Friday, 19 July 2013

Evaluation of Embryo Development and Best Embryo for Transfer

Patients who have been undergoing ovarian stimulation for in vitro fertilization (IVF) must take daily injections of hormones to stimulate the growth of follicles in the ovaries.  Within these follicles are eggs (oocytes) that must then be retrieved so that they can be placed in a laboratory for fertilization by sperm.  The primary reason that IVF is so successful is not only through the development of multiple embryos, but by selecting the best embryos from this group for transfer into the uterus.  In some ways, IVF is like cramming many months of trying to conceive into one cycle, because of the many embryos that result, but it enhances the probability of success by choosing the best embryo(s) from the group to be replaced.  In a natural cycle, typically only one oocyte develops and is ovulated, regardless of the quality.

Advances in embryo culture have made increasing duration of embryo culture possible.  In the early days of IVF, embryo transfer might have been performed after fertilization (day 1) or on day 2 after oocyte retrieval.  Technical expertise and research studies have refined optimal culture conditions so that embryos are now routinely cultured to day 3 or day 5 after oocyte retrieval, and some embryos are even transferred on day 6.  Ultimately, there are limitations to embryo culture in the laboratory, since the development cycle of an embryo requires that it hatch from its shell and implant in a receptive endometrium or fail to develop further.

In order to select the best embryo for transfer, an embryologist observes embryo development in the laboratory over the course of several days.  On the day following oocyte retrieval, the embryos are assessed for normal fertilization.  Problems in development can arise even this early fertilization is a chemical reaction that is initiated after a sperm cell penetrates an oocyte; however, the insertion of a sperm into an egg does not guarantee that fertilization will occur properly.  In addition, sometimes the defense mechanism of the egg, which should prevent more than one sperm from entering, fails, and the resulting embryo will be abnormal.

Factors that determine when to transfer embryos include the age of the patient, the number of embryos that are available to transfer to the uterus, the quality of the embryos in the laboratory, past history of IVF treatment and the outcome of those treatments. Determining the number of embryos to be transferred follows similar considerations, and guidelines for the number of embryos to transfer have been developed.

In order to choose which embryo(s) to transfer, several systems to assess the quality of the embryos have been developed, so that the best embryo can be selected.  In short, the system created to selecting embryos for transfer must be better than picking embryos randomly.  The most common method for choosing embryos is visual morphologic assessment using a microscope, an embryologist observes and takes notes on how the embryo appears using standard criteria.  By assessing embryos daily, the embryologist creates a record by which to compare one embryo against another so that they may rank in order of choice for transfer.  Since embryos are cultured individually in labeled droplets in a culture dish, a record for each embryo can be created over the course of days.  There are numerous standardized grading systems for evaluating embryos, but each laboratory may also set its own criteria for what indicates better quality embryos based on observed outcomes and may follow unique grading systems.

When evaluating embryos under a microscope, morphological items of interest may include the symmetry of the cells, the evenness of cell size, the number of cells, the number of nuclei in each cell, the amount of fragmentation of the cell, the quality of the shell (Zona Pellucida), and the clarity of the cell’s cytoplasm.  For an embryologist, learning to identify and judge these characteristics requires extensive training, and expertise in morphological assessment is gained over time, so it is not surprising when patients don’t even understand some of the terms reproductive endocrinologists sometimes use when talking about embryo quality.

In general, we do our best to convey to patients meaningful information about the quality of their embryos prior to transfer.  Since most people don’t have an education in human embryology, it can be difficult to convey this information in a satisfactory way. The ideal embryo to transfer on day 3 after oocyte retrieval has eight cells.  Sometimes other embryos may have more or fewer cells.  Most people seem to understand that slow development is likely to decrease the probability of success, but not as many understand that faster development (having more than 8 cells on day 3) may not actually be better.  We suspect that 8-cell embryos are generally the best (excluding other considerations) because the resulting pregnancy rates seem to be the highest when 8-cell embryos are transferred.  This observation does not have an easy explanation, but is just something that has been observed.  However, it also does not guarantee success, and it does not always indicate that 8-cell embryos will the best embryos of any group of embryos available for transfer, just that the probability of success may be highest when this specific situation is observed.

When other factors are considered, such as the other morphologic characteristics listed above, other embryos may be found to be superior in implantation potential.  There are theories as to why the 8-cell embryo is likely to be among the best embryos of any group: there are “checkpoints” in cell development, much like a turnstile – the checkpoint that prevents cell division likely does so because proper conditions for cell division have not been met, possibly because the cell is abnormal, and checkpoints that allow too much progress (more rapid cell division) might not be conducting proper quality control and have failed to slow down abnormal cells or premature cell division.

When there more good quality embryos on day 3 than are appropriate for transfer and when the quality of these embryos is so similar that it is difficult to choose which among them are the very best, embryos are generally cultured for two more days.  Typically, embryo transfer is not performed on day 4 because the stage of development on day 4 is difficult to assess using a microscope: on day 4, embryos are typically at the “morula” stage, which is amorphous and lacks clear characteristics by which to judge development.

On day 5, embryos have ideally become “blastocysts.”  Blastocysts have two cell types: an outer ring of cells that are destined to become the placenta and an “inner cell mass” that eventually becomes the developing fetus; because blastocysts typically have more than eighty cells, it becomes impossible to look at each individual cell to assess it, as is done on day 3.  Instead, a quality “staging” system is typically used.  A number score (1-6) to indicate the degree of expansion, like a shoe size, is assigned, and letter grades, A-D, like in school, are given, first for the inner cell mass and then for the outer cells.  So, a high quality blastocyst might be stage: 4AA or 5AB, for example.  A stage 6 blastocyst has “hatched” from the shell surrounding the embryo.  Since two different systems are used, it is impossible to compare day 3 and day 5 embryos.

Day 5 embryos are given letter grades to assess the quality of the two cell types based on morphologic assessment.  For example, numerous cells in a tight cluster are representative of a better quality inner cell mass than sparse cells that are loosely associated.  On day 3, letter grades are not typically given, which is often frustrating for patients.  Although we could assign letter grades to these embryos, it wouldn’t mean the same as on day 5, since different things are being assessed.  Also, assigning letter grades on day 3 might obscure the real quality of the embryo: for example, if two students take a math test and one scores a 96% and the other a 99%, but the teacher gives them both an “A” grade, are they really the same?  By grouping assessments into more encompassing, simplistic categories, important information is lost.  A patient can always ask the doctor to make such a comparison for ease of mind, but that assessment may obscure the true quality of an embryo.

In the absence of more specific information, such as the genetic make-up of a cell, which usually requires invasive procedures such as embryo biopsy, morphologic evaluation is the mainstay of embryo assessment.  Other methods of assessment, such as analyzing the culture medium in which the embryo has been growing to detect markers of embryo health, are being developed, but have not been shown to be more accurate in determining which embryos have the highest implantation potential.  Not all embryos, even when morphologically perfect, may be genetically normal, just as not all poor quality embryos are genetically abnormal, so selecting embryos based on morphology is not a perfect system.  In addition, it is possible that all embryos are abnormal and no pregnancy will result or that other factors unrelated to the embryos will interfere with successful pregnancy despite morphologic assessment.  However, selecting embryos based on morphologic criteria currently remains the best method to choose embryos without using more invasive techniques.

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