Embryo Quality and Failed IVF - 5 days in the IVF laboratory
The webinar is hosted by Laura Van Os, an embryologist from IVF Spain. In the webinar she focuses on the 5-day period after the insemination when the embryo stays in culture. Laura tries to answer the question why IVF cycles fail and if modern science can do anything about it.
Factors explaining IVF failure
Laura starts with listing the factors that can explain IVF failure. These can be:
- immunological factors,
- coagulation disorders,
- endometrial receptivity problems,
- all sorts of uterine and tubal pathologies or hormonal problems.
Of course one of the reasons why IVF cycle fails is the embryo quality itself. In this case, it can be either the environment surrounding the embryo which is failing or it can be the quality of the embryo itself which is curtailing a successful pregnancy.
Embryo quality = oocyte + sperm quality
Every conversation about embryo quality should be proceeded by a talk about egg quality and sperm quality. Unfortunately, there are not many markers for egg quality. It is impossible to determine egg quality and its ability to give rise to an viable embryo just by looking at it under a microscope. Another important thing to know is that not all eggs are mature (only the eggs at metaphase II stage are the ones that can be successfully fertilised). However, among mature eggs there might be some eggs with abnormalities. Some eggs with abnormalities will be excluded from the cycle but most of them will be inseminated (as these abnormalities are mostly harmless). And finally, even among the good-looking mature eggs it is very difficult to tell if they are of good quality or not. It is impossible to know if they can give rise to viable embryos or not.
Laura explains that contradictory to the eggs, the quality of a sperm sample can be simply assessed just by looking at it under a microscope. The main parameters which we usually look at in a sperm analysis are:
- and morphology.
According to the World Health Organisation, concentration in a normal sample should be about 50 million per milliliter. Progressive motility should be about 32 percent and normal morphology should be above or equal to 4 percent. When there is a little deviation from these parameters it is actually very easy to solve inside the IVF laboratory. Simple sperm problems can be solved just by conventional sperm selection techniques (density gradients, swim-up, etc.) plus an ICSI procedure. Then there are some very specific molecular problems which require different approaches. There’s a wide range of pathologies and each of them has a specific treatment.
Once we have the mature egg and the processed sperm samples, the next step is the insemination of the eggs. This can happen through various techniques and one technique is called ICSI. This means that we inject the sperm cell directly into the egg. The other technique is conventional insemination. Whichever technique is used, the day after egg retrieval doctors will evaluate fertilisation of the eggs. Fertilised eggs (zygotes) have two little bubbles inside of them, with male and female genetic material. This genetic material will unite to form an embryo.
An Embryo dividing
From then on, the embryo will start dividing. Ideally on day 2 it should have around 3 cells, on day 3 around 8 cells and from then on the embryo should keep growing exponentially to 16 cells, 32 cells, 64 cells, etc. Before this increase takes place, the cells in the embryo will compact. This is called the morula stage and this is how the embryo looks like on day 4. After this a little cavity will start to appear in the embryo, it will grow and grow and it will fill with liquid until finally we’ll have a blastocyst. It is composed of two main parts. Inside it has some inner cell mass that will become the future baby. It also has some outer layer of cells called the trophectoderm, the future placenta.
Number of oocytes retrieved vs. number of embryos
At this point Laura throws down a common myth that seems to be widespread among patients. She explains that the number of eggs retrieved in an egg retrieval is not always proportional to the number of blastocysts. Nor their quality. Actually the number of blastocysts per egg retrieval is variable among patients and even from one cycle to the other among the same patients. Some patients seem to be very discouraged when they don’t retrieve enough eggs. They think it’s impossible that they will become pregnant. However, this is really not always the case. As Laura says: all you need to become pregnant is one top quality embryo, not thirty of regular quality.
Embryo grading framework
Laura also explains that embryos are usually given grades such as A B C and D. It is a grading framework which is used in clinics worldwide. A and B embryos will always be transferred and cryopreserved, whereas D – quality embryos will never be transferred nor cryopreserved. When it comes to C-quality embryos, there are subtypes and situations so not all C quality embryos are the same. It is good to remember that embryo scoring systems are a simplification. The grading systems are useful to rank embryos within a cycle to know which embryo should be transferred in first place. Different laboratories and even operators within the same laboratory may be more or less strict when classifying the embryos.
The embryos’ (and blastocysts’) quality can be categorised in three different ways. The classical way is the morphological assessment at a fixed time point. It includes the trophectoderm quality, inner cell mass quality and the expansion degree.
Embryo monitoring using time-lapse systems
The most powerful technique used to assess embryo quality is called time-lapse. It is performed in incubators that have cameras inside them. Thanks to the cameras, doctors can look at the embryos anytime of the day just by opening their computer. They do not need to open the incubator and take the embryo out to look at it under a microscope. In this way, they do not disturb the stable culture conditions. Additionally, the time-lapse technique has individual patients’ chambers and it allows to perform a morpho kinetic analysis of the embryo. With time-lapse, doctors can predict the implantation potential of the embryos and select the best embryos for transfer. The embryos’ (and blastocysts’) quality can be categorised in three different ways. The classical way is the morphological assessment at a fixed time point. It includes the trophectoderm quality, inner cell mass quality and the expansion degree. The most powerful technique used to assess embryo quality is called time-lapse. It is performed in incubators that have cameras inside them. Thanks to the cameras, doctors can look at the embryos anytime of the day just by opening their computer. They do not need to open the incubator and take the embryo out to look at it under a microscope. In this way, they do not disturb the stable culture conditions. Additionally, the time-lapse technique has individual patients’ chambers and it allows to perform a morpho kinetic analysis of the embryo. With time lapse, doctors can predict the implantation potential of the embryos and select the best embryos for transfer.
Embryo quality and PGS – Preimplantation Genetic Screening
The last tool to assess embryo quality is PGT – pre-implantation genetic testing. Laura says that not all embryos are normal and some to them have genetic errors. The percentage of abnormal embryos increases with maternal age. When you transfer an abnormal embryo, in most cases and it will not implant. If it implants, it will lead to a miscarriage and in a few cases, the pregnancy will go on but the baby will have a disease. This problem may be solved with the PGT technique. Its main benefit is diagnostic value. Thanks to it, patients will know if their embryos are normal or not. Laura adds that PGT also helps to reduce time to pregnancy. Simply put, a patient avoids useless transfers and excludes embryos which are not going to implant or which are going to miscarriage.
Summing up, if you need to decide how to go on in a future cycle, you have to know which problem to address.
Laura Van Os says that thanks to high quality, modern technology professionals are able to fully assess the quality of their embryos. If it turns out that the embryo quality is not a reason for a failure, they will be able to focus more on the environment of the embryo and search for medical solutions of their problems.
Other resources worth reading:
Questions and Answers from the event
I had a 3BB transferred in April & this failed. I have a 3BB & 2 5BBs frozen with IVF Spain. Will the grading be the same when they have thawed?
Yes, the grading will be the same when they have thawed. So in this case probably we will transfer the 5BBs embryos because they are more expanded and they are frozen on day 6, that’s why we transfer the 3BB in the first place. According to statistics, the quality of the embryos doesn’t change after we cryopreserve them through vitrification.
I had an embryo frozen which had irregular cell division (from 1 to 3 cells) but went on to form a beautiful blastocyst. Can you give a view of implantation potential and the possibility of this leading to a healthy baby in a FET cycle?
It’s a good question. In the past, we used to believe that when there was a division from 1 to 3 cells, this was quite catastrophic. But we have seen in recent years that when these embryos reach the blastocyst state, what usually happens is that they leave these 6 cells “out of the embryo.” And we have seen when we performed PGT on these embryos that they are genetically normal. So this means that the embryos have some sort of repair mechanism so they can tell that maybe one cell in the embryo is not healthy and they just leave it out. So the rest of the embryos will be healthy and actually it has good implantation potential.
What is the link between morphological quality and likely genetic quality e.g. is a 5AA more likely to be genetically normal than, say, a 4BC?
Sometimes we’re surprised because we have embryos which are AA-quality and they look perfectly fine and however they are the embryos of the pool which are not genetically normal. So there is not always a correlation. If it is a very severe chromosome problem and a lot of chromosomes are altered, this will show in the early embryo development. But if for example, you have had trisomy of chromosome 21, even when you have a fetus in the sixth month of development it will be difficult to detect. So imagine an embryo which is just 5 days old. It will not show so easily on the outer side.
Are there more success rates with frozen or fresh embryo transfer (using donor eggs)?
The first transfer in a cycle has a higher implantation potential than the other embryo transfers. But you have to keep in mind that the first embryo which is transferred, namely the fresh embryo transfer, is the best embryo of the pool. That is so because we select the best embryo to be transferred first. The ones that are transferred afterwards, the cryopreserved embryos, are the ones which did not have the best quality. It’s true that the first transfer does have a little bit more implantation potential but it could also be because of this.
Would you recommend PGT testing for IVF with donor eggs? / Is it necessary to perform PGT on donor egg blastocysts?
If you actually look at the euploidy rate described for egg donors, I think it was 60%. So not 100% necessary but it will save time up to pregnancy. You have to think how important is it for you, how hard is it for you to go through the disappointment of an unsuccessful transfer? How much time, money and mental energy do you waste on this?
If the embryo doesn’t form a blastocyst until day 6 is it more likely to be genetically abnormal?
I think it is a little bit more likely to be genetically abnormal if it has a slower pace. But it is a little bit contradictory evidence.
Are you against day 3 transfers? It’s very confusing understanding the pros and cons of day 3 or day 5.
It is a personal decision. I understand that some patients decide on the embryo transfer on day 2 or day 3, for example, if they have only few embryos. But what we see regularly in the clinic, there are patients who’ve had sometimes from 5 to 15 transfers on day 2 without a successful result. And when they perform a single cycle leaving their embryos to day 5, although they develop to day 2 or day 3, only a few of them reach day 5. So you can waste a lot of time transferring embryos of day 2 or day 3. And when you don’t become pregnant you will never know the reason. Maybe they were just not able to reach the blastocyst stage or the blastocyst rate is really low. So definitely leaving embryos to day 5 doesn’t harm them and you will have a lot more information and you will waste less time.
I have 2 grade 3 embryos on day 3. However, they stopped to develop to the blastocyst stage and I lost the chance for embryo transfer. Please advise if it is worth doing embryo transfer on day 3 instead of the blastocyst stage.
If the embryos are not reaching day 5 in the laboratory, they will not reach day 5 inside your uterus. This is a common question and something that patients wonder a lot about but it is really like this. You will not become pregnant just by transferring the embryos on day 3 if they are not able to reach the blastocyst stage.
Would transferring an embryo with irregular cell division (but that made it to blastocyst stage) at the same time as transferring embryos with normal cell division put at risk the normal embryos?
I once read an article that I thought had a funny title and it said: ‘It’s better to be alone than with bad company.’ To transfer one blastocyst of good quality with one blastocyst of bad quality isn’t a good solution. It’s better to transfer alone one embryo of good quality. But that said, even if the embryo has irregular division, it might have correction mechanisms so it might be still of good quality. In any case, we do not really encourage transferring two blastocysts at the same time. So I would just go for transferring the normal embryo at once.
Are there any studies to suggest that a woman’s BMI impacts the egg quality and therefore the embryo quality?
Yes, there are. Actually sometimes when patients visit IVF doctors, their first recommendation is a lifestyle change and following a diet because this can definitely influence pregnancy rates. It’s more a medical question, a doctor could probably give you a better answer but yes, it does have an influence.
Can BMI be too low?
This is more a medical question so I could just give you my opinion. Probably a doctor can give you a better and well-informed answer. I do think BMI can be too low for a pregnancy.
I had 2 cavitating blastocysts transferred in May (good and top quality); this failed. I also had 2 failed FETs last year from the previous successful cycle in 2014. I am now considering egg donation, I am 42. I collected 6 eggs in May which are cavitating only. Is this a sign of poor egg quality?
Yes, it does seem like a sign of poor egg quality. But maybe at your age what is lacking here is a PGT. The embryos seem to reach the blastocyst stage, these were day 5 embryos, probably at day 6, they would have been fully expanded. I don’t know what their morphological quality was. But at the age of 42, it is very likely that the egg quality has an influence in this. So you would need many embryos transfers to have a euploid embryo which would make you pregnant.
For my 1st transfer, I had 2 3BBs and 1 5BB blastocyst. 1 was being left for review until day 6 and turned out to be a 5BB. You mentioned on my first question that on the next transfer you would use the 5BB, but earlier you said the best quality embryo would be transferred. So why was the 5BB not used originally? Is a 5BB better than a 3BB?
The 3BB would have been a 5BB on day 6. But the thing is this embryo had one more day of culture. So now we have the embryos when they have had one more day of culture. Here I’m lacking the Time Lapse information. So it might have been that the embryos were in Time Lapse because we were using this system and then we had some additional information about the embryos and their cell-divisions. So that could be an explanation. But right now if I had a 5BB and a 3BB which are already cryopreserved, just knowing this about them and not knowing anything more, we would first transfer the 5BB because it has a higher degree of expansion.
I had good graded 6AA and 5BC embryos on day 5. However, PGS showed abnormal chromosomes on both embryos. What can be done to improve quality, if there is any chance?
This question we get a lot and I think it is more medical. I’m sure that a doctor will know better how to answer you because sometimes they decide to change the stimulation protocols to try to increase the embryo quality. But from the laboratory perspective, unless there is some sperm quality or fertilisation problem, there is not so much that we can do just by knowing this. Because women are born with the number of eggs they have, the euploidy rate is a little bit determined by age.
Will adenomyosis and endometriosis have an impact on embryo implantation and if yes, what is the treatment for this?
Again this is a medical question. I wish I could help you but this should be answered by a doctor. I’m an embryologist, a biologist. Just feel free to send this question to our medical team so they will be able to give you a proper answer.
Can a small amount of alcohol before and during IVF treatment affect egg quality and transfer?
It’s hard to say and I would not know how to give you an answer. It’s true that drinking can have an influence on sperm quality but in case of egg quality and transfer quality I would not know what an answer to give you.
What is the difference between PGT and PGD?
In PGT we are testing the number of chromosomes and if these chromosomes are complete. And PGD is really specific because we are checking a single gene. So PGD is only used for couples who already know which disease, in particular, they want to check. And PGT is used for everybody because we all may produce chromosomally abnormal embryos. So PGT shows chromosomal abnormalities in embryos such as trisomies of chromosome 21, for example, Down syndrome, Turner syndrome, common syndromes. And PGD is only testing if you have one particular disease.
If embryo stops to grow on day 3, is it caused by egg or sperm quality?
There are some cases where all the embryos stop to grow around day 3. This can be due to sperm quality because the embryo genome starts to activate around this point. So if the embryos always block particularly at day 3, it can be due to the sperm quality.
If in PGT the result of trophoderm is normal, can the ICM be still normal and lead to a healthy baby?
Yes, this is possible and there are many studies about it. And in a low percentage of cases, it could be that the placenta is not representative of the ICM. It is really very low parentage of cases but it’s possible.
Do you have a view on why a genetically tested normal embryo doesn’t implant?
If a genetically normal embryo doesn’t implant, you have to think that you did everything possible regarding the embryo. So you can exclude the embryonic factor and probably there is something else which is preventing the implantation. So there might be something related to your uterus, immunological problem, implantation window, something like that.
Is IVF Spain doing PGT testing on-site? How many days does it take to get the results back?
When we perform PGT, it takes like 15 days to get back the results and you know if embryos are chromosomally normal or not.
Which one is the best predictor of implantation: ICM or trophectoderm?
Trophectoderm is the best predictor for implantation.
Can you recommend any supplements etc. to improve egg quality?
Again that’s a medical question so it will be forwarded to the medical team that can give you a right answer.
Does ICSI have an impact on the embryo quality instead of IVF?
No, it doesn’t. We know this for sure because sometimes in egg donation programmes in our clinic we fertilise half of the eggs through ICSI and half of the eggs through conventional IVF. And what we see in the result is that they basically have the same development, we see no influence. Very often the best quality embryo comes from ICSI, not the IVF. So we do not see any difference. We do this very often in egg donation cycles.
I have had an IVF cycle where the follicles grew in different batches – so there were only 3 mature eggs at egg collection. What can be done to try and ensure consistent follicle growth? I used Noresterone and Evorel patches pre-cycle and I was wondering if the oestrogen in the Evorel patches could be responsible for the inconsistent follicle growth?
This question is really related to the area of stimulation. So we will take note of the question and we’ll try to give you a proper answer when we have forwarded it to the doctor.
What things will you advise to improve egg and sperm quality?
Generally speaking, of course, a healthy lifestyle. For sperm there are many things, like antioxidants, not wearing too tight underwear, not smoking, not drinking too much. So basically a healthy lifestyle. And as we said, neither too high nor too low BMI.
Which is more important in the embryo quality, the first or the second letter (like 5AB or 5BA, which embryo is of better quality)?
The most important is the second letter. So actually the 5BA would be considered to have more implantation potential than a 5AB. However, 5AB and 5BA are really close to each other. And if you remember one of my slides, embryo scoring systems are a simplification.
So an A grade sometimes can be really close to a B grade.
We have transferred NGS tested aneuploid embryo last year. However, it resulted in a biochemical pregnancy which shows that it has implanted at some level. We are told that the embryo didn’t have enough energy. Still not satisfied with the explanation. How will you explain it?
The embryo did implant in fact if you have a positive HCE. So it’s a biochemical pregnancy. And I guess your doctor was trying to explain how the embryo did manage to go through this implantation potential. However, it did not manage to complete the whole process that was necessary for the embryo to keep growing and follow all the first embryological steps common for embryogenesis. What is the reason for the biochemical pregnancy? I won’t be able to tell you. There are many reasons why biochemical pregnancies may occur. Like we said last time, the embryo was euploid so you could at least exclude the embryonic factor.
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