertheless, the Food and Drug Administration (FDA) and the primary medical association that oversees ultrasonography – the American Institute of Ultrasound Medicine (AIUM), have thus far refused to endorse 3D and 4D ultrasounds offered by commercial studios. The concern is less about the technology itself, and more about how it is applied. While the sound wave levels used for a 2D and a 3D/4D are reportedly of the same frequency, (it’s the computer diagnosis that creates the image differentiation), there is more built-in oversight in the medical community performing diagnostic ultrasounds.

For instance, is the person performing the commercial fetal portrait properly trained? Right now, it is up to the 3D studio to make sure that the person controlling the knobs and holding the transducer has undergone the same training standards required for ultrasonography at an ob-gyn office. Professional (non-physician) ultrasound practitioners undergo nearly three years of training, including 12-18 months for didactic and 12-18 months of clinical practice in order to gain the key certification from the American Registry of Diagnostic Medical Sonographers (ARDMS).

Moreover, there is concern that a commercial portrait ultrasound will reveal a developmental problem with the fetus that should be observed and discussed only through a physician/patient relationship. Another concern is that enthusiastic parents will forego a routine medical ultrasound after obtaining an elaborate portraiture one. In response, many commercial ultrasound studios are requiring patients to bring proof of a prior medical diagnostic ultrasound.

Finally, knowing a bit about the technology helps parents make an informed decision. In the medical community, the standard is to expose the fetus to the lowest possible exposure level for the shortest amount of time, usually 10 minutes or so. Because frequent ultrasonography at higher levels can produce a heating effect in bone and tissue, the aim is to minimize exposure. Yet some commercial fetal portrait studios offer deluxe packages involving a 45 minute video ultrasound.

A spokesperson from 3DBabyVu insists that the potential for physical damage to the fetus via a wrong decimal level setting is literally and virtually not possible, at least with the standard GE Voluson machines, which provide a cap to the frequency level. Yet he admitted that the same machines have two other settings for cardiac mode and vascular mode to examine more robust adult tissue. If patients choose to purchase a dynamic 3D or 4D image package offered by one of these enterprising studios, we strongly recommend that you learn as much as possible and even consult with your ob-gyn if you are at all confused. Also, it is best to confirm that the sonographer at the commercial studio is ARDM certified. Because the practice of fetal portraiture imaging is self-regulated, it is the patient’s responsibility to be aware of current research and be as informed as possible prior to using this new technology.

We occasionally get asked by same sex couples if it is possible to create an embryo, and hence a baby, by using the DNA either from 2 sperm or 2 eggs, instead of the DNA from a sperm and an egg. In mammals, such a feat hasn’t been possible until recently. A paper in the scientific journal Nature (22nd April 2004) reports the birth of 2 mice; each created using the DNA from 2 eggs and with no sperm.

Creating offspring using only female genes and with no paternal contribution is a common phenomenon in nature and in fact is a method of reproduction employed by almost all plants and animals. Mammals however have not been able to reproduce in this way. This impediment is attributed in large part to a process called genomic imprinting.

Experimentally, when mouse embryos are created using only the DNA from 2 eggs, the resulting fetus is well formed, but only a rudimentary placenta develops and the pregnancy fails. This is because the placenta is created mainly by paternal genes, and without the involvement of a sperm, we can’t get a normal placenta.

But if we have 2 copies of almost every gene (one from Mom and one from Dad), why can’t the maternal genes make a placenta? Biologists think that it’s a conflict of interest for Mom’s genes to make the placenta. Since the placenta in many ways is a parasite that fights for Mom’s resources, Mom’s placental genes are deliberately inactivated or switched off and it’s left to Dad to make the placenta. This process of deliberately inactivating a set of genes from one parent, so that the other parent’s genes are left to do the work is called imprinting. These genes carry with them a history of their origin because they are endowed at conception with a maternal or paternal imprint.

One negative consequence of imprinting is that when an imprinted gene is defective or otherwise does not work, the inactive, but perfectly good gene from the other parent can’t be called upon to help out. Diseases like Prader-Willi syndrome and Angelman’s syndrome which have variable physical, mental and behavioral effects on afflicted individuals are caused by defective imprinted genes.

So what happens when an embryo is created using 2 sperm and without maternal DNA? In this instance, as would be expected, the placenta is normal and fully formed, but the fetus is typically deformed and most notably lacks a head. It could be said that without a Mom, mammals lose their heads.

In the Nature paper, the mice without a father were created after exhaustive attempts: 2 live born from 457 reconstructed eggs. And the researchers used a trick to get around the imprinting issue. To make each embryo they used a mature (ovulated) egg and an immature egg from a newborn mouse in which the genomic imprint had not been established (imprinting occurs as eggs grow and mature). This allowed them to overcome the absence of the paternal imprinted genes since there were few or no imprinted (and therefore inactive) genes in the DNA from the immature eggs. The process was not very efficient in creating live offspring, but one of the resulting mice reproduced normally after reaching adulthood. The second mouse was used in tests to determine its DNA normalcy.

Each year Pacific Fertility Center® sends a delegation to the annual meeting of the ASRM the American Society for Reproductive Medicine. This prestigious conference draws researchers and practitioners from around the world, and this past event in San Antonio in October 2003 was no exception. Over 6,000 people attended from 32 different countries.

We have provided this summary of highlights to share with Fertility Flash readers. This tiny sampling by no means reflects the scope and depth of the 1800 scientific research papers that were presented. Human Nuclear Transfer From a popular press’s point of view, the most talked-about paper was Dr. Jamie Grifo’s research on human nuclear transfer. Each day of the conference, a new headline appeared with the world “clone” or “clone-like” even though Grifo and his Chinese colleagues, who reportedly tried the process unsuccessfully, insist that the process is not cloning. They fused the DNA from the oocyte of an infertile woman with a donor oocyte from which the DNA had been removed, and then fertilized the “reconstituted egg” with sperm. This experimental procedure has not yet produced a live birth, and the FDA prohibits this type of research in the U.S. It was recently banned in China as well. It is an incredibly complex procedure that is not likely to ever be commercialized due to the fact that so many embryos are rendered non-viable. OK to Go Patients who have just undergone Embryo Transfer after IVF are no less or more likely to conceive if they immediately go to the restroom. A study revealed that there was no difference in pregnancy rates between those women who had to go immediately and those who waited. Relax about SSRIs Women undergoing infertility treatment who take prescription medications in the category of Selective Serotonin Reuptake Inhibitors (Zoloft, Prozac, Paxil, etc.) have less to worry about. Children conceived by women on SSRI medication were no more or less likely to have problems. 911 Decline Infertility patients from New York treated in the midst of the September 11, 2001 tragedy suffered from a higher rate of pregnancy loss than those treated prior. The results of nearly 400 patients who underwent an IVF procedure before and after September 11 were examined. Individuals placed in the “before” or “after” groups showed no significant differences in age, number of eggs retrieved, or number of embryos transferred. Clinical pregnancy rates were also comparable between the two groups. However, there was a nearly 25% lower delivery rate for the patients with a pregnancy test after September 11. This study again points to the significance of psychological factors that impact outcomes of infertility therapy.

Telomeres Predict Poor Prognosis Scientists are noticing a correlation between short telomeres and egg quality. Telomeres are small pieces of DNA at the ends of chromosomes, that shorten naturally as we age. Telomere length could someday be used as a test of fertility potential.

Joe Conaghan, PhD	Eldon Schriock, MD

Drs. Joe Conaghan, PhD and Eldon Schriock, MD along with other PFC professionals attended the ASRM meeting and are committed to continually evaluating the latest research and using proven treatments to improve patient care.