Beta-3 Integrins and Embryo Implantation
One of the mysteries that confound reproductive biologists is the issue of why human embryos implant into the uterus at relatively low rates as compared to other animal species. This is evident when looking at implantation rates at the time of In Vitro Fertilization treatment. The chance that any one embryo will implant in the uterus varies with female age such that at age 40, only about 5-10% of transferred embryos will successfully take hold and create a viable pregnancy. Even when looking at donor egg-derived embryos from 21-28 year-old donors, the rates of implantation are about 30-45% per embryo. One mechanism to explain the failure of some embryos in implanting – perhaps the primary mechanism – is chromosomal abnormality. If an embryo does not carry a perfect set of 23 pairs of chromosomes, the embryo will likely stop developing, often before implantation can occur.
Implantation of embryos is a complex process. Initially, the embryo has to attach its placental cells to the surface cells of the uterine lining (the endometrium). This is a process that is mediated by a complex of proteins expressed both on the surface of the embryo and on the surface of the endometrium. Expression of the uterine proteins is under the influence of the ovarian hormone progesterone. There are estimated to be over 300 genes that are either turned off or turned on in the endometrium during the “implantation window,” the 3-4 days during which the endometrium is receptive to an embryo attaching. Most of the products of these genes and their role in implantation remain to be identified. In a small percentage of cases, failure to properly secrete one or more of these proteins may be a cause for implantation failure of normal embryos.
One protein produced by the endometrium during the implantation window that has some evidence for a scientific basis for a role in implantation is the cell-to-cell adhesion molecule known as beta-3 integrin. Integrins are a class of cell surface proteins that appear to act in all types of cell-to-cell recognition and adhesion processes. The beta-3 class of these proteins has been shown to be produced in response to progesterone in the endometrium and are purported to be one of the key proteins for adhesion of embryos to the endometrium. Failure to express this protein appropriately has been theorized to be a cause of unexplained implantation failure. Why some women do not produce beta-3 integrins is usually unknown. However, some proposed causes include presence of blocked fallopian tubes filled with inflammatory fluids (hydrosalpinx), endometriosis, and poor progesterone production.
In order to diagnose whether or not a patient is producing beta-3 integrins, an endometrial biopsy must be performed 8-10 days after ovulation, as determined by LH surge testing. The biopsied endometrial sample is then sent to a laboratory that performs immuno-histochemical analysis on the tissue. The tissue is fixed to a slide and treated with antibodies to beta-3 integrins. These antibodies then are further treated with a second color marker antibody, so that endometrium-secreting beta-3 integrins will light up under the microscope. The tissue is scored by manual analysis by a medical technologist specifically trained to analyze beta-3 integrin expression.
In June of this year, I had the opportunity to visit Adeza Biomedical, a Cupertino-based laboratory that offers beta-3 integrin testing. I was impressed with the facility and the scientific integrity of the staff. I was also impressed with the labor-intensiveness of the analysis process. They receive specimens every day from infertility clinics across the country and are usually processing 6-12 specimens daily. They also send a portion of the biopsied tissue to a local pathologist to determine if the configuration (histology) of the endometrial tissue indicates it has been obtained within the implantation window or whether it is “out-of-phase.” As it turns out, a high percentage of tissue samples (40-45%) at Adeza are reported as negative for beta-3 integrins. A smaller percentage of these negative specimens are “out-of-phase”. So most of the specimens failing to show beta-3 integrins production are “in-phase”. It is unclear why this lab finds such a high rate of their test samples showing negative results for beta-3 integrins when the true incidence of lack of beta-3 integrins in most patients should be low. It may be that either the lab is setting the scoring level for a positive result too high or it may be that the patient samples really reflect a selected population of women who truly have low expression of beta-3 integrins. Unfortunately, there is no clear answer to this.
Previously, we had been less inclined to perform endometrial biopsies. Even if we found out there was a lack of beta-3 integrins, we wouldn't know what to do to induce their expression. However, we are beginning to find that we can often induce the expression by treating beta-3 integrin-negative patients with the aromatase enzyme inhibitor, letrozole (Fertility Flash; A Closer Look at Letrozole; May 2006, Vol. 4, Issue 4). Many women, especially if the histology on the original biopsy is “in-phase,” will have a positive biopsy result after treatment with letrozole.
Biopsies are typically performed 8-10 days after an LH surge in a natural cycle. Repeat biopsies on letrozole (taken days 3-7 of the cycle) are also performed at this time. We usually will use some local anesthetic in the cervix prior to passing a small plastic tube through the cervix to scrape out some endometrial tissue. Mild cramping may occur. The cost of the biopsy is $125.00 and the cost of the tissue analysis by Adeza is $400.00. It takes about 4-5 working days for the results to be received.
If you would like more information about this test, visit www.adeza.com and select the E-tegrity logo. You can download a patient brochure from this website. If you would like to know if this testing is appropriate for you, please ask your PFC physician.
Carolyn Givens, M.D.
Carolyn Givens, MD was the first in San Francisco to successfully initiate a pregnancy using intracytoplasmic sperm injection (ICSI). She currently co-directs Bay Area Pre-Implantation Genetic Diagnosis Program (PGD) and is director of PFC's PGD program. Dr. Givens' excellent care and over 12 years of experience is recognized by her peers who repeatedly single her out as a “Best Doctor” in national surveys. See www.bestdoctors.com.
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ASK THE EXPERTS
Sperm Samples at Home
Pacific Fertility Center Team
Left to Right: Front: Philip Chenette, MD, Isabelle Ryan, MD, Carolyn Givens, MD
Back: Joe Conaghan, PhD, Carl Herbert, MD, Eldon Schriock, MD
Question: Can I collect my sperm sample at home?
Answer: Yes, sperm samples can be produced at home and brought into our office provided that you follow some simple guidelines. Most importantly, the instructions for producing a sample must be followed as if you were producing a sample in one of the two dedicated rooms in our office. You should shower in the morning and wash the genital area with soap and then rinse with plenty of water. Most of the samples we receive are produced by masturbation and you should be careful to wash your hands immediately before and after the collection. If you need lubrication and/or a condom to produce the sample, these must be supplied by PFC. Most condoms and commercially available lubricants are toxic to sperm in some way, but we can supply you with materials that we have tested and that we know do not kill sperm. You can take them home if that's where you'll produce your sample. Similarly, we must provide the container into which you will collect; again to ensure that it is sperm friendly.
The most important part of producing the sample at home is getting it to our office within 60-90 minutes of collection. Your semen sample contains sperm but also many enzymes that are important in the natural process of reproduction. One part of your reproductive tract, the seminal vesicles, produces enzymes that coagulate the semen immediately upon emission. This allows the viscous sample to remain within the vagina, a process that might be an evolutionary vestige of the copulation plugs that are seen in other mammals and that prevent the female from mating with a second male. Within 5-20 minutes however, other enzymes in the semen (this time from the prostate gland) liquefy the clotted semen, liberating the trapped sperm so that they can enter the cervix. Sperm in the first fraction of the semen are bathed in prostatic secretions and have better motility and survival than sperm in latter fractions which are bathed in vesicular fluid, since the seminal vesicles emissions are last in the ejaculatory sequence. This is why we always ask if any part of the ejaculate was lost during collection. If the first few drops of semen don't get into the collection cup, we may have lost the best sperm and we may underestimate the quality of your sample.
All of these enzymes in the semen make it a hostile environment. Sperm trapped or left in semen will die relatively quickly, but sperm washed out of this enzyme bath can survive easily for 4 or 5 days in the laboratory. Semen can also cause uterine contractions, which is why we have to process sperm samples and remove it before performing your intra uterine insemination. Getting your semen sample to the laboratory within 60-90 minutes of collection allows us to assess your sperm before the enzymes can do any damage.
It is important that you have an abstinence period of at least 48 hours but not more than 7 days before giving us a sample. Samples produced after 2 days abstinence will usually have the highest numbers of motile sperm with the greatest forward velocity, when compared to samples produced after shorter or longer abstinence. Waiting too long between ejaculates is the biggest mistake we see, possibly because some men think that they can save all their sperm for the day of their big test. However, older sperm begin to die if ejaculations are infrequent and we see the percentage of live sperm decrease with increasing abstinence. Also, please remember that abstinence means no ejaculation, not just no intercourse!
Once your sample has been collected, it is important to avoid exposing it to extremes of heat or cold before bringing it to us in the laboratory. Don't put it in the refrigerator while you take a shower. Don't leave it on your dashboard in the sun while you pick up your dry cleaning. And don't leave it in the glove compartment, forget about it for a week, and then deliver it to the lab. The sample will be fine at room temperature, and you don't have to break the speed limit in trying to get it to us.
You will need to have made an appointment with us so that we know you will be bringing in a sample, and when you arrive in our office, a member of our staff will check your specimen in. We need to be sure that it is labeled properly and we will get some details from you regarding your abstinence period and how and when you produced the sample. And we will check your identification (usually your driver's license). This last step is important in establishing the identity of the sample and is part of a “chain of custody” procedure that we use with all samples passing through our facility. We will examine and if appropriate, process the sample within 30 minutes of receiving it, or immediately if the sample is already 1 hour old. Hopefully we won't be calling you to say that we need to repeat the test!
Joe Conaghan, PhD
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My Journey to PFC
It's apropos that the title of my article is “my journey to PFC”. When you move from New York, to London, to San Francisco, are a part of two decades of assisted reproductive technology, and even take the time to fit in a stint at the San Francisco Symphony, you have indeed taken a “journey”.
I was born in New Rochelle, New York and was the eldest of two girls. However, outside of blueberries, hot sandy beaches, and the Easter Bunny, I remember little about my childhood in the United States. At the age of five, my family moved to London and I grew up in the very pleasant town of Enfield. Ultimately, I found my way into nursing. Interestingly, my sister became a sculptor – our parents certainly allowed us to follow our own paths!
1980 marked the start of my journey to PFC. After working for a while at an OR near London, I embarked on a six-month vacation to the United States. When I visited San Francisco, I just loved it. The city was so friendly and open; it was a very different experience. I returned to London where I worked in the operating department of the Royal Free Hospital, but my heart was in San Francisco and, four years later, I moved to the Bay Area.
My first jobs in San Francisco were as an usher at the San Francisco Symphony and a receptionist at a Homeopathic Clinic – a lovely break from the stresses of OR work. I enjoyed my time at both places, but clearly, nursing was ingrained in me. It was only a matter of time that I would find myself back in a healthcare setting. In 1986, I was an IVF Coordinator at an OB/GYN clinic in Berkeley, which was one of the first to have an in-office IVF program. Working with fertility patients was an empowering and positive experience and I learned a great deal in the 4 years I spent there. From there I went back to the OR, wondering if it was my calling. I worked at a surgery center in San Francisco and enjoyed the work and really liked the people.
However, on a whim, I responded to a job listing for an IVF coordinator at the San Francisco Center for Reproductive Medicine (SFCRM). I was told that the position had been filled. A few days later, I decided to call back and ask if I could leave my resume on file with them. They told me that the position had once again opened up. I took the job, and the rest, as they say, is history.
Working at SFCRM was a challenging and exciting experience. It was a rewarding and eye-opening experience—I had the opportunity to see a practice go from a tiny, empty office with bare walls to a busy full-service fertility clinic. I wore many hats during the early stages — I was an IVF coordinator, I purchased equipment, wrote protocols, and on occasion tackled problems with the antiquated plumbing system. Everyone worked exceptionally hard to get SFCRM off the ground and it was a true team effort. Drs. Herbert and Chenette joined SFCRM in 1992 and the practice grew and grew.
In 1999 Drs. Carolyn Givens, Eldon Schriock, and Isabelle Ryan and Drs. Carl Herbert and Philip Chenette joined together to form Pacific Fertility Center as it exists today.
My responsibilities have evolved over the years but my perspective and appreciation towards those who help individuals overcome infertility never wavers. I am truly fortunate to have an incredible team of nurses, medical assistants (MAs), and clinical coordinators. They work very hard together, and are key in providing care to patients as they navigate their treatment at PFC. Each one of them has gone “above and beyond” what I have asked. I think that truly speaks to the type of people they are.
As Director of Nursing, my role is far more “hands off” than it used to be. I do miss the more extensive patient contact, but with such a positive and supportive nursing team, I know our patients are in good hands. Some of my major responsibilities include: helping to ensure that we are compliant with the FDA, maintaining our accreditation and chairing our Quality Improvement Committee. I always have to keep in mind that my department does not function on its own – all departments depend on each other and have to work together to provide the best patient care.
What has made my experience in ART so exciting is that it is never static. When I first started in IVF the greatest improvement was moving from egg retrieval by laparoscopy to the current ultrasound guided method. Since then, there have been so many innovations that it is hard to remember them all. The changes and improvements quickly become the new standard of care.
I can only imagine what the future holds. It's amazing how many changes I've seen over the years. Thankfully, I've been fortunate enough to work in an environment where change for the better is ongoing and accepted. This gives me confidence in knowing that we're doing our best to help our patients.
Karen Volpe, R.N.
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Oocyte Freezing Hype
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, Vol. 3, Issue 1) 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
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