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

Blastocyst
 

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.)

STEP SIX: EMBRYO DEVELOPMENT

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 embryos that have fertilized normally and are ready to grow and develop into embryos that can be transferred.

At the end of fertilization, normal embryos 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 embryos to grow and develop. This is key for the growth of embryos. Embryo 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 embryo is within the tube and develops and divides 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 embryo 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 embryo 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 embryology 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 embryos so that they don’t continue their development. Finally, the embryology lab temperature must be maintained so that when the embryos are taken out of their warm environment to be checked, their temperature does not drop abruptly. 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 embryo can proceed with development to a stage where they can be transferred into the uterus. The embryo actually can be transferred at any point in its development, but time and research has identified the two optimal embryonic 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 embryo has developed into a 2-4 cell embryo, in normal development. Sometimes embryos will have progressed further than 4 cells but that can either be because the evaluation of the embryo was later in the day (48hrs) or the embryo is developing faster than expected, which could be an indication of an abnormal embryo. Embryos 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 embryo as viewed through a microscope. Almost all labs will count the number of cells that the embryo has on that particular date, up to the Morula stage where individual 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 embryo. That is, the amount of fragmentation or debris located within the cell. These fragments are pieces of tissue that have been extruded in cell division. 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 embryos 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 embryos are evaluated for fragmentation and symmetry, and the combination of those two factors lead to a grade. Some clinics break these down and give two grades for each embryo; 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 embryos in the hope of standardizing this among IVF centers within the United States. In their system, cleaved embryos (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). Embryos are then given a score of Good, Fair or Poor.

The biggest disadvantage of current embryo 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 embryos and only Grade 3 embryos have a decrease in pregnancy rates. This method does little to evaluate the internal quality of the embryos, which we know is really the main determining factor for embryo health or viability. That technology is yet to be developed. Embryo 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 embryonic cell. When looking at embryos to decide which to transfer, not only must the embryo appearance be taken into consideration, but its development rate must be considered as well, as an indirect measurement of embryo 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 embryo 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 embryos do have differences in rate of division. In a natural (non-IVF) cycle, the embryo 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 embryo transfer cycles, or 1 embryo transfers, many clinics are now culturing to Blastocyst stage before doing the transfer. This is because culturing to Blastocyst stage leaves less embryos to choose from and MAY indicate a healthier embryo, 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 embryos (7-8 cell, grade 1) because I know that many embryos will not make it to Blastocyst, and that is not necessarily because the embryos are bad. In doing PGS, I have seen Blastocysts turn out to be chromosomally abnormal embryos whereas an embryo that did not survive to Blastocyst had normal chromosomes (PGS usually is done with Day#3 embryos and takes two days to get the result so by the time the result comes back, the embryos are Day#5). I have also seen ugly poor looking embryos (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 embryos 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 embryos to start with so that there will be embryos 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 embryo.

In the next entry we’ll discuss embryo 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: Embryo Transfer". Thank you for joining me today!

Edward J. Ramirez, M.D. F.A.C.O.G.
Medical Director, Monterey Bay IVF
Monterey, CA
http://www.montereybayivf.com/

45 Year Old Danish Couple On Sixth IVF Attempt: Should They Alter Meds & Blastocyst Transfer?

Question:

Dear Dr. Ramirez,

We are a 45 year old couple from Denmark on our sixth IVF attempt. The first three attempts produced 5/5/9 eggs with 225-275 units of Gonal-F and a fertility rate of 100%/60%/60% respectively. Eggs for transfer were three 8-cells on try one on day three, two 8-cells and one 10-cell on try two on day three and two morulas and a 10-cell on the third attempt on day five.

We then increased Gonal-F to 375 units and got 12 and 13 eggs in the next two attempts and a fertility level of 85%-100% and decided to go for Blastocysts and had two BC's and a Morula in the fourth attempt on day six and one BC and a Morula transferred the last time on day six. On the recent attempts we've been supplementing with Ovitrelle prior to aspiration (as well as folic acid, acupuncture and considerate food and no alcohol of course). After aspiration a dose of 16mg Medrol was administered for four days, then 8mg and then 4mg, as well as 81mg of Aspirin for the duration.

Our questions are: 1)should we continue to go for Blastocysts for transfer, and 2) do you have any suggestions as to an altered protocol perhaps in terms of increasing the dose or frequency of Medrol or any other meds that might help us?

Thanks in advance for your reply. T. From Denmark

Answer:

Hello T. from Denmark,

The good news is that your wife's ovarian response is still good and strong. She has done very well on her current stimulation protocols. The problem that you have is not so much the external quality of the embryos formed, they have been good, but the internal quality of the embryos. We know that with age, the quality of the eggs and thus the embryo quality deteriorates. That is probably what is leading to your failure. We rarely see pregnancies after the age of 43 in a woman using her own eggs. However, there was a case in New York of a woman who was successful at 49 years old, and is currently the oldest to become pregnant with her own eggs using IVF. It did take her two and 1/2 years of trying, however. Statistically, your chances of pregnancy with IVF are less than 1% per attempt based on age factors alone.

In terms of whether to do D#3 or D#5 transfers, I don't think it makes any difference. One is not better than the other. The embryos that would make it to blastocyst would still have done so in the uterus. In fact, I think the uterus is a better culture environment than the lab. I generally transfer at D#3 for this reason. In fact, in your case if you were my patient, I would transfer ALL embryos back on D#3 to maximize your chances.

In terms of what other protocols, I use Medrol starting at the beginning of the cycle (D#2) taken as 16 mg until the transfer then decreasing to 8 mg thereafter. I stop with the pregnancy test. I also use aspirin 81 mg per day starting at the beginning of the cycle and heparin 2000 units twice per day injections starting at the beginning of the cycle. I also use a "mixed" protocol of Gonal-f or Follistim + Menopur/Repronex for a total FSH dose of 600IU to start. In most cases, the patients stay at that level but some will decrease based on their response. In your case, your wife does not need more meds since she stimulated well, but a mixed protocol might be advised.

Your only option is to keep trying or move to donor. I am amazed that you have done so many cycles already. Most in the U.S. will not do that many cycles due to cost issues.

Good luck with this upcoming cycle & never lose sight of your goal...it can be achieved if you are open to options.

Edward J. Ramirez, M.D., FACOG

Executive Medical Director

The Fertility and Gynecology Center

Monterey Bay IVF Program

http://www.montereybayivf.com/

Monterey, California, U.S.A.