Science Pulse    How Embryos Make the Grade

For the most part, the process of gauging the quality of an embryo involves a lot more than what meets the eye. Embryo evaluation, a standard practice in fertility clinics worldwide, allows PFC embryologists to provide you and your physician with valuable information about your embryos, and about your fertility.

When eggs are retrieved from a patient’s ovaries, they are surrounded by thousands of helper cells that prevent us from seeing eggs directly or making any comments about quality. For patients having ICSI (Intra-Cytoplasmic Sperm Injection), we strip away the helper cells, but even then, very little information on quality can be ascertained. Only when eggs are of particularly poor quality are we able to observe obvious differences from healthy eggs. The vast majority of eggs do not show any characteristics to indicate quality. Therefore, the embryologist will not typically convey any information about egg quality.

For most patients, about 70% of their eggs will fertilize, regardless of egg quality (see Fertility Flash Vol 3 Issue 3 for more details). And fertilization is not necessarily an indication of things to come. A high rate of fertilization does not suggest better embryo quality.

The first step in embryo evaluation is examining fertilized eggs for the presence of two pro-nuclei (PN) in their centers. Each of these pro-nuclei contain the DNA from one parent, and in 95% of fertilized eggs they are unremarkable. In a small number of fertilized eggs however, we do see nuclear abnormalities such as asymmetry of size, any number other than 2, or failure to align in the egg center. These unusual abnormalities do suggest abnormal embryos, and we make patients aware of these. Each embryo will be noted for its number of pro-nuclei, e.g. 2PN or 3PN.

All of the fertilized eggs or embryos are kept in the laboratory for a minimum of 48 hours prior to the embryo transfer procedure. Only eggs that are grossly abnormal, such as those fertilized by more than one sperm, are discarded. Even eggs in which we see no evidence of fertilization are kept in case they fertilize late. No further observations are made until close to the time of transfer as the embryos must be left undisturbed in the laboratory incubator.

While incubating, the fertilized egg begins to divide. This first round of cell division happens within 12 hours of fertilization and thereafter, the cells in the embryo divide in two about every 16 hours. This continuous process of cell division is a very important indicator of embryo health as the embryos have only 4 to 5 days to make enough cells for implantation in the uterus. When we look at the embryos on their third day of life, we expect to see about 8 cells. Many embryos will have close to this number, and some will even have more, but embryos that are significantly delayed with 4 cells or fewer will have very little chance of establishing a pregnancy. Counting the number of cells in an embryo is the most important part of assessing their quality. (Article continued on back) We will give patients this cell count for each of their embryos.

A much less important aspect of determining quality involves observing the integrity of the cells. Some embryos will have uneven or asymmetrical cells and some will have one or more cells that are disintegrating. Cellular fragments that result from this disintegration are only an indicator of quality when they are severe, or when most or all of the embryo has broken up. Fragmentation is a normal feature in embryos and only about 20% of embryos have no fragments at all. However, the absence of fragments does not guarantee pregnancy as there are many other factors involved in embryo quality.

This degree of fragmentation and cell asymmetry is given as a grade, usually 1, 2 or 3. Grade 1 embryos look beautiful and normal in every way. Grade 2 embryos will have a small degree of fragmentation and or unevenness, but are still considered high quality. Only if an embryo is in real trouble and has more fragments than cells, will we assign the dreaded Grade 3. These embryos very rarely implant after transfer and are not considered viable enough to freeze regardless of how many cells they contain.

While cell number is a very important predictor of embryo quality, grade is mostly most useful in deciding which embryos to transfer or freeze. Grade allows us to rank embryos when there are several embryos with the same cell number, and grade is only loosely associated with quality. Embryos that are given a poor grade are unlikely to implant after transfer, but other factors are much more important in establishing a pregnancy. A patient’s age for example is the best predictor of pregnancy, regardless of embryo grade. Also there is no correlation between embryo grade and genetic status. Genetically abnormal embryos are just as likely to be Grade 1 as genetically normal embryos. Moreover, a pregnancy from a Grade 3 embryo has no more increased risk of birth defects than a pregnancy from a Grade 1 embryo.
- Joe Conaghan, PhD, HCLD and Carolyn Givens, MD
Joe Conaghan, PhD, HCLD, PFC's ART Laboratory Director, is internationally recognized for his work with embryos. His background includes involvement in the first PGD on human embryos. His high standards and extensive experience brings national recognition to our laboratory. He also trains fellow embryologists for licensure and is an inspector for CAP, the licensing body for IVF laboratories in the USA.
Carolyn Givens, MD has been performing embryo transfers for over 12 years. She was the first in San Francisco to successfully initiate a pregnancy using ICSI or intracytoplasmic sperm injection. She currently Co-directs PFC’s Pre-implantation Genetic Diagnosis (PGD) Program. Dr. Givens’ excellent care is recognized by her peers who single her out as a "Best Doctor" in national surveys.

Photo Gallery    Grading Embryos

Photo 3 Pro-nuclei:
This is an abnormally fertilized egg with 3 pronuclei. One nucleus is from the woman’s DNA, but the other two pronuclei are from two different 2 sperm (each sperm contributing 50% of their DNA) that penetrated this egg. The resulting embryo will typically die within 48 hours because it has 50% more DNA than normal.

Photo Grade 1:
This embryo has even, symmetrical cell sizes. There are no cellular fragments. This is a beautiful grade 1, 8-cell embryo.

Photo Grade 2:
The embryo is developing slowly. It has 5 cells, however it should have 8 cells at this stage. Each of the 4 cells visible in this view are different sizes. This is a grade 2 embryo.

Photo Grade 3:
Although this embryo has 8 cells, many cellular fragments are present. These are scattered throughout the embryo. Cell sizes are uneven. This embryo is a grade 3. One positive characteristic is the flattening of cells against each other. This shows that the embryo is alive and attempting to develop.

Conception Health    The Benefits of Prenatal Diagnosis

There are two different prenatal diagnostic tests, chorionic villus sampling (CVS) and amniocentesis. CVS is a procedure in which a small amount of tissue (chorionic villi) is obtained from the developing placenta at approximately 10-13 weeks of pregnancy. The tissue is then evaluated for chromosome abnormalities, and if indicated, specific genetic diseases. The primary advantage to CVS is that this test can be performed much earlier in pregnancy than amniocentesis. However, CVS does not detect neural tube defects (spina bifida, meningomyelocele or anencephaly). Therefore, patients who opt to pursue CVS undergo an AFP blood test and a high-resolution ultrasound later in pregnancy to screen for these defects. Also, approximately one percent of all CVS results will show a mixture of normal and abnormal chromosomes, which is called mosaicism. The majority of the fetuses in these pregnancies are normal, however additional testing, including amniocentesis, may be indicated.

CVS can be performed one of two ways depending on the location of the placenta within the uterus. The transcervical method is performed by inserting a thin catheter, guided by ultrasound, through the vagina and cervix to reach the chorionic villi. The transabdominal method is similar to amniocentesis. Using ultrasound, a thin needle is inserted through the mother’s abdominal wall to obtain a small amount of tissue. In either case, this placental tissue is then sent for analysis.

Amniocentesis is typically performed between 16-20 weeks of pregnancy. Under ultrasound guidance, a thin needle is inserted through the mother’s abdominal wall into the amniotic fluid surrounding the fetus. A small amount of fluid is then taken and analyzed for chromosome abnormalities, neural tube defects, and if indicated, specified genetic diseases. The main benefit to amniocentesis is that although it is performed later in pregnancy, it is possible to test for genetic disorders, including chromosome abnormalities and specific genetic diseases, AND neural tube defects, such as spina bifida, all at once.

Whether patients choose CVS or amniocentesis, it is possible to obtain the same information with either procedure. However for patients who choose CVS, it is necessary to do a follow up blood test and detailed ultrasound in the second trimester to rule out neural tube defects. It should be noted that the results from this blood test and ultrasound are not as conclusive on neural tube defects as the results from an amniocentesis. Because both procedures are considered invasive, meaning that it is necessary to enter the womb with either a needle or a catheter in order to obtain cells, there is a small risk of miscarriage due to the procedures. The risk for either CVS or amniocentesis is approximately 1/200. Diagnostic results from either procedure take about ten days to be completed.

Regardless of whether you are considering CVS or amniocentesis, genetic counseling is an important step in your overall decision-making process and in assessing your risk factors for genetic disorders. Genetic counselors are available to discuss in further detail the benefits, limitations, and risks for prenatal diagnostic testing in order for you to make the best decision for you and your family.

Kendall Glynn, MS, CGC, Certified Genetic Counselor, California Pacific Medical Center

Ask the Experts    How Do I Buy Sperm?

A.
When it comes to finding a sperm bank (check with our New Patient Guides for a list of recommended banks) and choosing a donor, you will have plenty of options. Typically, individuals have very specific ideas about the physical, intellectual and sporting abilities they would like in a donor and the sperm banks do a very good job of providing a wide variety of donors. They also ensure that the donors are healthy, disease free and have good quality sperm, though some banks have better quality sperm samples than others. All of this information will be made available to you, ensuring an informed choice.

Unfortunately, we do not see patients making donor choices based primarily on sperm quality. From a medical standpoint this is an important factor. You would be wise to choose a donor with high numbers of sperm with good motility. Motility tells you how many of the sperm are alive. Unfortunately human sperm samples contain a lot of dead sperm and freezing those samples will kill even more sperm. After thawing, most sperm banks will guarantee that at least 35-40% of the sperm will be alive, but it’s worth taking the trouble to find samples that will thaw with motility of 50% or more. To calculate the total number of live sperm that you are buying, multiply the sperm count by the motility. We expect this number to be at least 20 million sperm, but the more the better. This is especially important when choosing donor sperm for intrauterine insemination (IUI) as sperm are quickly attacked and killed by white blood cells (the foot soldiers of the immune system) when placed inside a woman’s body. So the more live sperm we have, the greater the chance that one will make it to fertilize the egg.

Once you have chosen your donor, and are satisfied that he has great sperm, your final decision will be whether to buy the sperm processed or unprocessed. If a fresh sperm sample is frozen without being processed, it will be cheaper for you to buy, and easy for you to take home to do your own vaginal insemination. This type of sample is usually referred to as Intra Cervical Insemination (ICI) prepared, and it is essentially neat semen to which they have added cryoprotectant. Sperm banks will also offer IUI prepared sperm at a higher price. This refers to specimens that have the dead sperm and seminal fluid removed before freezing. You would typically only buy this type of sperm if you were having your Physician perform your insemination. Your Physician will place the sperm directly into your uterus and thus closer to the site of fertilization. It is important to understand that ICI prepared sperm cannot be placed in the uterus as the seminal fluid may cause contractions that could be painful and also counterproductive to the sperm trying to swim up to reach an egg.

When buying donor sperm for an In Vitro Fertilization (IVF) cycle, we suggest buying ICI prepared sperm. It is less expensive and our laboratory will have to process the sample regardless if it is ICI or IUI prepared sperm for use in IVF.

If a sperm sample thaws with less live sperm than guaranteed by the bank, (an event that we occasionally see) we will give you a report that you can take to the sperm bank for a refund. Their liability however, is limited to the amount you paid for the sample. We therefore recommend that you buy more than one vial of sperm at a time, and suggest that you buy sperm that was frozen on different dates. This will minimize the chance that you will end up with sub-optimal sperm on the day of your insemination. Couples undergoing IVF with donor sperm should always have a minimum of 2 vials on hand for their cycle, even though we usually only need one.

If you have sperm left over after your cycle, you cannot return it to the sperm bank for a refund. You can continue to store it at PFC and you will be billed annually for the cost of storage (currently $400 regardless of how many vials you have stored). The sperm banks will also store the sperm for about the same storage fee, or you can ask for it to be discarded if you no longer need it. Bear in mind however, that the same donor may not be available the next time you want to get pregnant. If you are hoping to have two or more children that will be true genetic siblings, you may want to stockpile some sperm from your favorite donor. - Joe Conaghan, PhD

Patient Odyssey    Results of Our Two “Grade 2” Embryos

Our journey began with high tech ovulation monitors, hoping for the best every 28 days. We lost faith more than once, struggling through the monthly disappointments. Eventually we went for fertility testing – sperm count and motility for him, ovary and uterine health for me. Everything looked fine. Still no results.

Next we sought out a Naturopath. More tests plus a daily regime of herbal remedies. No luck. Then we turned to our trusted OB for a round of IUI – what we affectionately called the “turkey baster”. Still no results. Next we looked to Eastern medicine and engaged an acupuncturist specializing in infertility. More herbs. Every day. No results. Finally, in the summer of 2004, we began researching Bay Area fertility clinics. We assumed it would be the last step in our journey, either way.

In September 2004 we began working with PFC and embarked on our first round of IVF. Scott learned to give shots, and Cara dutifully produced five eggs of various sizes, only two of which turned into embryos, both of very low quality. We hoped for the best but knew the chances were minimal. The pregnancy test ten days later confirmed we weren’t pregnant. It was disappointing, to say the least.

We met with Dr. Ryan and discussed what to expect for our second round. If we had a similar experience in round two, which was likely, we needed to consider the alternative of an egg donor. This was not good news. To learn that IVF may not allow us to have our own children was extremely discouraging. We took several months off to prepare for the second round. We felt it might be our “last chance” of having a baby that was biologically ours.

In January 2005 we started our second round at PFC with a modified stimulation protocol. This time Cara produced eight eggs, two of which became “viable” embryos and a third that was marginal. Even though the two viable embryos were “Grade 2” and were only six or seven cells, they were at least considered “reasonable.” We hoped, prayed, and tried to remain calm and positive.

Then the “magic” began. Within a few days of the egg retrieval Cara had a dream we would have twin babies – a girl and a boy. And a week before our first pregnancy test a close friend dreamt we were pregnant. Then his wife announced she had a premonition we were going to have twins. We stayed hopeful.

After ten days of waiting we received the results of our pregnancy test, and it was positive! Plus Cara’s hCG level was high enough to indicate there could be more than one “bun in the oven.” At our 6 week ultrasound we announced to Dr. Ryan that we had twins (before the ultrasound). When she found two embryos (7mm and 9mm long) with two happy heartbeats, it was as if we were being told something we already felt we knew.

We are now entering our 4th month of pregnancy. Every day we envision our babies being born healthy and happy. We are so grateful to Dr. Ryan, the entire PFC staff, and the many people that have supported our journey. We remain in awe of both medical technology and the magic of the universe for helping us to create two new lives. Cara and Scott France, Pacifica, CA

Thank you for your interest in subscribing to Pacific Fertility Center’s free monthly newsletter. In order to better protect your privacy, we have a new secure subscription/log in form. We respect your privacy: Your email remains confidential and will not be shared or sold. Please click here to change your subscription preferences.

-- Best regards from all of us at Pacific Fertility Center.