The first human pregnancy from an embryo that had been frozen and thawed was achieved in Australia in 1984, 6 years after the birth of the first IVF baby in the UK. The method used to preserve that embryo is called “slow freezing” and it is still the preferred method for preserving embryos throughout the world today. Slow freezing is a reliable and established technique that has served the IVF community well for over 20 years. The procedure has been refined throughout those years and it works, with slight modifications, for freezing all embryo stages and for sperm. However, despite many years of trying, slow freezing has never worked very successfully with oocytes. Frustrated by years of failures, scientists turned to an alternative procedure called vitrification in their quest to preserve oocytes. This approach is relatively new, but appears as through it will be preferentially used for oocyte preservation as we go forward. Vitrification kits are just beginning to get FDA clearance following scientific trials, and embryologists are being trained in the use of the new technology.
The main concern during the freezing of any cell is the removal of water without actually killing the cell. Since water expands in volume as it freezes, ice formation inside a cell would cause the cell to rupture and die. Therefore, cell water is traditionally replaced with a cryoprotectant (antifreeze) prior to cooling of the cell. This is achieved by sequentially incubating the cell in increasing concentrations of cryoprotectant. The cryoprotectant draws water out of the cell and itself enters the cell, all by osmosis. Once most of the water has been removed, the cell is cooled at the very slow rate of -0.3° C/minute until it has been cooled to below -30° C and is therefore fully frozen. Thereafter, storage of frozen cells is in liquid nitrogen (-196° C), which is a simple and practical storage medium.
Vitrification still requires the use of cryoprotectants and the cell is also ultimately stored in liquid nitrogen, but the journey from the incubator (at 37° C) to the nitrogen (-196° C) is much faster. The word “vitrum” in Medieval Latin means “glass” and the process turns the cell contents to a glass like substance instead of ice. Since no ice forms, the risk of rupturing the cell is eliminated. For glass to form instead of ice, the rate of cooling must be thousands of degrees per minute instead of the 0.3 degrees/minute that we use in slow freezing. Therefore, the process is sometimes referred to as ultra-rapid freezing, although the word “freezing” is really inappropriate here since the cell is not really frozen (i.e. no ice is created).
One of the big stumbling blocks during oocyte freezing was the sheer size of the cell (the oocyte is the largest human cell by some margin) and therefore its high water content. Just getting the cell to survive, (an oocyte has only one cell), was a huge stumbling block. Studies where 50-60% of the oocytes survived were considered groundbreaking, and still today there are few studies that have done better. Vitrification as a technique had been largely ignored by the IVF community as it was technically more challenging and used much higher concentrations of cryoprotectants. Cryoprotectants were thought to be toxic to cells. Today we know that they are safe and effective and do not contribute to cell death. It is possible that cryoprotectants may have deleterious effects on cells if they are metabolized, but virtually all freezing protocols utilize them at room temperature or below, where cell metabolism is significantly slowed or stopped. So, with success rates using traditional slow freezing failing to improve, vitrification has been given serious consideration as an alternative. In the few years since its introduction, vitrification has shown promising and excellent results in clinical studies (see Oktay et al., Fertility and Sterility, 2006, Vol 86(1), pages 70-80 a comparative review of slow freezing and vitrification results with human oocytes).
Making the transition from slow freezing to vitrification has been a challenge for the IVF community. As already stated, it is a technically challenging procedure, and training of embryologists in the technique has been slow. With slow freezing, embryos are placed in relatively weak solutions of cryoprotectant for as long as 15 minutes at a time. Then, they are usually moved on through slightly stronger solutions before being placed in large straws or vials which are then loaded into a computer controlled freezer for the long journey to -30° C. The embryologist can spend about 30 minutes with a set of embryos from the time that they come out of the incubator until they go into the controlled rate freezer. After 2 or more hours, the embryos can be placed in liquid nitrogen and the process is complete.
During a vitrification procedure, where typically only one oocyte or embryo can be worked on at a time, the transition from incubator to nitrogen takes only a few minutes. The embryo is stepped through solutions containing high and then higher concentrations of cryoprotectants where it shrivels and swirls in the extremely viscous medium. In the final stage, which is measured in seconds, the embryo is placed in an extremely concentrated cryoprotectant solution and then quickly loaded up into a tiny straw that is barely larger than the embryo itself. The straw is then sealed at both ends and plunged immediately into liquid nitrogen. The straw is so fine that it freezes in an instant, an important part of the vitrification process. The loading of the straw occurs at room temperature (25º C in the IVF lab) and it is cooled to -196º C in one or two seconds, giving a cooling rate of 6000-13000º C/min. The faster the straw can be cooled, the more successful the procedure. Performing this final step too slowly or too quickly can be the difference between success and failure and therefore requires extensive training.
At Pacific Fertility Center, we have been working on vitrification for over 2 years. Our initial interest was in oocyte freezing, but we were also interested in extending the technique to be used with embryos, and in particular to blastocyst stage embryos where slow freezing has not always worked well. Slow freezing has served us well over the years for embryos being frozen 1, 2 or 3 days after an oocyte retrieval, but blastocysts (5 or 6 day old embryos) did less well. With an industry wide transition to blastocyst stage embryo transfers, we looked at vitrification as an alternative method of preservation for these precious embryos.
A blastocyst is an embryo that has developed to the stage where it is ready to implant in the uterus. Instead of having a small number of loosely associated cells characteristic of earlier embryonic stages, it has 2 defined cell populations and a fluid filled cavity (or cyst). The cells that surround the cavity will form the placenta, and the cells within the cavity will develop into the embryo proper, or fetus and some of the extraembryonic membranes, such as the yolk sac. It is these interior cells that cause trouble during freezing since they are on the inside and difficult to expose to cryoprotectant. Slow freezing relies on cryoprotectant traveling through the outer placental cells, then the cavity, and finally into the fetal cells while water travels in the opposite direction. Fully dehydrating these fetal cells has always been a challenge and an embryo where these cells do not survive freezing and thawing will not result in a viable pregnancy. And with slow freezing, embryos tend to collapse in on themselves during dehydration, making it difficult to assess survival after thawing.
After investing heavily in vitrification training and implementing a successful oocyte vitrification program, PFC began working on blastocyst vitrification in January of 2007. By March we had a program established and were delighted by how easily blastocysts seemed to tolerate the procedure. Often, blastocysts looked no different after vitrification when compared to how they looked before the procedure. This result was in stark contrast to slow freezing where blastocysts always look shriveled and deflated after coming out of the freezer. By July 2007, we had switched completely to vitrification and currently we are enjoying the successes that it is bringing to our patients and us.
Our vitrification team consists of 3 embryologists: Mariluz Branch, the team leader, with Erin Fischer and Liz Holmes. Because of the technical challenges involved, we have to be cautious with involving other embryologists. So one of the three team members must be on duty every day (our lab is open 7 days a week). I am grateful to this team for their flexibility in accommodating our needs. By the end of the year we expect to have 2 more embryologists on the team, and then the final 3 in 2008.
Vitrification has been an exciting and challenging technique which we have embraced and conquered in 2007. We look forward to the gradual elimination of slow freezing and the successes that vitrification will bring us in the future. Joe Conaghan, PhD, HCLD
Joe Conaghan, PhD, HCLD, PFC's ART Laboratory Director, is internationally known for his work with embryos. He helps train and certify embryologists via the American Board of Bioanalysts, (ABB) of which he is a member of the Board of Directors. He is an inspector for CAP, the USA licensing authority for IVF laboratories. He teaches reproductive technologies at San Francisco State University and is chair of the College of Reproductive Biologists.
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Fresh Embryo at Blastocyst Stage: The cells are elongated and pressed against one another. The inner and outer cells are clearly visible, as is the cavity.
Two Vitrified Embryos at Blastocyst Stage After Warming: Though their appearances differ, both embryos implanted and created viable pregnancies.
This embryo looks perfect, as if it was never frozen. The outer and inner cells are clearly visible, as is the cavity.
This embryo has rounded, more dissociated cells resulting from shrinkage during incubation in cryoprotectant, (as cells shrink they pull away from each other). The cavity is small, but visible.
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The Trans fat, found in processed foods, may play a role in infertility. Implicated in prostate cancer, heart disease, and diabetes, and long thought to be a significant hindrance to good health, trans fat has been associated with ovulation disorders, according to a new publication1.
Trans fats are created in food processing. To avoid rancidity in foods, manufacturers heat oils under pressure to convert natural unsaturated fat to partially saturated fat, adding hydrogen molecules to change the bonds between carbon atoms in the long fatty molecule. Saturated and partially saturated fats are sometimes called partially hydrogenated fats. Saturated fats melt at a higher temperature, and are more stable on the grocer’s shelf. Partially saturated fat is resistant to oxidation and damage, melts at a higher temperature, and does not take on rancid odors and taste. Crisco, partially saturated cottonseed oil, was the first commercial product to be produced with the technique in the early 1900s.
Foods prepared with partially saturated fats can contain up to 45% trans fats. French fries, cheeseburgers, fried chicken, cookies, and chips are common offenders. An order of large French fries can contain 15g of trans fat. Oreo cookies contained trans fat until a lawsuit in 2003 induced Kraft Foods to alter its recipe.
Ideal for a manufacturer interested in long-term storage, saturated fats are not so well tolerated by the human body. Raising levels of LDL and lowering levels of HDL cholesterol, saturated fats have been implicated as a prime cause of the rising risk of coronary heart disease through the 20th century. According to the Nurses’ Health Study2, each 2% increase in trans fat calories doubles the risk of coronary artery disease. Since trans fats carry no health benefits and are potentially risky, experts have recommended reducing trans fats to trace amounts in the diet.
Infertility has been associated with trans fat intake. A study published in the January issue of American Journal of Clinical Nutrition from a group of researchers at Harvard University found that women with ovulation-related fertility problems tended to eat more trans fats than fertile women. Obtaining just 2 percent of total calories from trans fats was associated with a doubled risk for this type of infertility. The study showed that each 2% increase in dietary trans fat calories was associated with a 73% increased risk of ovulatory infertility3.
It has been difficult to separate out the effects of total fat and trans fat, since a diet high in trans fat diet is often high in total fats. In contrast to trans, higher total fat is known to decrease the risk of ovulation problems, improving ovulation, whereas women with a diet high in trans fat have an increased risk of ovulation disorders.
Dietary fats have been linked to markers of inflammation, a possible mechanism of trans fat effects4. In a randomized crossover study, 50 men consumed diets for five weeks that varied in trans fat content. Inflammatory protein markers were higher in men after the trans fat diet, showing that dietary fatty acids can modulate markers of inflammation.
The data is preliminary, but concerning. Since trans fats have no benefit and carry potential risks, they are best limited in the diet. Labeling requirements now include listing of trans fat content for foods. Lawmakers in several major US locales have passed regulations banning trans fats. Tiburon, California, on a voluntary basis was the first city to have trans fat free restaurants. Restaurants in New York City and Philadelphia are barred from using trans fat containing frying oils and spreads. The ban will be expanded to all restaurant foods next year. California is considering a statewide ban on trans fats.
Reducing processed foods and avoiding trans fats in your diet is an excellent goal for all, but patients with infertility may have special concerns. While more research is required regarding infertility and diet, there is no question a healthy diet is important. A diet of diverse and balanced carbohydrates, proteins, and fats, including omega-3 fats, will provide personal and possibly reproductive benefits for years to come. Philip Chenette, MD
1. Chavarro JE et al., May 2007, A prospective study of dairy foods intake and anovulatory infertility, Human Reproduction, 22 (5): 1340-1347.
2. Hu, FB et al. 1997 "Dietary fat intake and the risk of coronary heart disease in women". New England Journal of Medicine, 337 (21): 1491-1499.
3. Chavarro JE et al., January 2007, Dietary fatty acid intakes and the risk of ovulatory infertility. American Journal of Clinical Nutrition, 85 (1), 231-237.
4. Baer DJ et al., June 2004, Dietary fatty acids affect plasma markers of inflammation in healthy men fed controlled diets: a randomized crossover study. American Journal of Clinical Nutrition, Vol. 79, No. 6, 969-973.
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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
We hope to have embryos left after transfer and need to consider storage. Can you help us understand what determines your storage fees?
Many Pacific Fertility Center patients have surplus embryos at the end of their IVF cycle. If you chose to freeze your embryos, you will need to consider how long you plan to store the embryos before being used for a frozen embryo transfer. Patients who are finished building their family, but are not interested in destroying the surplus embryos, may choose to freeze them, offer them for adoption or donate them to research. These options are included on the consent forms, which must be signed prior to transfer.
Once you choose to freeze embryos, you need to factor in the annual storage fee. Pacific Fertility Center strives for lower fees, but must be able cover the underlying costs of services. Storage fees include expenses from the following sources: storage tanks, liquid nitrogen, leased floor space, embryologists and staff hours, equipment maintenance, annual inventory, information dissemination, forms, billing, legal fees and liability.
Let’s begin with the storage tanks themselves. At PFC we have 3 state-of-the-art embryo tanks: two tanks hold a total of 1376 spaces each. Every one of these spaces can hold up to 5 straws of embryos and each straw holds 1 to 3 embryos. These two tanks are full. Recently, we purchased another, larger tank, which holds almost 1500 patient spaces. This tank is already almost half full.
Once these tanks are filled with liquid nitrogen, they are extremely heavy. Because of their weight, they cannot be clustered together in the same room, but must be strategically placed to spread out the weight over the center’s floor. In addition, they must be stored in a secure, locked location. Every time we add a tank, an appropriate new space must be located. With square footage at a premium, this is not an easy task.
Storage tanks must be monitored. Gauges and seals must be functioning and the temperature must be kept at the optimum level with the addition of liquid nitrogen. The tanks are fitted with an alarm, which sounds if there is a problem. This alarm automatically sends an alert to the embryologist on call 24 hour a day, 365 days a year.
All embryo straws are labeled and a file is maintained for every patient who has embryos in storage. This extremely important aspect of storage is taken very seriously. A thorough inventory is completed every year. This is a time-consuming process as every straw must be located and identified. Patient addresses are kept up-to-date and confirmed annually when the invoice is sent or when patients notify the center of an address change.
If patients fail to notify us of a move and/or abandon their embryos, we make every effort to locate them. When they repeatedly fail to pay their invoice, we may be forced to send their billing on to a collections agency. During this process, we continue to store their embryos. As a last resort, we will go before a judge, show proof that we are unable to contact the patient after multiple attempts over a reasonable period of time, and request permission to discard the abandoned embryos.
One of the most frequently asked questions is “When am I going to be billed?” You will be billed based on the month that your storage begins. Patients often forget they have a back-up sample of frozen sperm and are “surprised” when they receive an invoice indicating they must pay their storage fee.
PFC is always available to answer any questions you may have regarding the storing of your embryos and sperm. For disposition questions, please contact Alexis Von Austin, Tissue Bank Manager at (415) 249-3636. For questions regarding an invoice, please contact Rosemarie S. Tagle, Billing Supervisor at (415) 249-3651.
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