Inside the ART Laboratory: Reducing Multiple Pregnancy

It has long been recognized that the success of assisted reproductive technology (ART: this term is used to include in vitro fertilization (IVF) and related techniques), depends on the quality of the ART laboratory. The modern ART laboratory is a place of precision and meticulous attention to detail. The ART laboratory is the location where eggs are cultured, embryos grown, and micromanipulation, such as insertion of sperm into eggs to achieve fertilization, is performed. Temperature, humidity, and air flow, all need to be monitored and carefully controlled. Culture medium, the nutrient rich soup in which the eggs and embryos are grown, needs to be adjusted to precise specifications. Many improvements have taken place in the ART laboratory since ART began to be practiced and all of these have lead to healthier embryos and increased success rates.

We have become much more skilled in growing embryos in the ART laboratory. Our success has improved the live birth delivery rates for standard IVF from <18%per cycle for women under 35 years of age in the early days (Edwards, 1985) of IVF to over 40%today (SART, 2009). One of the main changes has been in the formulation of culture medium. When we began IVF we used media that had been formulated for growing mouse embryos. Some of these media were too nutrient rich and some too nutrient poor. Experimentation in the mid 1990s led to an understanding of the conditions to which early embryos are exposed in the human oviduct and uterus prior to implantation. These experiments led to changes in culture medium to reflect the natural environment (Gardner, 1998). Many laboratories now use "sequential media" in which the mixture of amino acids and sugars to which the embryos are exposed are changed sequentially during the different stages of embryo growth. Culture medium also needs to be supplemented with protein in order for fertilization to take place and for embryos to develop. These protein supplements have become more sophisticated with time. In early days of IVF we used patient serum as the protein source. While this had certain advantages, the serum components were subject to wide variations depending on patient physiology and what that woman had had to eat the day of her blood draw. Current technology uses either purified human serum albumin or a synthetic serum substitute composed of several protein components.

Improvements in embryo culture have certainly increased embryo quality and our ability to grow embryos for longer periods of time in culture so as to better choose the embryos to transfer, but these improvements also have a down side. As our ability to grow embryos has improved and the chance that each embryo will implant has increased, the likelihood that more than one embryo will implant has also risen. When we transferred embryos 25 years ago our chance of live birth was low and the chance of multiple birth was lower. But as the potential for each embryo to survive has increased the likelihood for all embryos being transferred to survive has also increased.

Multiple pregnancy has been the most intractable problem within the practice of ART. To deal with this problem, we have steadily reduced the number of embryos that we transfer (Jain, 2004, Stern, 2007). The reduction in embryos transferred has led to a decrease in the rate of high order multiple deliveries-deliveries of 3 or more babies-but has done little to decrease the number of twins. Twins remain a significant problem in ART. In the 2009 national ART outcome report found on, the national rate of twining for women under 35 years of age using their own eggs in a fresh embryo transfer cycle was 32.9%. The mean number of
embryos transferred in each cycle in this group was 2.1 suggesting that a large percentage of transfers of 2 embryos result in twins. Even for women 38-40 years old for whom the potential of each embryo to survive is lower, there was a significant twin rate of 20.9% in 2009.

In some countries the government has enacted legislation that addresses the number of embryos that can be transferred to patients in any one ART cycle. Sweden, Denmark, Italy and Spain and the United Kingdom are countries that have legislation that restricts transfer of more than a specified number of embryos. In some countries this number is 2 or 3 embryos, in Sweden no more than one is permitted. In the U.S. this sort of legislation doesn't exist and reductions in numbers transferred are encouraged by guidelines promulgated by the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology, the professional organizations that set standards for the practice of Reproductive Medicine and ART (The Practice Committee, 2009). Proposing legislation on the number to transfer in the U.S. is problematic because, in contrast to many other countries, the U.S. government doesn't cover the expense of ART under a national healthcare system. The majority of people in the U.S. who undergo these procedures do so through self pay. This makes encouraging patients to transfer one embryo more difficult. Patients often see a twin pregnancy as a bonus. For the cost of a single cycle they can have two babies instead of one. Unfortunately, the much higher risks to both baby and mother of twin pregnancy over singleton pregnancy are not always appreciated by patients intent on getting pregnant and the inclination to push for transfer of more embryos in the face of emotional and financial pressures can be strong.

The only way to consistently reduce the number of twins is to always transfer a single embryo. Transfer of one embryo would not be a problem if we could always successfully choose the right embryo to transfer to achieve a pregnancy. Unfortunately, despite other improvements in ART we have made little progress in selecting the one embryo most likely to become a baby. Techniques for selecting embryos for transfer has changed little throughout the history of ART. An embryo is chosen for transfer by the way it looks under a microscope in the laboratory. The embryologist evaluates the embryo's "morphology", it's form and structure. The embryologist looks at several aspects of the embryo to determine whether this morphology is of high "quality". We look at the number of cells into which the embryo has divided. We look for embryos that have few fragments, pieces of cellular material that are separate from fully formed cells. And we look for symmetry, cells of the embryo that are of a consistent size and shape. Other embryo qualities factor into the assessment. These factors could include the granularity of the cells of the embryo, the presence of clear fluid sacs, or vacuoles, within the embryo, the rate at which the cells are dividing, and other features. If the embryo has been kept in culture for 5 or 6 days post fertilization, the fluid volume within the "blastocyst" that was formed factors into embryo quality as do assessments of the parts of the embryo destined to be the fetus (the inner cell mass) or the placenta (the
trophoblast). Assessment of embryo morphology is a subjective skill learned by an embryologist over time from looking at large numbers of embryos. It is thus subject to variation and inaccuracy and is highly dependent on experience. Making these assessments even more complicated is that fact that there are qualitative differences in the microscopes, optical systems and magnifications used for these assessments in different laboratories.

Selecting embryos to transfer on the basis of morphology has limits even when assessments are made consistently. It is well known that although embryo quality associates with success, there is not a one to one relationship between good morphology and implantation. Some very poor embryos have become beautiful healthy babies while many excellent embryos fail to implant. In any cycle there could be two embryos of excellent quality each of which could become a viable pregnancy but either of which might not. The challenge when we are trying to decide about which of these to transfer is to choose the right one. Attributes such as the genetic and metabolic health of the embryos do not always correlate with morphology resulting in a need for other methods of assessing embryos. Recent research has attempted to address this issue and to come up with more sophisticated methods for determining which embryo to use. To be useful for this purpose, these techniques need to be rapid, non-invasive, and accurate. The most intensively studied methods have been preimplantation genetic screening and proteomics.

Preimplantation genetic screening was used for many years in an attempt to improve success in women 38 and older and for those patients with repeated failed cycles of ART. The theory behind this procedure is that success in embryo transfer depends on identifying the embryos that have normal DNA. Embryos are "biopsied" to remove one or more cells and these cells undergo biochemical tests to determine whether the chromosomes containing the DNA are normal. This procedure has fallen out of favor with most clinics as the result of several randomized clinical trials showing that it does not improve success (Mastenbroek, 2007).

Proteomics includes a group of methods currently being trialed to identify healthy embryos by the nutrients that they use from the culture media and the waste products that they release into that media (Katz-Jaffe, 2009). Embryos are grown in culture and then that culture medium is removed and assessed for factors that might indicate health and correlate with success. A method for assessing metabolic profiles using near infrared-spectroscopy is one such attempted technique. Although proteomics has been greeted with great excitement, we do not as yet have a method that is ready for routine clinical use. Investigations into these methods are ongoing. As we move into the future, we will continue to improve our ability to grow embryos in the laboratory. Finding methods to determine which of these embryos to transfer such that a single embryo can be transferred with high likelihood of success, will continue to be the goal.

Edwards, RG. In vitro fertilization and embryo replacement: Opening lecture. in In Vitro Fertilization and Embryo Transfer Ed Seppala, M and Edwards, RG, 1985. Annals of the New York Academy of Sciences volume 442. pp 1-22.

SART. IVF success rates, national data summary. 2009 data

Jain,T, Missmer, SA, Hornstein, MD. Trends in embryo transfer practice and in outcomes of the use of assisted reproductive technology in the United States. New Engl J Med 2004:350;1639-45.

Stern, JE, Cedars, MI, Jain, T, Klein, N, Grainger, DA, Gibbons, WE Assisted reproductive technology practice patterns and the impact of embryo transfer guidelines in the United States. Fertil & Steril 2007:88 (2);275-82.

The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology. Guidelines on number of embryos transferred. Fertil & Steril 2009: 92;1518-19.

Gardner, DK, Lane, M Culture of viable human blastocysts in defined
sequential serum-free media. Human Repro 1998:13(3);148-59.

Mastenbroek, S, Twisk, M, van Echten-Arends, J, Sikkema-Raddatz, B,
Korevaar, JC, Verhoeve, HR, Vogel, NEA, Arts, EGJM, de Vries, JWA, Bossuyt, PM, Buys, CHCM, Heineman, MJ, Repping, S, van der Veen, F. In vitro fertilization with preimplantation genetic screening. New Engl J Med 2007:357(1);9-17.

Katz-Jaffe, MG, McReynolds, S, Gardner, DK, Schoolcraft, WB. The role of proteomics in defining the human embryonic secretome. Molec Hum Repro 2009:15(5);271-7.

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