By fertility experts from Spain.
Watch the recording of the Online Patient Meeting with Jose Luis Pablo Franco, M.Sc., IVF Laboratory Director at Fertility Madrid & ART Vitoria, who is answering the most common questions on embryo quality and PGT-A testing.
Jose Luis Pablo Franco presented an embryo of very good quality from day-1 to day-5 during his presentation. The first picture shows an embryo in day-0 when the gametes are put together, the sperm and the oocytes with conventional IVF or ICSI. Then during day-1, the embryologists check fertilization of that oocyte, they want to see which ones are well fertilized. On day-2 of embryo development, the embryo is divided, it’s a four-cell stage embryo. On day-3, there are 8-10 cells, day-4 is already called a morula stage embryo. It doesn’t give too much information, therefore, embryologists don’t take this one too much into account. The last one is called the blastocyst stage, it collapses and then develops. Together with some enzymes, it will get rid of the zona pellucida. Zona pellucida is a layer that protects the embryo during those first days, but in the end, it will disintegrate.
The day-3 embryo has different features, Jose explained that we see the number of cells, the symmetry of those cells, either even or uneven. We see if they have one nucleus or more per cell, it’s better to have just one nucleus per cell. Another feature could be fragmentation, which occurs when a cell divides into two cells. The cell leaves a part of that cytoplasm outside, and if you have a lot of this cytoplasm outside, this can harm the embryo, it won’t let the embryo develop properly. These are the main features that are taken into account when we speak about the day-3 embryo.
When we’re talking about day-5, the blastocyst stage, there are two parts to differentiate. The first one is the inner cell mass, which has to be of optimal size and shape. The size is a cumulus of compacted cells, and the inner cell mass is going to become an embryo in the future.
Then we have to take a look at the rest of the cells, so the inner cell layer has to have a lot of even cells. Based on these two parts, the embryologists will be able to grade the embryos as A, B, C or D.
Grade A and B are the best embryos for transfer. Grade B has a little more fragmentation, and if you take a look at the blastocyst, you will see that the inner cell mass is not that big, and the trophectoderm doesn’t have as many cells. If you take a look at grade C, there is even more fragmentation, and grade D embryos are not used.
When we’re talking about day-3 embryos, we cannot differentiate between cells and fragmentation. If you see the blastocyst, you cannot differentiate the inner cell mass and the trophectoderm.
It’s an incubator that takes a picture of each embryo every five minutes. That means we don’t need to take the embryos out of the incubator to check and take them under the microscope. Embryos are always in stable conditions, such as temperature, CO2, O2, that need to be controlled for good embryo development. Thanks to incubators, we also have a lot of information to try to select the best embryo for transfer, the embryo with the best chance to implant.
If you look at fertility and the miscarriage rates in your 40s, the possibility of pregnancy is going down fast, while the chance of miscarriage is going up quickly. When we are talking about morphology, we need to remember that if we have a grade A blastocyst, and you’re 30, and then you have a grade A blastocyst when you are 42, the pregnancy rates are not the same. The morphology is the same, but we don’t know what is going on inside this blastocyst. This can be checked with PGT-A (Preimplantation Genetic Testing for Aneuploidies).
As Jose mentioned before, we need to take into account the morphology of the embryo, but this won’t tell us anything about what’s going on inside those embryos. Therefore, this is a technique that can tell us that. This genetic test can identify embryos that have the normal number of chromosomes, which means they are euploid and those that have abnormal cells, called aneuploids. It’s not possible to fix those aneuploidies, the only thing we can do is to check the embryos that we have.
PGT-A is usually indicated for women who had more than 3 miscarriages, with previous implantation failures, which means they have had 4 or 5 good quality embryos transferred, but they didn’t get pregnant. Maternal age is also important. When you are 39 and above, you have a higher chance of miscarriage. An abnormal FISH test is also a good indication for PGT-A, this test will show us whether the chromosomal composition of the sperm is normal.
Another test that we often use is PGT-M (Preimplantation Genetic Testing for Monogenic Disorders). It is recommended if any of the intended parents is a carrier of disorders such as Steinert myotonic dystrophy, Duchenne muscular dystrophy, Huntington’s disease, and fragile X syndrome. This technique is used to analyse the embryos to prevent offspring from the disease their parents have.
Biopsy for PGT‐M is usually performed at day 3 of cleavage‐stage embryo development when the embryo is at the six to eight‐cell stage. On day-3, we choose the cell that we want to take out. We select the one with one nucleus, we make a hole with a laser, and we take that cell out. Sometimes, two cells are taken because, in this case, one cell was for PGT-A, and one cell was for PGT-M. It’s possible to do both. If a patient is in her 40s and has a monogenic disease, we can analyse the embryos for that disease, and after that, we’re going to check the chromosomes, so we can do both at the same time.
During day-5 blastocyst biopsy, we make another hole with the laser. We wait until the trophectoderm comes out a bit, and we aspirate one cell, we continue to aspirate it and perform two pulses of laser to make the blastocyst shrink. When it is shrinking, it’s easier to take out more cells, it’s best to take around six or eight cells from that blastocyst, then it is vitrified, and sent to the genetic lab. The results are ready in about more or less one week.
We used to transfer embryos on day 2 on day 3 but we’re not doing it anymore. We have been doing the transfer on day 5 or later for 8 years already. Why? Because on day 3, you’re going to have more good quality embryos but not all of them are going to reach the blastocyst stage. Nowadays we have better laboratories we have to have better laboratories. The embryo culture media is better now. The conditions of our labs are better, the incubators are better so we really trust our labs right now. We’re not scared about letting them grow to the blastocyst stage. If one good quality embryo on day 3 cannot the blastocyst stage, and if we had transferred that embryo, it would have not implanted or 90% of them would have not implanted. Definitely, I prefer day 5 because they have to reach that day. If this is not a good embryo, it’s not going to reach that day. The embryos that reach the blastocyst stage have better pregnancy rates.
This is something that we need to explain to our patients when we have our first contact. When there is a proposal is to perform an IVF with a PGT-A, we have to explain the results that we’re going to have. We are going to have three kinds of results: we have a white one, a black one or grey one. The white one is that we can have euploid or normal embryos, the black one is the abnormal or aneuploid embryos and the grey one is something called mosaicism. What does it mean? This means when we do a biopsy on day 5 embryo at a blastocyst stage, we take 6-8 cells. When we analyze those cells, some of them are going to be euploid and some of them are going to be aneuploid. These embryos are not the first choice for transfer. We have to transfer euploid embryos. But if you don’t have anything else and the only embryo that you have is a mosaic embryo, we need genetic counseling. We have to see how many chromosomes are involved, which chromosomes are involved and what’s the percentage of normal or abnormal cells. We have to take a lot of possibilities into account and see what’s the best option. Sometimes our recommendation can be not to transfer because it has too many things but, of course, the final decision belongs to the patients. Fortunately, this occurs very rarely but it’s something that we need to explain to our patients. We are saying we’re going perform a PGT-A so this way we are going to transfer all the euploid embryo so your pregnancy rates are going to be really high. But what happens if you biopsy three embryos, two are abnormal and one is a mosaic embryo? Patients need to know this because we don’t like to have mosaic embryos, of course, because it’s something difficult to explain and something difficult to make a decision of what to do with that.
This is a kind of difficult question to answer. As every patient is different, you have to look at so many things. You have to see if we have done more cycles, the quality of the embryos that they had in those cycles if you perform PGT-A, what’s the result of that PGT-A, the maternal age, etc. You have to take into account many things. One good example came to my mind: a good friend of mine had a baby when she was 38 and then she tried naturally and had two miscarriages after that. She came to see me when she was 40 years and 6 months old. We talked about a lot of things and decided to do an IVF with PGT-A because of the miscarriages and her age. We did four cycles, 15 embryos, all of them abnormal. She had the baby three years before but now only two years later, 15 embryos were aneuploid. We sat down and talked for a long time and I suggested maybe it was a time to stop, to think about other things, forget about fertility and enjoy your baby. Because she was losing time with his two years of cycles. You’re always thinking about having a new baby and it was perhaps time to stop and think about another thing. She stopped and then she had another natural miscarriage. She came to see me when she was 42 years and 6 months old and she said “I want to try it again” and I asked “Are you sure? You’ve been through so many cycles?” She wanted to do it again so we had 14-16 more embryos to analyze. All of them were abnormal. This is a rare case. We don’t really see this often because it is too much money and, psychologically, it is too hard. But she wanted to do it, she needed it to do it. But now we have stopped for sure. We’re not going to make any more cycles. She stopped 4-5 months ago and thought about other possibilities that we talked about, oocyte donation. She’s now waiting for her embryo transfer with oocytes from a donor and let’s see what happens. That was a good example for the today’s topic because I would say that 20 out of the 30 embryos that we analyzed were grade A so the embryos were beautiful but the problem was their machinery that we talked about wasn’t in a good condition and that’s why she didn’t get pregnant or she had the miscarriages.
I showed you on the slides and you could see the differences but the most important difference between day 3 or 5 is that on day 3 we take out one cell from an embryo, from an 8-cell embryo more or less. In a blastocyst, we take 6-8 cells from a 200-250-cell blastocyst. The first thing is that the volume that you are removing from those embryos is much higher on day 3 so that could harm the embryo and could harm their development to reach the blastocyst stage. Then, of course, when you have more cells, you have more DNA to analyze and the results are more reliable. On day 3, you’re going to have more embryos for biopsy because some of them are not going to reach the blastocyst stage. If a patient pays per embryo biopsy, you’re going to pay less when you perform the biopsy on day 5 because you’re going to have fewer embryos and those embryos are better than those on day 3.
I’m going to start with the second question. How long we need to wait for the results? Normally, it takes one week. We biopsy the blastocyst, we vitrify the blastocyst and then we have to wait around one week, sometimes less than that. We tell our patients that we have to wait one week for the results. After we have the results, we have to prepare the endometrium, if they want to do it in the next cycle. So we prepare the endometrium, thaw the embryo and we put it into the uterus.
Regarding the first question, I prefer to do it in a fresh cycle. Sometimes we have to do it because we don’t have a result from that first biopsy and we have to repeat the biopsy of that embryo. We don’t really like to do that because if it’s frozen, you have to warm that embryo, you have to perform the biopsy again and you have to verify it again. It’s too much for the embryo. It’s not a good thing to do. For sure, I prefer fresh. What we do is wait till the blastocyst stage, biopsy that embryo, then you’ll vitrify the embryo and wait for that result for a week. Just in case we didn’t get a good result or we couldn’t analyze for whatever reason that’s when we have to perform the biopsy on “a frozen embryo”.
The good thing is to have a grade A or grade B blastocysts for a biopsy but in case you have a hatched blastocyst, I think you mean that the embryo got rid of its zona pellucida totally, is not easy to do a biopsy on it. It is because the zona pellucida always helps you to perform the biopsy. Is it worthy? I don’t know. It depends on the cycle if you have a lot of grade A or grade B blastocysts, if you see any possibility for those blastocysts to have a good result and implant in the future, I think it could be a good thing to evaluate. But it always depends on what you have. If you have a lot of good quality embryos, this is not a really easy blastocyst to biopsy.
That’s the best thing to do and it is good to have it first. You need a karyotyping before the PGS because you can find something that you need to study or even if you are young and your karyotype is normal, we don’t need to perform a PGT-A but it’s always needed before the PGS. It is the right way to do it.
I don’t know if you have yet performed the embryo transfer or not but sometimes it’s difficult to answer this kind of question. When you’re 40, I would say your chances for pregnancy, if you don’t analyze the embryos, would be around 25-30%. We take into account all the embryos that we have. This is statistically talking. We have to take all the blastocysts with patients in their 40s so if you don’t do PGT-A, you don’t know if they are euploid or aneuploid. You transfer all of them and so the aneuploid ones are not going to implant or if they implant they’re going to finish in a miscarriage. Only the euploid ones are going to implant. So your chances of pregnancy would be around 30%. If you analyze those embryos and transfer one analyzed embryo, the pregnancy rate will rise to 60-70%. But you have to have that euploid embryo. We can analyze those five embryos and I’m telling you about 60% of pregnancy rate but if those five embryos are not euploid, we’re not going to transfer anything so your possibilities are zero. If you are asking me if I recommend you to do a PGT-A right now I would say try at least 2-3 blastocyst and see what happens. Then with the rest, you can try the PGT-A. As I said, I don’t like to warm the embryo, then vitrify it again and then warm it again for the second time. I would say try maybe 2-3 embryos I see what happens and then if you with rest you can think about performing PGT-A.
The answer is no because there are other things that we don’t really know about. The chromosomes of the embryo are a really big part of the chance to get a pregnancy but there are more things that we don’t know about. Is it worth it? As I said, I like to use this technique when you have an indication for that. If you are 32-35, I won’t propose you PGT-A because the percentage of euploid embryos is higher than the aneuploid embryos. If you analyze the embryos and you have euploid embryos, the chance to get pregnant is about 60-70 % so it’s a really good chance to get pregnant. But is it worth it? Like I say, it depends on your case, your age, your embryo. You are 40. It doesn’t guarantee you a pregnancy and it could be worth it in the future. Try with 2-3 of those embryos and then you will see in the future. Talk to your clinician about what to do with the rest of them.
Certainly, it is an invasive technique. I’m not worried at all about the technique – doing the PGT-A, doing the biopsy. I’m not really worried about that but still, it is an invasive technique. The risk for that is that the blastocyst can be so sensitive that it cannot resist the biopsy. After that, you have to vitrify the embryos so they have to survive after that. The thing is that blastocysts are strong and they persist a lot but, of course, there is always a chance of harming that embryo because it’s an invasive technique. If you have done a lot of biopsies, this is nothing that I really worry about. But it’s a good question because it’s an invasive technique. If you are 39 or 40 and you only have one embryo, sometimes we think we weren’t going to perform a PGT-A but, in the end, we only have one blastocyst, we can just transfer it and see what happens. I’m not recommending it if you’re 43-44 because of the risk of aneuploidy is too high. If you are 39-40 and you only have one embryo, and because of the risk of the embryo being too sensitive for a biopsy, you can consider just transferring it and checking what happens.
Of course. The first thing I want to say here is that we only perform the biopsy on embryos that have a chance to implant. We don’t perform the biopsy on the embryos like grade C or D. It’s related to good morphology to PGT-A and, of course, you have more chances if your embryo is a grade A. You biopsy that embryo and your result is an euploid or normal embryo. Your chances could be better but the thing is that if you have the result of our euploid embryo it means that you have a lot of chances to get pregnant. But, of course, then we have to take into account the morphology, of course. If we have 2-3 euploid embryos, normal embryos, first I’m going to transfer the best quality embryo.
This is related to maternal age. It’s not the same to analyze ten embryos from a 35-year-old woman as 10 embryos from a woman in her 40s. In your case, you are 40, I would say that, more or less, 3-4 of your embryos are going to be euploid, they’re going to be normal. The rest are going to be aneuploid. If you are 35, I would say about five embryos are going to euploid and the rest is going to be aneuploid. This always depends on the maternal age or something else like a normal FISH technique that we perform on the sperm that I told you about or perhaps sperm fragmentation, but, basically, it depends on the maternal age.
Of course, it is good. We have to differentiate here if we are not talking about PGT-A, we’re just talking about morphology. We normally like to have embryos on day 5. It doesn’t mean that on day 6 there are worse embryos. Sometimes the embryos need a little bit more time. This also depends on the time when we perform the conventional IVF or the microinjection (ICSI). It doesn’t really mean that the blastocyst on day 6 is worse than the one that you have on day 5. Sometimes we are not sure about the quality of that blastocyst on day 5 so we leave them to day 6 and sometimes even until day 7. In a few cases, we don’t have anything else – sometimes the embryos need one or two days to reach good quality blastocyst stage. It depends on the cycle. I prefer to have a blastocyst on day 5 and then the ones that I have in this cycle on day 6 could be worse but this doesn’t mean that you can get pregnant with blastocyst from day 6. I don’t know if I was clear about this or not.
We prefer to have good quality embryos for a biopsy the reason for having a no result is grade C embryos. I’m not really sure about them. 20-30% of your embryos with no results is too high. I would say that max. 5-10% with no results should be OK, depending on the quality of the embryos. This is a really important thing, the morphology and the quality of the embryos. Here the percentage is too high. That could be because they’re grade C. During a biopsy, we have to take cells only from the trophectoderm (the panel B), never from the inner cell mass because the inner cell mass is going to become the future embryo. We have to take cells from the trophectoderm – I think is what you are saying here, the outside cell mass. I’m not saying this is not possible because the morphology is so important but I think that the percentage could be a little bit higher. Perhaps if trophectoderm is of this quality, maybe you should not biopsy those embryos. Maybe if the quality is not that good to take 6-8 cells to analyze them, you may not want a biopsy on those embryos.
So they analyzed 20 embryos and only 3 were euploid embryos and the rest was aneuploid. I would say not to transfer because you have too many aneuploid embryos. If they are not good quality as your embryologist said, the trophectoderm is grade C, and they biopsied the trophectoderm and they didn’t get the result, there is maybe something wrong with those cells. My recommendation would be not to transfer those embryos without the analysis. I wouldn’t do the biopsy again in those blastocysts because of their morphology and because you’re going to harm them a lot and you are not going to be able to transfer them for sure.
The reason to perform PGT-A is not to transfer the aneuploid embryos. Why are we doing the PTA? We perform it just to know about our embryos so we discard the aneuploid ones and we only transfer the euploid ones. There is some evidence from the US when we talk about mosaic embryos. Do you remember the gray result I mentioned? That you can have the aneuploid cells and euploid cells and, of course, there’s evidence that most of the rest of the cells of that blastocyst need to be euploid so that way when you transfer that embryo, you’re going to get pregnant for sure. If you have a 100% aneuploid embryos and you transfer them, and although there is nothing 10% certain in biology, I would say that you’re not going to get pregnant or you’re going to suffer a miscarriage. I would say that evidence should be more for mosaic embryos. Maybe they transferred because they didn’t have anything else and the patient wanted to try but we don’t do that. We perform PGT-A – we don’t transfer aneuploid embryos. So we don’t really have too many cases of that, that’s true. I would say you’re not going to get pregnant with aneuploid embryos or have a miscarriage.
Perhaps this is a different grading than we have here in Spain because I don’t understand the number that they put at the beginning but for sure I would start with BC. I guess this BC is great for the inner cell mass and the trophectoderm. I guess 3BC is better than 4BC so I’d start with that one. They are not really good quality embryos. Your sister was 34. If they are euploid, just try with them. Even though they have not really good quality, just try them because they are euploid. Start with a BC and then I guess the CC and then the 4BC. I think that that would be right. If they are euploid, of course, just try.
You’re trying with your own oocytes, right? The answer is you have to perform a PGT-A again on your embryos. I would not recommend transferring two euploid embryos at the same time. We don’t like that. We prefer to transfer one at a time. Why? Because you are 42 and maybe you look very well, maybe you look like in your 30s but the thing is that biology is cruel. Your body is always better off to have a single embryo pregnancy rather than a pregnancy with two embryos. It’s much better. You have type 2 diabetes – that’s even worse. You’re going to increase the risk of that pregnancy by transferring two embryos. If you have two euploid embryos, just transfer one and then keep the other one just like you did in the last cycle. It’s always better to have a pregnancy with just one embryo – don’t doubt it.
With PGT-A what we are doing is analyzing the embryo, the inside of it, the chromosomes. What are the other reasons? There’s the endometrium – we have to have a receptive endometrium. The embryo needs to be in contact with that endometrium, it needs to talk to it and see if the endometrium lets the embryo implant there. This is the part, the implantation conditions, that we don’t really know about. You can prepare the endometrium, you can try to make it more receptive but still once we transfer the embryo inside the uterus but still there are things that we don’t really know. Even though it is an euploid embryo, it can collapse for whatever reason and it gets blocked and can’t implant in the uterus. But the most important other reasons would be the dialogue between the embryo and the endometrium.
I mentioned the white, black, and gray biopsy results. There is another one. The morphology, the quality of those embryos could be one reason, maybe the reason is that we didn’t perform the biopsy well. We have to put those cells inside a small tube. This is something we have to do under a microscope and sometimes we don’t do it properly. Maybe for whatever reason, the DNA doesn’t come out from the nucleus and sometimes it’s something inner end of the technique. The thing is you have too many “no result” embryos.
I would say that the fragmentation test is important here if it’s bad. The problem here is that we use different percentages when we talk about fragmentation. Depending on the technique that you are using and depending on the country. They say it was 29% damaged – it is not that bad but I don’t know what the brackets are for considering normal or abnormal sperm. If your fragmentation is damaged, I think the PGT-A could be a good thing to do. Sometimes we use a special platform to see sperm that is not fragmented you know. There are different ways to treat the fragmentation but the thing is I don’t know if 29% here is considered bad or not. As for the blastocyst, I’m not really sure what the 3.2.3. or 4.2.2 mean. In Spain, we don’t follow this grading and it’s difficult to say. But you’re 38 so you are about to think about PGT-A so if the fragmentation is it’s not good, maybe it could be a good option to perform a PGT-A on those embryos. If you already have had a miscarriage, for sure in the next cycle I would try PGT-A
I would say for sure do PGT-A because this is going to be your first cycle and you already have had two recurrent miscarriages and one biochemical pregnancy where we can’t know what happened there. You’re 38 and close to 40 which is a good indication to perform PGT-A. I would do it for sure. Just in the case where you only have one good blastocyst, you can try without but if you already have had two miscarriages, I would go for PGT-A.