By fertility experts from Spain.
Watch the webinar with Dr Danny Daphnis, Scientific Director at Mediterranean Fertility Center, Chania, Greece, in which he is explaining embryo grading.
Dr Daphnis started his talk by explaining the main aim of IVF, which is to have the live birth of a healthy baby. Everybody’s trying to get a good result. The main criteria for a successful IVF therapy are: sperm quality, egg quality, embryo, and endometrial quality.
The hardest thing in all these aspects is that we have no way of actually testing the proper quality of either sperm, egg, embryo or endometrial receptivity. We only have indications. Dr Daphnis explained that:
‘When you’re talking to your gynaecologist or your IVF specialist or embryologist, remember that when they’re talking about good quality embryos or good quality eggs or very good endometrium, it’s always about indications. The only proof of a good embryo or a good endometrium is a pregnancy, a healthy pregnancy.’
Dr Daphnis, later on, explains that all embryologists and all doctors get trained on embryo transfers at the very final stage of their training. The reason is that it’s difficult. It’s the most important time of an IVF, it’s the most important time because if you get that wrong, all your efforts, all your attempts have gone to waste. Therefore, doctors and embryologists need to realize how important it is to do a proper embryo transfer and select the best embryos for a woman to transfer back. The timing of embryo transfer, and embryo grading, are key ingredients for a successful IVF treatment.
Most IVF centres nowadays perform a day-5 embryo transfer however, you can have a day-2 embryo transfer, day-3 embryo transfer or day-5 embryo transfer.
These three times are the best times to perform an embryo transfer. There’s no way to predict whether day-2, day-3 or day-5 is best for transfer. Dr Daphnis emphasized that not all embryos will reach day-5 or the blastocyst stage, not all embryos, are destined to become babies. Therefore, Dr Daphnis advises not to see blastocyst transfer as the best solution. Unfortunately, it is not always the best solution, it is the best solution if you have enough embryos on day-2 or day-3 of development and you’re going to have 1 or 2 good blastocysts on day-5.
If you have 2 or 3 embryos on day -2 or day-3, and it is allowed in your country to transfer them all back, it is not going to make any difference in your success rate if the embryo transfer takes place on day-3 or day-5. The only thing you will gain by waiting for the embryos to reach day-5 is to see if they continue to grow. However, people against blastocyst transfer will always tell you that a lady’s womb is a much better incubator than the incubators you can find in the labs.
Blastocyst transfer is not for everyone. Yes, it’s a very good solution, but it was originally made for embryologists o be able to select the best embryos for the patients.
According to Dr Daphnis, the first thing that embryologist want to see is an embryo with 1 cell with 2 pronuclei and 2 polar bodies. The 2 pronuclei are like 2 craters in the middle of the cytoplasm, and the polar bodies are 2 small balls that you can see just outside the cytoplasm.
Then on day-2, you expect the embryo to be of 2 to 4 cells. On day-3, you expect the embryo to be 6 to 8 cells. On day-4 is what we call a morula stage, and from then on, the embryologists stop counting, the embryo becomes a mass, and on day-5 or day-6, depending on your lab, we expect the embryo to be a good-looking blastocyst.
When it comes to embryo grading, Dr Daphnis stresses out that when we’re talking about the quality of the embryo, it all has to do with the morphology. Unfortunately, we do not have the technological means to know if the embryo is a baby.
If you have a big cohort of embryos, you would expect to have a range of qualities. Usually, you would have some good quality, medium quality and some poor quality.
Blastocyst grading is quite intricate, but it has made embryologist’s life quite easy. We can select with easiness the best embryos, the best quality embryos. The blastocysts are graded in 3 different ways, usually, you will hear the blastocysts are graded from 1 to 6, you’ll see a number at the beginning, then you’ll see it has two letters AA, BA, BB, CC, DD, etc.
First, Dr Daphnis explained what the inner cell mass and trophectoderm is.
When an embryologist grades the blastocyst from 0 to 6, it states how big the blastocyst is.
The first letter means how big the blastocyst is. It doesn’t mean that if you have a 0, which is a morula, it is a bad blastocyst, it’s just not gone as far, at the time, that the embryo was checked. That is why Dr Daphnis emphasized that development is not a negative aspect. It’s not good to have, for example, a 5 or 6 – a hatching or a fully hatched blastocyst because you want that part of the development of a blastocyst to take place in your womb, not in the laboratory. The first letter signifies how good quality the inner cell mass is. A is the best, C is the worst.
When the embryologists’ grade day-2, day-3 or day-5 embryos, their main aspect is to see if they have normal development if the number of cells and the morphology is good, the fragmentation is good. They select the best looking embryo, unfortunately, there is no way to know if a good-looking embryo is a baby.
All embryo assessments are morphological. We look at the embryos, we look at the outside, we try to grade them, we know how the development goes, and this is what we try to assess. However, we always take into consideration that most embryo assessments are subjective, they’re done by different people, by different embryologists.
‘I’m quite a strict person, I will never grade a blastocyst as AA, or I will never give 8 cell embryo a grade 1 because I believe that if I do give this spectacular grading, it’s just like saying to the patient you’re going to get pregnant, and I know that IVF is never 100%. Therefore, I tend not to be so gracious with my gradings.’
Blastocyst transfer is not always the best solution because not all day-3 embryos will grow on to become blastocysts. More studies have shown that around 40% of day-3 embryos will reach the blastocyst stage. That means you have quite a bit of embryo wastage, as we call it, so if you have 10 day-3 embryos, only 4 of them will reach the blastocyst stage.
Dr Daphnis advises that these are general statistics, it doesn’t mean that it applies to you necessarily, but you should always keep that in mind.
If the embryo does reach the blastocyst stage, it still doesn’t mean it will give you a baby. Therefore, it’s a good way for an embryologist to be able to select the best one, but that doesn’t mean that by doing a blastocyst transfer, we shall get a pregnancy. However, if there is a good quality blastocyst, then chances of pregnancy and implantation rate might increase.
Time-lapse incubators are incubators in which the embryologists do not have to open and close them to look at the embryos, they already have a camera inside them, so we can look at embryonic development throughout the time that we have these embryos in the laboratory. That means we can consider looking at embryonic development from day-0 to day-5 and seeing how they develop. We try to find patterns that might help us select the best embryos, and we try to see how we can make what we call a non-invasive selection of the embryos just by looking at how they’re developing.
Artificial intelligence (AI) has been thrown into all these time-lapse incubators. These incubators are trying to predict the clinical pregnancy outcomes using a light microscope. They’re trying to find the patterns in how the embryos are dividing. If, for example, they divide too fast, then it might be good, whereas if they’re dividing too slow, it might be wrong, if they’re dividing all together, for example, instead of going from 1 cell to 2 cells to 3 cells and then to 4 cells, then that might have a detrimental effect. All these things are being looked at, at the moment.
Finally, Dr Daphnis added that technology is thrown into embryology, and hopefully, all this technology will help us make a non-invasive selection of the best embryos to transfer back to patients to achieve a healthy pregnancy.
As I said during my talk, first of all, BB is considered to be a good quality. If you recall, the number two signifies how the blastocyst has developed, so 2 is considered an early blastocyst. Now, again an early blastocyst doesn’t mean a poor quality blastocyst. When you’re using a fresh donor egg, I do expect that the day-5 embryo will not be so well developed, that doesn’t mean that it’s a poor quality blastocyst. No, not at all, a fresh donor might give you a faster development, so instead of having a 2BB, you might have a 3BB or a 4BB, but that’s up to it. PGS testing with donor eggs, again, PGS is an invasive technique.
The lab, which does it has to be quite up to scratch with PGS and PGS, will not necessarily increase your chances, it’s just another way of selecting the embryos. In essence, I’m going to answer your question in simple terms no, a 2BB embryo is not a poor quality embryo, so please don’t feel that you were cheated in any way. Remember, an embryo is only one part of the equation, the second part of the equation is endometrial receptivity, and of course, unfortunately, luck plays a little bit of a role.
I know for sure that Greece does not allow known donation, it has to be an anonymous egg donor. Regarding using your own eggs, if you’ve never, ever had an IVF attempt, you need to know that AMH is just one parameter. Although people try to link it with egg quality, it’s not proven, it just says that you have a low ovarian reserve, it doesn’t mean that you cannot get pregnant with your own eggs because you have a 0.49 AMH. Therefore I would suggest speaking to an IVF specialist, see other parameters of your medical history because, as I said, AMH is just one small part of your medical history and your fertility score. I would suggest looking more into it before you go to donor eggs.
It depends on each laboratory what grade is their B or C. Usually, the best embryos you want them to be A or B. Some clinics do use a great C quality because as I said in my key facto, embryo assessment is subjective, and it does remain on the embryology’s eyes what is best, for example, I’m a very strict person, so A is very difficult for me to give, so if I give a B, C, it is a good quality blastocyst, it’s not a poor quality process so that always depends on laboratory and each laboratory has its own protocols.
Your transfer has been canceled because it was 15 millimeters and not 1.5, I’m guessing, it’s 15 or 1.5 centimeters. There have been papers on that. It would be answered better by a gynecologist but my experience says, and in all the papers that I’ve read, it is true that if an endometrium becomes more than 15-16 millimeters, you tend to abandon the cycle. It’s counterproductive to have too thick endometrium. Usually, a good endometrium is between 8 -11 millimeters.
No, not at all, it doesn’t make them worse because again, it depends on what each laboratory uses, what incubators they use, and what culture media they use. You can have up to early day-6, that’s the morning of day-6 to see the embryo, see the blastocyst and freeze them or transfer them at that point, so that’s not worse in any way.
That is usually done if you want to travel, and it’s difficult to arrange traveling dates, you tend to get a pill. Another aspect is some ladies need to have their ovaries quietened down, so a contraceptive pill usually, reduces estradiol levels, so your next cycle becomes better for us to start, but you don’t need to have a contraceptive pill, it all depends on each patient.
That is the most difficult aspect that I have dealt with by being an embryologist. There is no easy way to explain it, you have to be truthful, you have to explain what exactly took place. Usually, with the aid of vitrification, the last 10 years, this talk I have had maybe once or twice, so it is very rare, nowadays. In the old days, you used to have it more often, nowadays, vitrification has around 95% chance of surviving intact, whether that’s a day-2 or day-3, or day-5 vitrified embryos it’s called.
The answer to the question is yes, it does depend on the embryo quality, that’s why it’s important for embryologists and for IVF clinics to only freeze good quality embryos. It’s better to make a patient sad by saying, we transferred 2 good embryos, and you don’t have anything more rather than to make a patient happy during a fresh embryo transfer by saying you have 2, and we’re going to freeze 2. If those 2 that you froze are not of good quality, we have to give patients the same chances either it’s fresh or, it’s frozen and of course last it is important like I said before, nowadays, everybody uses vitrification. I doubt anybody uses slow freezing, so it does depend if they’ve used slow freezing then, chances are less.
Now, you’re picking straws, and I understand why you’re asking this question, it’s a very legitimate question. AB means that the inner cell mass is A and the trophectoderm is B, I’m just going to repeat myself here inner cell mass is what gives rise to the embryo, B is what gives rise to the placenta, so in essence, AB is better than BA because you want the embryo to be a better quality of placenta, however, both these qualities either AB or BA are very good and have high chances of implanting.
If the laboratory has the correct experience, it’s better to do a day-5, simply because on day-5, you have more cells to analyze, which means that we have more DNA, and we have a better assessment, whereas when we do a day-3 embryo transfer. If you recall a day-3 biopsy, if you recall on day-3 embryos have 6 to 8 cells, so it’s much less invasive to take 10 cells from 15 rather than to take 1 cell out of 6 or 7 or 8 cells, so mostly we tend to do a day-5 embryo transfer. You’d get more DNA, and you also have fewer embryos to analyze, and that means that the embryos that you do analyze, hopefully, are going to be of good quality as well.
It all depends on how you felt at the clinic that you had your IVF. When I say that 30% of failure is due to transfer skills, it’s not just the doctor doing this and putting the catheter there, although that is a very important aspect. I know for sure that in all 4 clinics that I’ve worked for, doctors were very very careful in doing embryo transfers, they were not allowed to do embryo transfers, and they actually scored themselves, so if they didn’t get many patients pregnant, they stopped doing the embryo transfer procedure, so it is a very important aspect, you mustn’t traumatize the patient, you must know how the patient’s cervix is. I should not answer this question necessarily because it’s a doctor’s question, but I’m just giving you my experience on this part. The catheter should go in, it should go out without the patient understanding anything, feeling anything, it should be completely a non-traumatic experience. If it gets traumatic, then the chances are lower in succeeding and the embryos implanting.
Everybody wants to get As, it’s like when you’re in school, you want to get the best grade, that is totally understandable. However, it’s not a sign that an A is going to get you pregnant, and B is not going to get you pregnant. Please, remember that we grade them according to beauty, to how they look, so an embryo which is 3BB has a very good chance of implanting, and if another embryologist is as strict as I am, then the B is probably a spectacularly looking embryo, so please do not get heartbroken if you have a 3BB or a 4BB or a 4BA, it is still a good quality blastocyst.
If you have better quality than that, then no, but if that’s what you have, then we need to discuss it with the patient. BC is actually not a bad quality because B, the first letter, signifies the trophectoderm, so that’s good quality. C, that means that the placenta is not that good, but again always take into consideration that pluses are dynamic entities, they do change, and we’ve seen this with time-lapse quite a bit, so I would transfer a BC, a CB I would be warier. I would talk to the patient, explain to the patient, and at the end of the day, speak to the patient and inform them if we have no other choice, then, unfortunately, we’ll have to go with the CB or CC if that’s the case.
No, it’s just expensive. Time-lapse incubators vary from 80 000 EUR to around 250 000 EUR, so as you can imagine, IVF clinics usually do not charge for time-lapse, and if they do charge, it is a very small amount. In order to make money, buying such an expensive piece of apparatus is not financially stable, so they would buy it more for the scientific aspect and, of course, to help the patients, but it is quite expensive, that’s why clinics don’t buy it.
Your lining is good. Number 4 does not signify quality, number 4 signifies how expanded the blastocyst is. I doubt 6 is the quality, it’s not the quality, 6 is a universal grading for blastocyst, 6 is how much it’s expanded. So, 4AA is much better than a 6A, remember this because 6AA means that the embryo has completely hatched out, it’s gone out of its outer cell and it’s just withstanding, you want that to happen inside your womb, not in the lab, so if 2 x 4AA is looking quite good I hope all the best and good luck.
I would recommend it in your case. You are a more specific case, and I understand your predicament especially, with your early miscarriage at week 9 and the four failed cycles. I would recommend for you to do PGD because PGD and please, remember this will not necessarily give you your goal, your aim of having a baby. However, it will give you vital information about your embryos, so it will give you information if your embryos are chromosomal of good quality to give you pregnancy or if they’re not. I hope that they are and I do recommend doing PGD in your case.
That’s a good thing. Let me explain each stage. When you see the zero in front, that’s a morula, 1 is an early blastocyst, 2 is what we call a cavitating blastocyst, so when you look at the scoring, it means that number 2 means it is cavitating. 3 is a blastocyst, 4 it’s expanded, 5 it’s hatching, 6 it’s hatched. You have a day-5 embryo, 2 in development, and then whatever the score is. Usually, 1 and 2 are very difficult to grade. If you remember in that very busy slide that I showed you, it’s very difficult to distinguish between the inner cell mass and the trophectoderm 1 and 2 embryos. So early blastocyst and cavity blastocyst, so in essence gardener who devised this grading system said, we should all grade 1 and 2 as CCs, so keep them in the medium because we can’t grade them properly. If you have day-5 embryos which are cavitating, that means that they’re at the blastocyst stage and they’re doing what they’re supposed to do.
I would, in this case, trust your clinic doctor, he probably had information from the embryologist, and the reason is simple with an Embryoscope, which is a time-lapse incubator, we don’t only see the end result, you see how the embryo develops throughout time. That means that the grade C blastocyst did not have a good developmental step throughout its development. I’m sure it was not difficult for them to just put another embryo to freeze, it’s easy for them, it doesn’t use much effort, so not to freeze it this specific embryo, I’m sure they did a favor, it comes back to what I said before embryologists should only freeze embryos which will give patients a good implantation rate nothing else, so it’s good that they made this decision, and they did not tell you that you have 2 frozen embryos just to keep your hopes up, but then at the end of the day you would actually get a good result.
The reason is simple when you go for 2 trips when you do a blastocyst biopsy on day-5, you cannot do an embryo transfer on the same cycle, that’s why you have to travel twice, whereas if you do a day-3 biopsy, that means that you can do the same cycle. Don’t do FISH, it’s very old. I did FISH back in 2004 when I was doing my Ph.D., and it’s a very old technique. If they’re going to do a day-3 biopsy, at least they could do ARRAY-CGH, which should give you all chromosomes whereas, FISH will only give you 5 or 6 chromosomes, that is a very outdated way of doing PGS, so I would advise against doing FISH. If they do ARRAY-CGH, at least on day-3 embryos, yes, but coming back to my previous answer to a patient who asked if you’re going to do a day-3 or a day-5, I would suggest doing a day-5 NGS, and unfortunately, go for a second trip.
Unfortunately, there is no way for us to improve egg quality and we can force fertilization, we can force division, but we cannot make eggs of better quality. You can attempt if you have a poor ovarian reserve or poor quality eggs to do what is called PRP to your ovaries, maybe that will help you get better eggs, however, there is no scientifically proven way to make your eggs of better quality.
When they do PGD24, that means that all chromosomes have been tested, yes, of course. There is always a possibility of embryos not surviving, especially embryos which have been biopsied because these embryos in order to do PGD, they have to be biopsied.
All protocols for thawing after vitrification are specific depending on each kit that you use. So an embryologist will know that, and it has nothing to do with the information that you need to know.
The minutes to transfer to your surrogate mother, again it depends on how each lab is set up, some labs want to wait for at least 3-4 hours, other want to do it within half an hour, so again it goes to the protocol, it has never been tested if it’s better to leave them for 3 hours in the incubator or to do the embryo transfer after half an hour, and so the answer to the second question is not proper, however, the is a chance that the embryos will not survive if they’ve been biopsied, even if you’ve had vitrification.
We used to say to our patients that you have around 70% chance of thawing, so I would say that is a good number, and the problem is that only the embryologists like myself know how to slow-thaw embryos nowadays. That’s also a key ingredient if the clinics do have these embryos, make sure that they have older embryologists who know how to thaw them. I think nowadays, nobody uses slow freezing, everybody uses vitrification, so the chances I would say are around 70%.
This means that the inner cell mass is very good quality on both embryos. I am just a little bit wary of the 6, if the 6 is according to Gardner’s classification, that means that both embryos are hatched, so that means both embryos do not have the outer membrane to protect them. Usually, hatched embryos do not vitrify as well as embryos that are not hatched, the zona pellucida, the outer membrane as we call it does provide extra protection for the survival of the blastocyst. However, if you have those embryos, of course, use them, the grading looks quite a good AB and AC, is okay, AB especially is much better, but I am a little bit wary about the 6 if it actually does signify that the embryos are hatched, please ask your laboratory.
This is a very old technique that we used to do. What we did is, we used the laser to make a couple of holes or to thin the outer membrane. I was just talking about it – the zona pellucida and that made it easier for the embryo to hatch because embryos want to get out of the zona pellucida when they’re in a lady’s womb. It hasn’t been proven that embryo hatching does have a positive effect on embryonic quality unless you’re using older age eggs and you measure the zona pellucida being very trick, otherwise, if you do it on donor eggs or just eggs, which do not have as thick zona pellucida then, it has no positive effect.
This is a question a gynecologist would answer, and there is a possibility that if your fibroids were too big, and your ovaries were above your womb, the doctor couldn’t get through during an egg collection to the ovaries, I have seen it happen. Just because they’re behind the womb and they do not move. I have helped doctors trying to move ladies inside when she’s sedated to push everything down, so the ovary can come down and be more accessible for the egg collection, but I have experienced, not often but rarely because the off chance that you cannot access the ovaries, therefore, I would not say that he didn’t do the IVF, I think that would be unethical, he might have had a problem in actually accessing your ovaries, I have seen it, and if you have fibroids, then he would be very wary of going through.
Again, it all depends on the size and where it’s located and where your ovaries are located. I don’t want to comment on that too much because again, I’m an embryologist, not a gynecologist, but I have seen ladies were we weren’t able to access the ovaries just because they were above the womb and they were behind or that you couldn’t find a way to go through, but it happened very rarely. I am guessing it has nothing to do with the embryo transfer that you had when you did your donor eggs because the fibroids were probably out of the way, out of the endometrium. It had to do with how accessible your ovaries were or ovary, I don’t know which one had the follicles during egg collection. It is a possibility, a small possibility, but it is there.
The maximum age for embryo transfer in Greece, unfortunately, at the moment it is 50. The Greek authorities have told us that they want to change the law to 53, but at the moment, it is 50, not 51.
No, it isn’t. Especially, if you use a laser, and you don’t touch the actual embryo. It’s not harmful, it has never been said that it’s harmful. An experienced embryologist who does the hatching if they are to do one is needed for it, that is it.