Journal Club Global: Embriões mosaicos ao Teste Genético Pré-Implantacional para Aneuploidia (PGT-A): o que fazer?

Bem-vindo ao Fertility & Sterility Journal Club Global. Discutiremos embriões mosaicos ao teste genético pré-implantacional para aneuploidia (PGT-A)

Article:
Clinical management of mosaic results from preimplantation genetic testing for aneuploidy of blastocysts: a committee opinion

Discussants:
Augusto Azzolini, PhD
Bianca Bianco, PhD
Matheus Groner, MD, PhD
Ciro Martinhago, MD, PhD
Carlos Alberto Petta, MD, PhD

Moderators:
Renato Fraietta, MD, PhD
Pietro Bortoletto MD

Hello, good evening. We are starting the event, we are letting everyone in. Let's wait a few minutes just to be able to enter everyone who registered for the event.

Ready, everyone is coming in. Wonderful, good. So, welcome to Fertility and Sterility Journal Club Live, it's our second event in Portuguese.

My name is Pietro Bortoletto, I am the Interactive Associate and Chief of Fertility and Sterility. And again, I am extremely happy to be with you in this Journal Club Live in Portuguese for the second time. Today's event is being organized and presented mainly by Dr. Renato Fraietta, but unfortunately we are having a technical difficulty, he will come in a little while.

But to start the event, I will introduce Dr. Matheus Groner, who is an urologist and works with Renato, to introduce the panel and to introduce the topic of discussion today. Matheus, please. Good evening, good evening everyone.

The idea is that we talk a little bit about the article that was selected, so it was up to me to talk a little bit about this article. Can everyone see on the screen? Can someone just give me an OK? Perfect. Thank you.

So, the idea is that we talk about the article that will bring to us how we should do clinical management of the results of PGT when the mosaic embryo comes. This is the opinion of a committee that was organized by ASRM. My name is Matheus Groner, I am a Doctor of Sciences at UNIFEST, and I am a Preceptor of Medical Residency and Human Reproduction at UNIFEST.

It is important to bring that I am a clinician, and this article brings a lot of questions from the point of view of the laboratory, of the method of examination, so it is good that I will already give my interpretation of a good clinician who is facing these mosaic embryos as a result. Fortunately, we have a well-mixed panel that will help us understand this article a little better. The article brings that PGT is a technique in growth.

We have some data that in 2014, around 14% of cycles did PGT. In 2019, we have reached 44% of cycles in the United States doing PGT. Studies show that we still do not have evidence that PGT will bring better outcomes, especially in women under 38 years of age.

And mosaic embryos as a result of PGT have a varied prevalence, from 2% to 20% of the results of all PGTs. It is important to emphasize that the initial fear was that these mosaic embryos would be a mosaicism, as a prenatal or pediatric mosaicism, which is diagnosed by the cariote. I will try to explain a little the difference between a PGT-A mosaicism and a prenatal or pediatric mosaicism, according to the cariote.

When we think about mosaicism in the cariote, when foli or amniocentesis is done in the prenatal or even peripheral blood in the neonatal, we have a cellular cultivation of these cells and a visual analysis of these cells. And if there is a percentage of cells with normal chromosome and a percentage with normal chromosome, we have a diagnosis of a mosaicism. When we are dealing with a PGT, the PGT-A is not a cariote.

There are several techniques to try to bring this DNA and try to analyze this DNA, the best known, currently the NGS, the most used. The DNA of this family is extracted and amplified, and bioinformatics algorithms are used to compare with the values of normality. There is no visual representation of the cells, and we end up bringing as a mosaic results that are intermediate, which did not reach the 3, which would be a trisomy, or did not reach the 1, which would be a monosomy.

Doing here, as these images below, we can see what is normal, which is the euploid, which would be 2. And here we can see a trisomy of 14 and a mosaic of trisomy of 14. The PGT-A mosaicism is not necessarily a real mosaicism. And there are several factors that can contribute to this mosaicism.

The very statistical variations of the method. The mosaic can be, in fact, a real euploid or a real neoploid, according to these variations. According to the advances of the techniques for the PGT-A, there was an increase in these results of mosaicism, leading us to believe that the technique used can also interfere with these results.

There are biases in this amplification of DNA to achieve the result, being able to lead to this presence of more mosaic results. The interpretation of the results, either by software or by professionals, can also bring subjectivity and a wrong diagnosis. It may have seen the contamination of the sample, and this contamination is a result of mosaic.

It may be the cell's own mitotic state that leads to this result of mosaic. Biopsy technique is also associated, and also some laboratory conditions. But the important thing is what we have in the literature today about the reproductive defects when we think about transferring a mosaic embryo.

The article brings a lot of things for us to discuss. Until the publication of the article, there were more than 2,700 transferences of documented mosaic embryos. This study from 2021, a retrospective study, showed that, yes, there was a lower rate of implantation of mosaic embryos, even compared to euploid embryos of the same morphology.

And there is twice as much risk of abortion when compared to euploid embryos. However, there are some biases in this retrospective study. These couples who transferred these mosaic embryos only transferred this mosaic, because there was only it.

Were they already couples of worse prognosis, and that's why they had these worse defects? And this mosaic embryo can be a real neoploid, which would also bring a bias in the selection of this embryo. Another study he brings, a study by Capaldo from 2021, a prospective study, and he tried to expand the interpretation. He labeled as euploid not only the embryo that arrives in the 2nd stage, but from 1.5 to 2.5, expanding the range that we would launch as a mosaic.

And he noticed the same initial defects as pregnancy in course and abortion rate between euploid embryos and embryos that varied from the range of 1.8 to 2.2, which would be the low-degree mosaics of this study. The article ends this part by showing us that it is still uncertain if the mosaic and which of these mosaics can really interfere in the initial reproductive outcome. The biggest concern also in the transfer is not only the outcome, but after pregnancy and if this will bring a disease, either in prenatal or in the birth of this baby.

So far, there is no association between the transfer of a mosaic embryo and a fetal risk at birth. This study by Treff in 2021 was a review of more than 25 publications and described less than 1% of the 2,769 transfers that had the diagnosis of the same neuploidy that was associated with this result of mosaic. The study also brings three reported cases in which fetal neuploidy was seen, coming from the mosaic embryo in the same locus.

A case of a mosaic embryo that had a normal neonatal phenotype, but confirmed mosaic in his exam, a baby with neuploidy and that had an altered phenotype with the correction of the aorta, and a neonatal neuploid with food difficulties. The article also brings two important discussions in the face of mosaic embryos. If these mosaic embryos can be, in the future, some neonatal that has a uniparental dysosmia.

Uniparental dysosmia is one of the causes of mosaic, it is a post-egotic neuploidy due only to one of the parents. It is a rare event and some loci, some chromosomes such as 6, 7, 11, 14, 15, 20 can bring abnormal phenotypes. In this situation, there is a case reported in the literature in the post-transference of mosaic embryos that led to a diagnosis of uniparental dysosmia.

This mosaicism can also be confined to the placenta and bring to these embryos a risk of obstetric pathology, such as growth restriction in truterine. This 2020 study of VIOTE evaluated 162 newborns and there was no difference in the weight at birth between these transferences of mosaic embryos and neuploid embryos. And there are still no studies on the long-term benefits, either in the pediatric population or later, of these embryos that were transferred with mosaic.

The study tries to bring a risk analysis, which embryos would be more at risk or not. According to the percentages of mosaicism, sometimes it is released in the result as low or high degree of mosaicism, and it was seen in studies that the low degree has a better rate of implantation and gestation at birth. As for chromosomes involved in this mosaicism, there is a concern about avoiding viable aneuploids, such as chromosomes 13, 18 and 21, but there is no evidence in the literature that helps in this choice.

If this embryo is a trisomy or monosomy mosaic, there is also no evidence of which is worse or better for the clots. If this mosaic is complete or partial, in the case of a partial mosaic, the chance of being a false positive is greater, the concordance with the internal cell mass of the embryo is less, so the rate of gestation at birth is usually a little better than when the mosaic embryo is complete. As for the number of chromosomes involved, if there are 3 or more chromosomes involved, the clots are worse when compared to 2 or less.

But if we compare the group in which there are 1 or 2 or more chromosomes involved, there is no difference in the clots. What about post-transference? We can now evaluate the result in prenatal with some screening tests, such as ultrasound or NIPT, but these screening tests will not be able to diagnose neuploidy during prenatal. And diagnostic tests can be indicated, such as a 10 to 13-week chorionic biopsy or a 12-week amniocentesis.

These, however, can diagnose neuploidy or fetal mosaicism. Other tests within these can be done to try to look for a specific disease, such as PIC, NIC, FICH, or the study of uniparental dysomia. There is no evidence in the literature to recommend these prenatal tests in transference of mosaic embryos, but the American College already guides that these tests should be offered to all pregnant women.

The American Genetic Society also guides the study of dysomia in these prenatal embryos transferred as mosaics. Genetic advice is important in this scenario, showing the risks and benefits of each of these prenatal techniques and the limitations of the available tests. The article evolves to bring the importance of starting to build and set some policies regarding the PGA result, regarding the mosaic result.

The laboratory must have a certain policy of when and how to report the result. Each clinic must have its own protocol of conduct in the face of the result. And genetic advice must be present before indicating the PGT-A, knowing all the possibilities that the couple may face.

To conclude, in short, the transference of mosaic embryos, the adopted policies must be clarified to the patient, in terms of consent, on all possibilities, when considering the transference of mosaic embryos. I have always argued that there are several explanations for this mosaic result, in addition to being an affected embryo. There is no way to predict adverse events or rank these mosaic embryos as the best or least likely to have worse outcomes.

There may be a lower chance of implantation and a higher risk of abortion when transferring a mosaic embryo. Less than 1% of newborns have shown signs of illness, but the data is still initial. More and more people have transferred mosaic embryos, and it is important for us to evolve this knowledge to bring more evidence on the subject.

Genetic advice is always very important, and psychological monitoring too, to be able to help in these choices. Clinics and laboratories that transfer mosaic embryos should monitor and publish these cases. It is building evidence.

Everyone has to help us to get more results and to have more evidence to help these couples. Wonderful. Thank you, Matheus.

And now that we have solved the technical problems, Renato and Augusto were able to join us, I will introduce Dr. Fraietta, who is the free and docent adjunct professor and vice-chief of the urology discipline and the integrated sector coordinator of human reproduction at the Federal University of São Paulo. Renato, good evening. Please, introduce the rest of our panel, now that we have solved this technical problem.

Thank you. Good afternoon, Pietro. It's 6.15 pm, right? It's already dark, unfortunately.

Pietro's voice is annoying like this. Good evening, everyone. First, I want to thank you for the presentation.

I feel very satisfied to be the host of this second webinar in Portuguese. I want to thank you and I will introduce the people who are here with us to debate. Matheus has already presented a summary of the article.

Dr. Bianca Bianco is a biomedical doctor with a degree in genetics and a geneticist counselor. She has a master's degree and a post-doctorate from UNIFEST. She is a professor of the medical genetics discipline and a member of the Integral Care Group for Rare Diseases at the University of Medicine, ABC, and a geneticist at the Instituto Ideia Fértil.

Dr. Carlos Alberto Petta, free and docent professor at UNICAMP and responsible for the clinic Fertilidade e Vida. Dr. Ciro Martinhago, geneticist and specialist in medical genetics at the Brazilian Society of Medical Genetics. He is a doctor in reproductive genetics with more than 20 years of laboratory and clinical experience.

Dr. Augusto Azzolini, master's and doctor in sciences at the Federal University of São Paulo, coordinator of the GeneX clinic's reproductive laboratory. I want to thank you in advance for your presence and for those who are listening to us, also grateful for your participation. Let's go.

I'm going to start with the subject, it's a very intriguing subject, that's why it was chosen for us to be able to debate. So, I'm going to start by talking a little bit about PGT-A, before talking a little bit about mosaicism. Petta, according to the article published in Human Reproduction last year, the article Good Practice Recommendations on Add-ons in Reproductive Medicine, which talks about several adjuvants for vitro fertilization practice.

One of the conclusions of this recommendation is that the pre-implant genetic test for neoploidia is not currently recommended for daily routine practice. Petta asks, does PGT-A increase the rates of live births? Can you please talk about the literature, your experience, your opinion? Good evening, everyone. Thank you, Renato, for the invitation.

It's a great pleasure to be here with you. We have some clear things. If you look at the reason for the article, it is to give us clinicians, for example, a scientific substrate to expose to our patients what exists.

What exists today is that for women under 38 years of age, you will not improve the rate of live births. Period. What we have at the same time, the article itself mentions American data, which is what we see a lot today in private clinics, you go to your patient and say, you will not benefit in terms of live births by doing a genetic analysis of this chromosome because you are under 38 years old.

And then you start that, I wouldn't say clash, but it's an orientation, and more and more you see the couple, the patient himself, wanting the genetic analysis. So today we are a very controlling society, and it is very difficult to counter-argument. Sometimes I cite my example.

I say, look, I have embryos, my wife was 33 years old, I didn't do a genetic analysis. And sometimes what I hear is, okay, that's you, but I'm different. So I'll feel safer if I do a genetic analysis.

What can we have as pros? We can, for example, reduce the time until pregnancy, especially in those couples that have several embryos. So if you do a genetic analysis, you will select and only put the euploids, and you can reduce the time until pregnancy occurs. We also see many articles that if pregnancy does not occur soon, these couples give up, they are very frustrated from an emotional and psychological point of view, and often do not continue the treatment.

So this can be for those couples who have a large number, you reduce this number and reduce the time until pregnancy. It is logical that we always have to explain that this can be an aggression of the embryo. And then there is also the personal experience, because your laboratory is a technical procedure.

So just like surgery, you have people who operate faster, you have more skilled people, and you have more skilled embryologists, or not, to do a biopsy and damage this embryo as little as possible. So, at the same time, your laboratory has to reassure you that these biopsy results are still good. But we clearly live in an era in which it will be increasingly difficult to counter-argue with your patient, which he cannot do, or should not do a genetic analysis, because he can.

There is a psychological aspect, so if you have a failure, and you say, probably the most likely thing is that there was a genetic alteration in the embryo. And the first thing you hear is, ok, why didn't you test it then, for me to know what it is? I am here guilty, imagining that I did not rest enough, that the problem was mine, and now you say that it could be a genetic problem of the embryo, and I could have seen it before. So, in our day-to-day as clinicians, we really live in a dilemma, of showing what is scientifically plausible, and respecting or meeting the desire of this patient, who, despite all the head-ons, really wants to know what the genetics of this embryo are like.

Last point, today we freeze a lot of eggs, but we also freeze a lot of embryos. And this is a very important point, because if you are freezing someone's embryo at 36, 37, where you do not have an indication to do a genetic routine analysis, it may be that this woman will defrost this embryo only at 42. And if she is not pregnant at 42, her chance of finding euploid embryos at 42 is much smaller.

So, in my view, keeping embryos for the future, and in general for the future, it seems advisable to me that you at least know that these embryos are euploids, because if they are not, if there is one or only two, this woman will have the opportunity to join more euploid embryos when she is 42, 43. Right. The other question I was going to ask you, you already talked about being able to select the embryo, to be able to anticipate, if possible, to choose the embryo that can lead to this gestation earlier.

Very good. And the importance of information. Bianca, let's talk a little bit about the more technical part.

I even relied on article 1, which is what we are referring to. The article talks about the NGS technique, to what extent the identification of mosaicism by NGS is a problem. That is, to infer through a bioinformatics algorithm to measure the amount of DNA by amplification and comparison in contrast to the standard karyotype, which is done by visual observation.

Good evening. I also thank you for the invitation. I think this is our challenge, to make the equivalence.

When we talk about cytogenetics, we see the image. I'm seeing it, I know what I'm seeing. If I have doubts, I increase the number of metaphors.

If my doubt persists, I can still proceed, for example, to FSH, that I will see a mark. So I'm seeing, is there a mark? Is there no mark? And I can analyze a large number of cells, of metaphors, of interphasic nuclei. And when we talk about PGT-A, it is a sample that has a restricted number of cells, and we make this equivalence, I think it is very difficult.

In fact, we are not seeing exactly, I don't know exactly how many cells were biopsied, I know the amount, I can quantify the amount of genetic material, but these algorithms, at the same time that they help us, they can confuse us. So if we have degraded material, I can have noise, and this can, for example, confuse our analysis. Cytogenetics, as it is a technique that has existed for a long time, we already have very well established concepts.

So for us to talk about mosaicism, for example, analyzing 40 cells, 40 metaphors, I have to find three metaphors with similar alterations, and then I will have a cure, a detection capacity of 8% of mosaicism. These parameters are still missing when we talk about algorithms and when we talk about analysis by NGS. And then we have other confusing factors, because we have different platforms, different algorithms, and different patients, that we can have all this combination that can confuse our analysis.

So I think it's challenging, we really have to discuss to find the concepts. As we have established in the older techniques, we have to find the concepts that we can apply now in these new technologies. I think they are robust technologies, very sensitive, that some people say, we should not believe.

I think so, I believe in these techniques. Several works show the accuracy. So I think they have credibility, but they are so sensitive that maybe we are interpreting, as I said, these noises, these small variations, as an importance that may not have.

And then, how are we going to interpret this? I think we need to walk a little bit together, the clinic and the laboratory, for us to be able to establish these concepts. Excellent. And you can't keep taking more wrong cells to analyze, unlike a karyotype, for example.

Very well. Augusto, are you listening to me? Are you in the room, Augusto? I'm coming, doctor. Hi, Augusto, how are you? Augusto, now for you, about Table 1, and also a little bit about what Bianca said, the article puts as contributors to the result of mosaicism, statistical variation, artefact of test and noise, amplification of DNA, contamination, mitotic state, variation in embryonic biopsy technique and laboratory conditions and embryology.

What is the participation of these factors in the mosaicism rate, Augusto? First of all, thank you, Dr. Renato. I'm very happy for the invitation. I am very grateful to be here by your side.

I have participated in many meetings at the São Paulo School, also by Dr. Ciro's side, who has learned a lot from my career. And also to put myself in this position as an embryologist in the technique that directly impacts the result. As the doctor has already said, and Dr. Bianca too, these factors can contribute, yes, in a way to increase or impact mosaicism.

In terms of amplification, which the article mentions, I see as a problem in the laboratory a volume of dilution. The laboratories usually expect 2.5 microliters of the solution where we send these cells. If I do a much greater dilution when putting these cells, the preamplification of this material is all done on top of this volume.

So, if I cause a much greater volume when I send it, maybe the preamplification is being very diluted and the final result will be affected. Also, if we think about contamination, I make a very big parallel with the sex diagnosis analysis, where men do not enter to make this diagnosis. It is usually done only for women to prevent the contamination of XX, the XX cell.

Also, in the laboratory, we have to have very well-established routines of quality control. The dilution, the flow always connected to make this amplification, a cell, the operator, the endologist, can generate a contamination and in this preamplification, in a small cell volume, a normal mine cell can also be analyzed and generate noise, perhaps in the embryo that is altered. Or even a bad denudation.

If in the biopsy it is the cells of the cumbus, maternal cells, it can also generate noise. But I think that, among all the techniques, what most impacts, and is what some articles relate to, is the execution of the biopsy technique itself, to remove insects. Basically, we can do it in two ways, as the articles quote, which is the pooling or flicking, which is pulling the cells, or doing the flicking, which is scraping the cells of the biopsy pooling with the holding, or the association of the two.

The articles do not cite differences between them, in the range of opioidism, of mosaicism, however, the quality of the cells removed must be well evaluated. The integrity, if we are measuring these cells, and the quantity. And this goes a lot from the training and the look of the embryologist.

It is no use removing very few cells to preserve the embryo, and a genetic diagnostic laboratory does not have enough material to analyze, but it is also no use removing many cells for the laboratory to have enough diagnosis and the embryo not to be preserved. So, this is very much part of the experience of the embryologist, and the correct timing to do this biopsy. The moment of expansion, many times, doctors are anxious to biopsy the embryo on day 5, but they do not have the necessary expansion to remove these cells.

So, forcing a biopsy at this moment can be harmful to this embryo. So, yes, it is a direct impact on the result of mosaicism. Very good.

I'll go back to Bianca again. Bianca, I want to know how do you classify the degree of mosaicism? What is the percentage that is low degree? How much is high degree? How much is neoploid? How do you divide and use as a parameter those of low degree? Can we think of transferring those of high degree or not? How do you make this division? Well, if we have a variation in the images, less than 20%, we will consider this embryo as neoploid. If these variations are greater than 80%, it is aneuploid.

And in the middle of the way, if we have from 20% to 50%, a low degree mosaicism, from 50% to 80%, a high degree mosaicism. This can vary a little bit according to the platform. We can consider 30%, 70% to classify as low degree and high degree.

It is difficult, this classification, and even more difficult, the decision of how to treat this. But I have a tendency to prefer, of course, those of low degree. Then we would expect, if this is a true mosaicism, that we have a smaller amount, perhaps, of altered cells, and that maybe this can, not necessarily, but that this can have a smaller impact on the phenotype that this future child and this individual will have.

Those of high degree, we think we can consider with caution, depending mainly on chromosomes that are involved. Those alterations that, for example, involve chromosomes that are viable, compatible with life, in this case, I believe they should be avoided, the transfer of these high degree embryos, these high degree mosaics. I find it interesting, over the years, we are getting a little more confused.

So, the percentage, we would transfer those of low degree, but over the years, we are also, according to the situation, transferring those of higher degree. Of course, we will learn over the years. Dr. Ciro Martinhago.

Very well. Dr. Ciro, the article states that he, the survey done of more than 2,700 embryos, transferred mosaics, documented, involving prenatal and postnatal results, neonatal, and less than 1% of aneuploidy. I want to hear your opinion, having less than 1% of aneuploidy in these prenatal and neonatal.

Is this an incentive to transfer mosaics? What is your opinion, Ciro? Perfect, Renato. First, publicly, I would like to thank you for the invitation, for the kindness, for us to talk a little bit about our daily lives. We discuss a lot on WhatsApp about these little reproduction problems, but I think it's very cool.

Without a doubt. I think we can expose this type of daily problems to colleagues, doctors or biologists, who work in the area. Very well.

Renato, in a general way, summarizing the history of mosaics, I usually say that the patient who has only mosaics and this embryo to be transferred, the suggestion is to freeze this embryo, leave it stored and make a new cycle. This is the first recommendation that we make for a couple who only has mosaic embryos to be transferred, without getting into controversy about high, low chromosomes, etc. Obviously, some chromosomes are not viable.

We remember the chromosome 21, for example, which is a high probability of giving birth to a baby with Down syndrome. Taking into account the mosaic, Renato, it is extremely difficult to have a sensibility to say that the low or high mosaic embryo will be born with a mosaic of 2%, 3%, 10%, 20%, whatever. What leads us to believe, in a general way, as you said, is to be very optimistic about the current results.

I have a great concern about what we call segmented mosaic, which is not the whole chromosome, it is just a segment of this chromosome. Depending on the chromosome, Renato, and depending on its size, it can generate a baby and it can generate malformations and even birth, as happens in chromosomes 5P, 4P and other chromosomes. However, segmented mosaics are shown to be the most promising in giving birth to healthy babies.

Statistics show that both high and low mosaics are giving birth to healthy babies. Possibly, this should be a technical artifact, especially by NGS, as Bianca said. In general, Renato, what we suggest to couples, the concern with uniparental dyshomia, although Nathan Treff has shown in embryos, about 0.06% of embryos have uniparental dyshomia.

What does that mean? It means that this baby, this embryo, has two chromosomes of the same parent, and not one of each parent as it should. Depending on the chromosome, this can be catastrophic. Prader-Willi syndrome, and so on, have been showing this for decades.

But, in general, putting 0.06% of embryos, we know that it is a birth, born alive, in the general population, it would be 0.03%. It's a double in embryos, but we don't know this in nature as it should be. So, I see today, Renato, this new technology with good eyes, mainly this technology that is being analyzed by SNIP, it was through this technology that Nathan was able to show polyploidy, how many embryos have uniparental dyshomia. But, in general, the transfer, mainly from the low mosaic, taking into consideration mainly the chromosomes 21, 18, 13, and the sexual ones, which I don't suggest, 15 would also be included in this story, the transfer in any way, always shows the patient exactly that the basal risk that the couple has naturally would be 2 to 3%.

And once the mosaic embryo is transferred, this risk adds less than 1%. I mean, if he were to try naturally, he would have practically three times the risk, any couple, than transferring the mosaic embryo. Now, never say, Renato, that there are no risks.

This is the question. Look, this chromosome never happened to be born, this or that. And as it was very well spoken, even by Matheus in the class, I mean, we don't even have a prospective assessment of these children.

We have children who were born healthy, some of them were prenatal diagnosed, remembering that NIPT is not prenatal diagnosed, cariotic is not prenatal diagnosed, the ideal would be NGS or microwave in myotic liquid or birth, and that we can't somehow establish true mosaicism in a complex organism, such as the human being, because I myself have several patients who sometimes the mosaic is in the skin, it is not in the blood, it is in the brain. So we may come across in the future an eventual deficiency, a deficit at school, something like that, which we still don't know today. However, only the future will tell, Renato.

And through these transfers, we are bringing more and more knowledge so that we can advise these couples to transfer these embryos or not. Just to finish, a recent work in 2024 shows that patients above 42 years of age, it is more viable to transfer this mosaic embryo than a new cycle for this patient. Remembering that this 42-year-old patient will usually generate 4 or 5 embryos, possibly one of these would be normal.

This, of course, based on data from American clinics. Below 42 years of age, the suggestion is to keep this embryo, and wait for information that comes in the future through work like this. In the long run, right? I will ask some more succinct questions.

Does the mosaic embryo implant less, Petta? There is no microphone. So it implants less and aborts more. This is the clear answer.

And then we get exactly to what Ciro was talking about. If you have an euploid embryo that has a greater chance of implanting and aborting less, you start with the euploid embryo and the discussion of the mosaic is if there is no other option or if you do not have an analyzed embryo to know what you will put if one or the other, right? And that was the next question. Putting together the risk increase, which is double abortion.

Augusto, following this same line, talking a little about embryonic development, we do the biopsy. The biopsy is done in the trophectoderm and the presence of altered cells in the trophectoderm and the child is born normal. Does this mean that the embryo has the ability to regenerate? From the biological part that we see in the daily routine, I also draw these parallels.

We do embryonic cryopreservation and vitrification and there is some degeneration of the blastoma. There is a possibility of recovery. We do the biopsy, it remains integrated, even of low quality, it still has viability.

But I read an article and I saw that around 70% of embryos that were cultivated, extended cultivation from the 5th to the 12th with embryos that were mosaic, they saw that they were alleoploid after the 12th day. There are also some discussions in animal models that speak of a possible downregulation of the alleoploid cells. There would be a lower metabolization of the alleoploid cells and a selective apoptosis of these cells.

So maybe there would be a bias. Maybe Ciro and Dr. Bianco can help me. Maybe there is also a discussion about the possible monosomal and trisomal rescue.

That would be a trisomy and the cell excludes a chromosome and it leaves this division. Or this cell being monosomal and this single chromosome being divided. These are still debatable theories.

Possibility of explanation for this mosaic of a normal embryo. Do you want to comment, Ciro? Very quickly, Renato. This regeneration is very interesting because the most accepted theory is that alleoploid cells have a much lower cell division than alleoploid cells in general.

Trisomy of the 13th, it seems that the embryos arrive in the blastocyst earlier. There are some exceptions. But in general, this is what happens.

And it happens, Renato, that at the end of the story a lot of alleoploid cells into aneuploid cells. Because alleoploid cells divide at a much faster rate than the aneuploid itself. And these altered cells are generated in this embryo, which would be this theory of regeneration.

This is the first thing. Second, Renato, in my opinion, many of these mosaics are being generated post-biopsy in that sample only, and not in the embryo. So this is a pitfall that we don't know how to fix.

Maybe freeze this material and postpone it. Bianca, Ciro even commented that if the couple has a mosaic embryo, if possible, repeat the cycle and always look for an euploid embryo. In which situation do you recommend? Of course, it depends on each embryo.

But the question is when to consider transferring mosaic embryos, when there is no more available euploid embryo, the couple does not want or is not able to do any other stimulation to achieve an available euploid embryo? This lack of option of not having euploid embryos, I think it is a routine, what we hear the most, what we see the most every day. But we also have financial options, but we cannot ignore the psychological and religious ones. Patients who say they are tired of the treatment, it is exhausting, so I don't want to do it all over again.

Let's discuss the transfer of this embryo, the religious questions too, what am I going to do with this embryo, discard it, it is not an option for me, this is an important issue. The perception of the benefit of the patients knowing that other people may have transferred or nothing happened, they did not implant or the gestation did not go ahead, so if it doesn't hurt me, well, it won't hurt me either, so this is also an important thing. But I think the most important thing is to try to discuss the limitations of the test.

I always say that what is more important than the test is what it does not see. So I always try to discuss these limitations, what are we going to see, what are the possibilities of the result, and what can happen with that transfer. And then I look for literature data to know if there are born with a mosaic, or even if we can make an analogy with this transfer of this embryo, and make clear the consequences of this transfer.

It is never simple, it is never an easy decision for either side, not even for us who are talking to the patient, not even for the patient this decision, but I always say that we will exhaust the doubts as much as possible. So we will talk, and they will have doubts, and we will talk again until they can, the couple or the person, can make the decision. So, look, I will face this risk, or better not, I will try another cycle, or I will wait a little, or I will go to other possibilities.

So we have to consider all of them, but in general, the lack of options is the most common. And that's why we are here debating about this, a really intriguing subject. Matheus, we talked about prenatal results, neonatal results, and what, according to the article, and what you have from literature, how is the evolution in the long term of children born caused by transference of mosaic embryos? I think the issue is that we don't have it, we don't have this science yet, we don't have this evidence, this long-term monitoring, transference of biopsied embryos is a new thing, and of mosaic embryos is even newer.

The article even brings this as a fragility of this long term, and the importance of building this evidence. So, from the moment that we accept, we as professionals, and the couple too, accept this embryo, it is very important to have a segment for us to be able to publish and raise, and have a science that for now is still a failure. So, I think we can't say to this couple that, even if it is born, and it doesn't have any initial genetic diagnosis, that doesn't mean it won't have a problem in the future.

Dr. Ciro already brought some examples of this. So, I think it is a dark path that must be divided and must be followed, because if we don't follow and don't publish, we don't have evidence, we don't build. Scenes from the next chapters.

Ciro, going back to you, you transferred the mosaic embryo and got pregnant. What do you recommend for exams and when? And taking advantage of the question, does this differ when we transfer a non-tested embryo or a tested and euploid embryo? Is it different? Are the tests different? And when to do these tests with the pregnant woman? Excellent question, Renato. Especially about today's prenatal diagnosis, we have invasive, non-invasive.

So, when it comes to prenatal certainty, always amniotic fluid. Chorionic velocity, NIPT, are screening tests. If we consider only chorionic velocity, despite being a month earlier than amniotic fluid, we have around 1% to 2% less assertiveness in the diagnosis exactly because of the mosaic.

And the mosaic can be in natural gestation, 2% to 3% of all natural gestation are mosaic. The mosaic is not a monstrosity that is being created within human reproduction, but in nature. This is very common, and it is believed that we all have a mosaic to some extent.

The first thing I always say to the patient is if you want to be sure, not getting into interruption controversy, some couples opt this way, others not so much, the diagnosis needs to be done in amniotic fluid. And to detect the mosaic, the same technique is used in the embryo, the NGS, the most sensitive diagnosis technique for mosaicism. On the other hand, Renato, NGS does not detect parental dissonance, which would be important in cases like this.

So it ends up being a micro-A, the gold standard to detect at the same time mosaic and also parental dissonance, or even any other disease. Remembering that the micro-A has a resolution of dividing the DNA in more than 800,000 points. There may be an alteration, a small deletion, duplication in the genome that has nothing to do with the mosaic.

So Renato, it is super important to clarify this for the patient. And this normal test, even with high coverage, will not show that this embryo has nothing. And always remembering to show the couple that there are other concomitant diseases.

There are diseases of the general population that can affect any baby. I had a couple who transferred the normal embryo, the baby had a problem in the foot test, and I said, Doctor, there was a genetic problem, didn't you do a genetic test on the embryo? I didn't. In fact, the chromosomal part was tested, not the others.

So, in short, prenatal, the miniotic fluid, microA and NGS, ideally microA. Petta, the article talks about double transfer of embryo, being one mosaic with another euploid, or then with a mosaic embryo and another untested. What is your opinion about this? Renato, I think you should remember, we were together in the Regional Council of Assisted Reproduction Medicine and we had a lot of requests from patients wanting to transfer the embryo with a down, an aneuploid embryo with a down, and the doctor asked for an authorization from the CRN.

Our conclusion is that the embryo belongs to the patient and the couple. I think this is our basic principle for northeast medicine, because we don't have numbers to say if it will get worse, if it will get better, or what will happen. In theory, if you put an embryo that has a higher risk of abortion, both become pregnant and one will become more likely to have an abortion, it can harm the one that was doing well.

So, from a practical point of view, it doesn't make sense if you have an euploid embryo and you put a mosaic together. But what the article itself says is that it is the right of this couple. The embryo is theirs, so we as clinicians, as doctors, have to guide.

In this, I think it is very important to have genetic guidance, independent genetic guidance, because this is also an important thing, because then you say, you said what I could put, and when you have independent genetic guidance in writing, everything that can happen and what cannot happen, you are making it clear in several parts everything that we can think. If you ask if the embryo is mine, if I have an euploid, I first put an euploid. If I have an unanalyzed one, I first put an unanalyzed one.

But you know it is not like that in people's thoughts and beliefs. And they have the right because the embryo is theirs. Can we do this? We can.

We must advise, we must protect that all this is informed and made sense. Excellent. And based on all that we talked about, the last question I ask to Matheus, all this involves psychological issues, it messes a lot with emotions.

So, Matheus, what is the importance of the professional psychologist in these cases, Matheus? Look, I think it goes even further. I think the couple that is going through all this process already deserves psychological support. Because we who experience this on a daily basis already know the ups and downs they will face.

Imagine a couple who has a problem. So, I think it is essential. And when you think about this issue of PGT and the results of PGT, I think even more, because it will mess with things beyond science.

Dr. Bianca already brought this, the issue of culture, the issue of religion. So, how much these choices will weigh on the emotional side of these couples. So, I think it is essential a joint follow-up with a psychologist, even more so if it is a psychologist who also understands this clinical part within reproduction, I think it will only be beneficial, without a doubt.

Well, we are finishing here, straight to the end, I will only ask each of you to make a statement, any consideration you want to conclude. Please, 30 seconds each. Let's start with Bianca.

Let's go. I think we need to discuss more and share more of our experience, which is often not yet possible to publish. Personal experience has no scientific value, but we add to our personal experiences until this has a scientific value and we can build these consensuses and guidelines for these patients.

Thank you. Petta, your final considerations. Renato, we are in a journey.

We discarded the mosaic embryo, today we are transferring the low-degree mosaic embryo, we are already discussing transferring some high-degree mosaics. While this is very important, it improves the technique and gives us more reliability in what we are talking about. And this journey, in fact, is just beginning.

That's it. Ciro, your final considerations. Well, Renato, in my opinion, the trend of the future of genetic analysis itself, increasingly with other technologies associated, artificial intelligence, whatever it is, fluid analysis, culture analysis, I think the trend, Renato, is not to call the neoploid, viable and unviable embryos anymore.

It is to start speaking a universal language so that everyone understands, not only the patient, the other medical colleagues. I think this will be the trend, the trend will be to add other variables along with PGTA and say, look, this embryo is viable or it is not viable, maybe even with some percentage, as artificial intelligence is already bringing. We are already living this, we are living this, but for the future.

Augusto, your final considerations. What the partner said, thinking that biopsy will not cease to exist over time, it is a technique that is implemented in a well-established laboratory, I think it is worth the investment, the dedication of the laboratories to formalize it, to perform it with excellence, and it must be a technique that must be evaluated in monthly, semesterly, it depends on the year, and mosaicism is a strong indicator of the quality of your biopsy. If it is very high, maybe it is a signal to your laboratory that something is happening.

Well, thank you very much. Matheus? Speaking specifically of mosaicism, what worries me the most is that, to a certain extent, the mosaic embryo is a possibly altered embryo, either with mosaicism or neoploidy, or it is a variation of the technique. It is a mistake of the technique and we are taxing a possible good embryo in that sense.

So I think that's what we have to build a lot, and we are building from now on, to be more sure, to talk to the couples, to see if it is worth or not the transfer of these embryos. I think we are evolving, and we have to continue this evolution and publish about it for us to know more. I want to thank, I enjoyed it a lot, I want to thank everyone's presence, everyone who debated, they are people of high stature, from several institutions, so it was important for us to debate, and this debate continues.

There are several subjects, several other themes. I want to thank Fertility and Sterility, Pietro, for this opportunity, it is the second time that I coordinate this Journal Club in Portuguese. Thank you very much and see you next time.

I pass the word to Pietro. Thank you Renato, Ciro, Carlos, Augusto, Matheus and Bianca, a very rich and super interesting discussion. Today's discussion will obviously be shared on the podcast of Fertility and Sterility, on the site of Fertilityandsterility.com as well, and we hope you share it with your colleagues in Brazil who cannot be here with us tonight.

We hope to have this event several times a year, and if you are interested in any topic or to be a panelist in this discussion, please send an email to me or Renato, get in touch. But until next time, thank you and good night to everyone. Good night.

Good night, thank you. Good night, bye.