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A Step By Step Guide To The IVF Process: Step Six -- Embryo Development


Dear Readers: This is the sixth part in the series I have begun to help answer what In Vitro Fertilization (IVF) is and how it works with my world-wide Blog audience. What you read here is what I also provide my patients with on a daily basis. I plan on going into some detail but in a way that is understandable to the normal (lay) audience, and not the medical or scientific one. I also hope that this will not only clarify what you will go through, but explain why things are done a certain way and what the goals of each step are. I also want to convey that IVF is actually a replacement for some of the “natural” steps required to get pregnant and not some miraculous high tech fertility treatment that gets patients pregnant artificially, as many think it is. It is somewhat of a miracle that we can do as much as we can, but there are still lots of things/steps that we cannot do or influence. I hope this discussion will benefit you. This series will continue to be posted over the next few weeks in installments. (For earlier installments in correct order scroll down to the beginning of July 2012.)


At this point, we have gotten the eggs out of the ovaries, put them with the sperm or injected the sperm into them and fertilization has occurred. Each of these steps has lead to a decrease in numbers from the original number of follicles. Not all the follicles had eggs retrieved from them, not all of the retrieved eggs were mature and therefore could not be fertilized, and not all of the mature eggs fertilized or fertilized normally. So now we are left with a cohort of empyos that have fertilized normally and are ready to grow and develop into empyos that can be transferred.

At the end of fertilization, normal empyos are in a 2PN stage, or two pronuclei and is one cell. These are placed into special media that provides the proper nutrition for these early empyos to grow and develop. This is key for the growth of empyos. Empyo culture media has been researched and developed over the evolution of IVF technology, researching and finding the proper combination of chemicals to mimic the intratubal environment. In the natural process, the empyo is within the tube and develops and pides as it makes its way down toward the uterine cavity. So the culture media has to match the media that would be found within the tube. In addition, the environment, such as gas ratios and temperatures, have to match as well. So the requirement for empyo development is to have the proper nutrition within the Petri dish, the proper temperature within the incubator and the proper gas mixture within the incubator as well. Also, the empyo needs to be protected from airborne contaminants outside of the incubator. These factors are so critical that they will influence the success rates of an IVF clinic.

Many empyology labs, such as mine, are designed and constructed to the standards of a “clean” room, such as that used in Silicon Valley for making silicon chips. The airflow within the room is isolated. In other words, the airflow is completely separated from the air outside of the room so that there is little contamination from in the outside. In my lab, the airflow is set to be a positive flow, meaning the air pressure within the room exceeds the pressure outside the room so that when the door is opened the air pushes out rather than allow the outside air to push into the lab. In addition, the airflow within the lab is cleaned by a HEPA filter and Charcoal filter to filter out small particles and chemicals, called VOC’s or volatile organic compounds. VOC’s are the fumes or gasses you can smell after a room is painted or a road is repaved, but there are VOCS’s that can’t be smelled as well. These chemicals can kill or injure empyos so that they don’t continue their development. Finally, the empyology lab temperature must be maintained so that when the empyos are taken out of their warm environment to be checked, their temperature does not drop apuptly. When choosing an IVF clinic, it would be wise to ask about their lab set up and, if possible, tour the lab to see how well the environment is. Many centers have certification by the College of American Pathologists (CAP) which means they have undergone a survey, which includes evaluation for many of the aforementioned criteria for labs, and have passed and received certification of their lab.

If all of the above quality controls are in place the empyo can proceed with development to a stage where they can be transferred into the uterus. The empyo actually can be transferred at any point in its development, but time and research has identified the two optimal empyonic ages as day #3 or day #5. These are the two ages that most IVF centers in the world use in their transfer protocols.

So, by the day after fertilization (48hrs after retrieval) has taken place, the empyo has developed into a 2-4 cell empyo, in normal development. Sometimes empyos will have progressed further than 4 cells but that can either be because the evaluation of the empyo was later in the day (48hrs) or the empyo is developing faster than expected, which could be an indication of an abnormal empyo. Empyos are also examined for external features and a grade is given. There is no universally accepted standardized method for grading but they are all essentially similar. They are all based on the external or “morphological” characteristics of the empyo as viewed through a microscope. Almost all labs will count the number of cells that the empyo has on that particular date, up to the Morula stage where inpidual cells are no longer visible. They will also give it a grade (A,B,C or 1,2,3) based on the clarity of the empyo. That is, the amount of fragmentation or depis located within the cell. These fragments are pieces of tissue that have been extruded in cell pision. Although cells with high amounts of fragmentation have been related to decreases in pregnancy rates, that is not an absolute. I have had the worst looking empyos lead to pregnancy, as have many other clinics. But, we know that most successes come from cells that either don’t have any fragmentation or a minimal amount. Finally, most clinics will look at the symmetry of the cells; do they look fairly equal or are there larger and smaller cells. In my practice, we use a simple 1,2,3 grading system where the empyos are evaluated for fragmentation and symmetry, and the combination of those two factors lead to a grade. Some clinics peak these down and give two grades for each empyo; one to represent fragmentation and one to represent symmetry, and some use letters instead of numbers. In 2010, the Society for Assisted Reproductive Technologies released guidelines for grading empyos in the hope of standardizing this among IVF centers within the United States. In their system, cleaved empyos (those before morula stage) are evaluated for the number of cells present, fragmentation (0%, 1-10%, 11-25%, >25%) and cell symmetry (perfect, moderate asymmetry and severe asymmetry). Empyos are then given a score of Good, Fair or Poor.

The biggest disadvantage of current empyo evaluation methods is that it is essentially a beauty contest, so as I explain to my patients, Grade A or 1 or Good is “beautiful”, Grade B or 2 or Fair is “average” and Grade C or 3 or Poor is “ugly.” We know that most pregnancies come from Grade 1 or 2 empyos and only Grade 3 empyos have a decrease in pregnancy rates. This method does little to evaluate the internal quality of the empyos, which we know is really the main determining factor for empyo health or viability. That technology is yet to be developed. Empyo chromosomes can be determined by removing one of the cells and checking for its chromosomes, a procedure known as Preimplantation Genetic Screening (PGS/PGD), but this still does not evaluate the structures within the cytoplasm, or outside of the nucleus where the chromosomes lie, which are the structures that provide the energy for the empyonic cell. When looking at empyos to decide which to transfer, not only must the empyo appearance be taken into consideration, but its development rate must be considered as well, as an indirect measurement of empyo health. I’ll discuss this decision more in the next step.

To summarize the stages of development, at 24 hrs after egg and sperm have been put together (day of retrieval), the empyo is a 1 cell 2PN; at 48 hrs (Day#2) it is 2-4 cells; at 72 hrs (Day#3) it is 6-8 cells; at 96 hrs (Day#4) it is usually a compacting Morula; at 120hrs (Day#5) it is a Blastocyst. There is some variability to this development scheme as empyos do have differences in rate of pision. In a natural (non-IVF) cycle, the empyo is usually at the Blastocyst stage when it reaches the endometrial cavity. In IVF the transfer is done either on Day#3 or Day#5. Because of the political pressure to do more single empyo transfer cycles, or 1 empyo transfers, many clinics are now culturing to Blastocyst stage before doing the transfer. This is because culturing to Blastocyst stage leaves less empyos to choose from and MAY indicate a healthier empyo, but the latter conclusion is not an absolute.

In my center, I have specific criteria to determine whether or not to proceed to Blastocyst. One of these criteria is that there has to be a minimum of 8 good quality empyos (7-8 cell, grade 1) because I know that many empyos will not make it to Blastocyst, and that is not necessarily because the empyos are bad. In doing PGS, I have seen Blastocysts turn out to be chromosomally abnormal empyos whereas an empyo that did not survive to Blastocyst had normal chromosomes (PGS usually is done with Day#3 empyos and takes two days to get the result so by the time the result comes back, the empyos are Day#5). I have also seen ugly poor looking empyos (4 cell, Grade 3) lead to pregnancies when transferred at Day#3 that would not have survived to Blastocyst. My reasoning is that Blastocyst culturing is not a perfected technology yet. A clinic that puts only a few empyos at risk to develop to Blastocyst is basically risking the cycle by not having anything to transfer. For that reason, I want to make sure that there are enough empyos to start with so that there will be empyos to transfer. In addition, it is still my personal belief that the uterine cavity is a better culture environment and media than what we have available in the lab. For this reason, most of my transfers are on Day#3, but I have colleagues who now transfer mainly Blastocysts. So, when looking at these transfer options with your doctor, ultimately the decision has to be made based on pregnancy rates at Day#3 vs Day#5 transfers and not just because you only want to transfer 1 empyo.

In the next entry we’ll discuss empyo transfer and the decisions that go into this step, as well as, the critical parts of the transfer technique.We will continue this discussion soon with the next installment, "Step Seven: Empyo Transfer". Thank you for joining me today!

Edward J. Ramirez, M.D. F.A.C.O.G.

Medical Director, Monterey Bay IVF

Monterey, CA

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