By fertility experts from Spain.
Alsu Saifitdinova, PhD, embryologist at the International Centre for Reproductive Medicine, explains miscarriages and embryo implantation failures and if they can be investigated and prevented using PGT-A embryo testing.
A long time ago, we divided all preimplantation analysis into two groupings. PGS, which is a pre-implantation genetic screening, and PGD, a pre-implantation genetic diagnostics. These two groups were based on those methods that were used about ten years ago, and sometimes it was confusing when it comes to a particular goal of using this analysis. After that, when some new approach was involved in preimplantation genetic analysis, it was even more difficult to understand which particular analysis is necessary to do for a particular family.
That’s why it was an international decision to divide all pre-implantation genetic testing into those 3 groups that I mentioned before. Among them were those that analyzed aneuploidies separately and now it completely covers the term PGS, so PGS means PGT-A, but PGT-A also can include some information about chromosomal abnormalities if we will think about sex chromosome abnormalities for example.
PGT-A is about the same as it was with PGS, this term was used some years ago for a long time, and now we cal lit just PGT-A instead of that. PGD goes for different types of PGT, which intend to analyze a monogenic disease that is PGT-M or construction of chromosomal rearrangements like PGT-SR.
In the beginning, it’s not obvious, but unfortunately, the chromosomal abnormalities are one of the features of human embryos, and it comes from the nature of chromosomal behaviour during oogenesis and spermatogenesis. We should remember that oocytes are quite old cells, so they are usually a little bit older than a woman that have these oocytes and at this stage, they could make some mistakes in chromosome separation and during cell division. A division which happens with oocytes when it prepares to transfer the genetic information to the next generation, and at this time it is possible that some chromosomes could be lost and then these oocytes can be fertilized.
We will have new embryos that would have one cell at the beginning, but it would have an old number of chromosomes, so some chromosome could be represented in 3 copies or 1 copy, or sometimes without some chromosome or even sometimes it could be 4 copies of the particular chromosome. In this case, the normal genetic information of the development of this embryo would be realized properly, and it causes some inherited diseases and is the most common reason for some defects in the early development, and this embryo wouldn’t have any potential for implementation or development in a healthy child. Analyzing chromosomal number at the beginning before implantation you would find those embryos which will have the implantation potential, so this one of the most important thing how to select those embryos which will give the healthy child and which can give normal proper pregnancy.
As I’ve already mentioned the first of all PGT-A a can help us select those embryos which have a proper chromosome number and another important thing that we also can select just one embryo. We don’t need to transfer more than one just in case if some will have some mistakes, so we can select those, which have the highest potential and then give the best condition for this embryo for the implantation. We wouldn’t put them together, we wouldn’t try to him to put 2 or 3, just in case if some of them could be incorrect and we could have some mistakes and chromosome numbers.
In this case, we would at the very beginning of the process of implantation, we will give the best condition for those embryos which have the best potential for further development. This is the most important thing because PGT-A excludes the possibility of miscarriages during the first trimester, actually, it has a really simple explanation just because when we analyze those embryos which stopped development during the first trimester, usually they have some chromosomal imbalance, so this saves us some time, and it helps us to find those embryos which have the best possible potential for development, then we give them the possibility to give pregnancy with singleton not trying to transfer more than one embryo. Another important thing that this helps us to exclude multiple pregnancies, so after that, the outcome of that IVF cycle will better than when we just tried to transfer unanalyzed embryos.
Sometimes people really worry about the viability of embryos after the manipulation with PGT-A. We need to take some cells from the embryo, and it was shown that at the very beginning during first cleavages the human embryos divide into eight cells, and each cell from this embryo have equal potential, so each of these eight cells can develop into normal organism without any mistakes, and so on. It was a really important finding which helped us to develop the PGT-A. Then we developed a new technique which is a little bit even less invasive because we now are analyzing just some cells from blastocyst stage and at this stage, the embryo has about 100 and maybe a little bit more cells, and we are doing the biopsy, and we just take not more than 5 cells or about 5 cells for this analysis.
All of these cells are not from inner cell mass, so they are not those cells that should make the embryo rise, these are the cells that surround embryo, and that helps it implant, but it is about a hundred of such cells which should help this embryo, and we just take a very small number of cells for analysis. It is important that should be performed strictly following the protocol, and all of the people who are involved in this process should be really experienced, so I think that it is important to choose a clinic with a long experience with highly qualified embryologist who can do it properly and in this case, there wouldn’t be any consequences for the viability of the embryo.
First of all, I must say that even young women already have not really young oocytes, so even a 20-year-old has oocytes of 20 years old. They are not just freshly divided young cells, so if we will compare with other cells in our organism for example blood cells or some other cells, skin cells they are really Young, they renovate every day, and some of them are not older than 10 days, and if you think about oocytes, they are really old cells and even at the age of 20 when we started analyzing these embryos, we can see that about 30% of oocytes would be aneuploid.
It was really puzzling when it was found because of course, we know that after 35 years old, we could see that the number of aneuploid cells increases but even if we analyze oocytes from a young donor, we can find that about 30% of them would be aneuploid. It would depend on the particular situation, of course, there will be some differences in the number of such cells, but unfortunately, we wouldn’t say that if this one is young she wouldn’t have any aneuploidies.
Our karyotype is a really important thing, it comes from the chromosome number from the parents, from a woman and man that want to have a baby. If they don’t have any rearrangements, they have a low risk of some kind of mistakes, but as I said the most common mistake that cures at this period is chromosomal number mistakes which are based on the age of the oocytes. So even those people who don’t have any kind of pathology, incorrect karyotype during oocytes maturation and this special cleavage which produces oocytes, the mistake can occur with high frequency in those who are a bit old. It depends on age but not on the karyotype.
When we are performing PGT-A, unfortunately, we’re analyzing only five cells. We are not able to carefully analyze the genome of the embryo, the nucleotides, all the other letters in the genetic code, so we just analyze the chromosome numbers, so we don’t know a lot of information about this embryo. We just can’t say that it will help all chromosomes, but unfortunately, apart from the chromosomal diseases and chromosomal mistakes, there could be some monogenic diseases, and they would have an impact on the development as well. We also have all other factors, which will have an impact on the realization of the genetic program. That’s why even if you have perfect PGT-A analysis, we wouldn’t say that you have 100% insurance that there wouldn’t be any genetic mistakes or genetic diseases or some kind of risk of miscarriages, due to different reasons. We try to select the best, and we just try to do the best but unfortunately with as always in nature, we can’t say that it will exclude risks.
I can’t say what is the particular cost for these tests just because in some situations it’s different. You can check it on the website of the clinic, and each clinic can tell openly the costs, and it just depends on the particular situation, and in our clinic, we are doing it for each embryo and using PGT-A for 24 chromosomes. We can’t offer any packages just because each embryo has the lowest price that is possible for analysis because I must say that now this analysis can be done everywhere, so it’s not a kind of some type of analysis where the price is really higher than the cost of the materials.
It’s not like it was 10 years ago when just some clinics could do it. Those tests can be done here, in our clinic or somewhere externally, of course, it will depend on the protocol. I must say that some part of the analysis could be done in another clinic, but if you are having embryos here, the biopsy will be done here, and then if you would send it far away, you should remember that during this transportation you would have just five cells in one cube, and if something happened with this cube, it could be melted, and well this thawing process could damage the DNA in the cube. If you want to send it for analysis somewhere far away, you should remember that you could have result saying that the analysis wasn’t possible to do the biopsy again.
Unfortunately, now it’s quite usual that you have to do a dry cycle, so you need to freeze your embryo, and it depends on the particular type of analysis. If we are talking about PGT-A using NGC, next-generation sequencing or if you are going to do some array CGH analysis you would obtain the cells just at day-5 or sometimes at day-6. So you have just some time for transfer of this embryo to the mother, but those hours are not enough for a full analysis of all chromosomes. But at this time when we analyze them using Fluorescence in situ hybridization (FISH) technique, we usually do it in a fresh cycle, but there is another distinction.
If we are doing Fluorescence in situ hybridization (FISH) technique we don’t have any possibility to do it in those two days because at the time it was a biopsy at day-3, and we can’t do all chromosomes just because it is just 1 cell, small nucleus and you need to check all chromosomes. I must say that in our clinic, we have the highest number in the world where we can analyze up to 12 chromosomes, so half of those in a fresh cycle. It’s possible to analyze using new techniques, but unfortunately, the price of this more advanced quality of analysis is time, and we hope that there may be a new method which could be a little bit faster and give us the possibility to obtain results in some hours. If it will be available, we will immediately start doing it in a fresh cycle because sometimes it is necessary just to do it in a fresh cycle.
There are some advantages of these because from a gynaecological side, you can prepare a little bit better for this transfer. Also, now when we have this situation with COVID-19, it is a little bit better if you could freeze your embryo and then wait before the situation will be a little bit better with virus environment, maybe wait a little bit before vaccine appears and then come and have this embryo transferred.
First of all, I must say that I would advise all couples that come for IVF to do karyotype because sometimes unexpectedly we can find some information about balanced chromosomal rearrangements in the couple. Some people have this chromosomal rearrangement, they carry them, and they don’t know it before they start the IVF. It happens quite often, and it is one of the important reason for failure in pregnancy, having chromosomal rearrangements. I would advise doing karyotype if you have any problem with having a pregnancy. So, you can exclude if there are some mistakes.
It is so important because if you have some mistakes in a chromosomal organization, you could have an embryo, which will look perfect just because during the first division, the cells would use the potential of their mother cells, and they would divide properly. They would organize improper blastocysts, and they would have the perfect potential for implementation and then if there were some mistakes in chromosomal rearrangements in their parents, they could have some small mistakes of the genome. In this kind of defects, they would have developed babies with some diseases, and it is even worst than not having children, and that’s why karyotyping is really important, and you should start with it if you just want to start the IVF cycle.
Another thing is that some people have a little bit higher rate of chromosomal mistakes in embryos and again it depends on genetics and unfortunately, it is really hard to find using usual methods. Sometimes, some genes that help cells to separate chromosome properly or some other types of cellular proteins which work usually normally in our bodies but if we are talking about oocytes for example which as I said are quite old cells. In this case, these mistakes begin to be a little bit more important, so for normal cells which divide really fast and it’s just mitotic division, some insufficiency of some proteins don’t have to be so important, so it doesn’t hurt them, but for oocytes because of the ageing, it becomes a bigger problem, so in those people, the rate of the embryos with chromosomal mistakes could be a little be high.
If you already have some experience with miscarriages, you need to have all the information about this pregnancy, from karyotyping these embryos just to understand the reason. If it is repeated, chromosomal rearrangement mistakes even if it wouldn’t be the same chromosome, it could be different chromosome, it can be a kind of prediction that it is better to do PGT-A to select the euploid oocytes before transfer.
At the age of 30, the percentage of euploid embryos is highest. It’s interesting, but in younger women, we have a little bit higher differences in the number of aneuploidies. At the age of around 30, we have about 70% of euploid blastocyst analyzed, so if you’re analyzing them at the stage of blastocysts, it will be about 70% of euploid cells.
For FISH analysis, it was about 5% of just mistakes, which came from the peculiarities of this technique. Now when we are talking about NGS technique actually I did hear any information about mistakes in this particular thing if you have really strong control of the identification of the material, you would exclude these mistakes, and if there wouldn’t be any kind of contamination, the mistake itself does not occur usually. Another thing is that sometimes after a week we cannot obtain the results and we calculate that of course, it depends on the time from the biopsy to the analysis, and it depends on the quality of the embryo and sometimes it is possible that we wouldn’t get any results. Some clinics prefer that they have about 1% of such situations, but we must say that in our clinic this is about 0.1, maybe even less but it happens sometimes. It is something inside the embryo where we would have all the DNA damaged before we started the analysis. It means that this embryo looks well, but it is already almost dead. In this case, we wouldn’t get any results.
I think it wouldn’t be so because as I said it was shown that in a 22-year-old the difference between the number of perfect oocytes is a little bit different, so, at 30-year-old donor, it would be 7 out of 10 euploid blastocysts, but if we are talking about 22-year-old it could be absolutely different. This difference’s very high, and there is some explanation that at this age the hormone system of a young woman is not so perfectly balanced so that’s why sometimes you could obtain 3 euploids out of 10.
As I said, it’s about 30%. So a healthy young donor will have about 30% of embryos that will have some different chromosomal abnormalities. It has been published and is well known that’s why it is one of the reasons why we would recommend to those who are not so young donors, and they don’t want to experience miscarriage but if you are starting a donor program to apply PGT-A to avoid it. You should remember, it is somewhere in the nature of human embryo development, and actually, these mistakes are not something unusual in general.
It will depend on the number of embryos which would be obtained for this particular person, and it was calculated that if you would have about 8 or 9 cells, we definitely would have at least 1 euploid. This is on average, but we also have an experience where there was just 1 cell from a woman at 42, and it was euploid, and after analysis it implanted. It really depends on the personal situation and personal condition and some personal genetic background as well, and of course, in general, the risk to have aneuploidy will be higher, but it doesn’t mean that it will not be possible.
Everything that involves mitochondria will cause some additional problems because of the copy number of this DNA from mitochondria. Unfortunately, we are still not ready to say that we can absolutely help in this situation. I must say that right now some labs in the world are working on this problem. It would be a technique based on the nuclear transfer from oocytes from a healthy person, without any mitochondrial genome mistakes and then transfer the nucleus from the mother.
Right now the problem is that we can’t transfer them without picking up at least some copies of the mitochondrial genome from the mother‘s oocytes. It’s still an experimental thing, so of course, we need to wait a little bit, and I am sure that one day we will be able to help those woman with mitochondrial disease.
When it comes to your son and if this syndrome is not really severe, it is a mild condition, the mitochondrial disease is really rare when it comes to transferring from the sperm when your son grows up. There are some cases of transferring of mitochondrial diseases from the sperm, but it’s really rare, I don’t remember the particular number, but it’s really rare.
There was some analysis on this. The most interesting was a study where they tried to compare the number of aneuploid cells after stimulation for IVF, and as a model, cows were used. It was found that it is not a really big difference between the IVF stimulated and not stimulated embryos. Then, there was a really interesting analysis in Europe when they washed out embryos after natural fertilization and analyzed them, and they found that again they have the same number of chromosomal mistakes. We know about it because we have also a big number of first trimester miscarriages during natural conception. It is not just the consequences of a kind of stimulation, it is a matter of human embryos and mammalian embryos development.