PGT-A testing in the IVF lab: The Embryologist’s Perspective

Carolina Andrés, Clínica Tambre, Spain: I’m a senior embryologist at Clínica Tambre, which is based in Madrid. I’ve been working for over 7 years at the clinic, and I’m thrilled to be here to answer all the questions that might arise.

PGT-A is something that we commonly use. Here in Spain, part of the treatments are covered by the public hospital system. Most of our patients, since I work at a private clinic, are of advanced maternal age, which is the main indication for this kind of procedure. I can’t remember the exact number of cycles, but we do it quite often. Most of our cycles are PGT-A, because there is a medical indication besides that.

Host: Sandy Christiansen, Clinical Embryologist & Fertility Educator and Advocate: Also important to note for the audience: depending on where you are, PGT-A might be considered standard protocol or part of your treatment. In some countries, it’s what we call a treatment add-on. Where I’m based in the UK, it is an add-on. It’s something you have to select, and therefore also pay for. In some countries, this can be quite expensive and possibly a limiting factor in accessing that care. Also, when I started my embryology career in Sweden, PGT-A testing was not legal or allowed. That was 16 years ago, and today it is still not allowed. It’s really interesting to see differences in practice.

Chrysa Karakosta, NewLife Greece: My name is Chrysa Karakosta. I’m from New Life IVF Greece, located in Thessaloniki, northern Greece. I am a clinical embryologist, but for the last 9 to 10 years, I’ve headed what’s called the international team at New Life. I still go into the lab, but my main focus is dealing with international patients.

PGT-A is something we do offer at New Life, but the Greek legislation is very specific about who is allowed to have PGT-A testing. In order for us to perform PGT-A, we need approval from the Greek National Authority of Assisted Reproduction. This is like the UK’s HFEA, which oversees and licenses all clinics. Patients must meet specific criteria to be permitted for PGT-A:

• Being over 40 years old

• Having 3 or more failed treatment cycles

• Having 2 or more miscarriages

• Or having some sort of abnormality in their karyotypes

They don’t need to meet all the criteria, one is enough. So yes, we do offer it. As Carolina said, we have many women of advanced age or international patients who have failed many times in their home countries and come to Greece or Spain hoping for a better outcome. So yes, we do quite a lot of PGT-A testing. Like Carolina, I can’t give you exact numbers, though.

Host: Sandy Christiansen, Clinical Embryologist & Fertility Educator and Advocate:  As embryologists, we’re all laughing here, for those listening in, because to do the testing on these embryos requires a really delicate biopsy procedure. In the UK, we have witnessed procedures, which means someone must witness every step: moving embryos from a time-lapse incubator into a biopsy dish, transferring the cells into a marked tube, and then freezing the embryo.

That requires extra staff, so in some clinics it can become more difficult to manage, especially depending on staffing. Those are the times when we definitely know how many PGT-A cases we have, and if it requires staff on weekends. It’s an interesting area. There are a lot of guidelines and best practices around this. To your point, Chrysa, people may need to have had multiple failed cycles, recurrent implantation failure, or recurrent miscarriages before considering PGT-A. Also, advanced maternal age is a key factor.

Some literature even suggests starting PGT-A from age 37 onwards. However, there are also guidelines, like those from the HFEA, that say PGT-A might not increase your chance of pregnancy at all. It’s rated ‘red’ for increasing pregnancy chances, but ‘green’ for reducing the risk of miscarriage. You have to read quite far down to find that detail, but it might be very relevant for some patients.

Carolina Andrés, Clínica Tambre, Spain: In Spain, there are recommendations from our main societies, which are the Spanish Fertility Society and the Spanish Association of Reproductive Biology (ASEBIR). They give recommendations, but there is no strict legislation. Even young patients who have had previous bad experiences, maybe several miscarriages, even though their age would not be within the group, we would do the PGT. We don’t have to go through any specific approval except for PGT-M or PGT-SR and so on, which is a different scenario. We don’t have the traffic light system like in the UK.

Host: Sandy Christiansen, MSc, Clinical Embryologist & Fertility Educator and Advocate: That’s the HFEA, our regulatory body in the UK – the Human Fertilisation and Embryology Authority. They license the clinics and gather all of the data. What’s really good is that they are your objective source of information. If you want to check out pregnancy rates, you go to them. Fun fact: these have finally been updated as of last week. Previously, they were still showing results from treatments started in 2019, so it has been a long time coming. We’re very pleased they have this.

They also provide best practice recommendations. There are no set guidelines, but they are based on what the literature says. Again, it’s not a strict rule. It is up to patients and their medical doctors to decide the best way forward. We know it’s in everyone’s best interest to help our patients get a healthy pregnancy as quickly and as safely as possible, with minimal add-ons and in the best way for them. That is always something we strive towards.

Chrysa Karakosta, NewLife Greece: Indeed. When we get PGT results, we can have a euploid embryo. This is a healthy embryo. We can have what’s called an aneuploid embryo, which is an abnormal embryo. It is not recommended to transfer such an embryo because it will either lead to a failed cycle, a miscarriage, or the birth of a baby that may have a problem.

A mosaic embryo is an embryo that contains both healthy and unhealthy cells. Nowadays, the great thing is that we have the technology to understand more about what sort of mosaic embryo it is – whether it’s a high mosaic or a low mosaic. This gives us the opportunity to consult with our cases and the geneticists and decide whether it’s worth transferring. You may not get the same result as with a euploid embryo, but they are what we call second-choice embryos after euploid ones.

Of course, we also have the “no result” embryos. Despite embryologists doing their best to biopsy an embryo safely, take the correct number of cells, and send them to the genetic lab, sometimes the DNA of the embryo isn’t good enough. The quality may not be sufficient to amplify the material and get a result. Sometimes we don’t even meet the lab’s criteria for analysis. That’s when we get a “no result.”

This means we have the choice to either transfer the embryo without knowing whether it is healthy, unhealthy, or mosaic, or to rebiopsy it and put it through the same process again. This is a very crucial decision that the embryologist needs to consult with the patient about. If the embryo is fragile, we may damage or lose it. Patients need to understand this from the beginning, especially if they have only one or two embryos, and we need to weigh the risks versus the benefits of the technique.

Carolina Andrés, Clínica Tambre, Spain: Yes. Following a bit of what Chrysa mentioned, no amplification or degraded DNA, those are the two scenarios where the results are not conclusive. They see that the chromosome gains or losses affect a lot of chromosomes. What they see in the amplification is very chaotic. In those cases, the PGT lab would report that to us, and that’s when we should consider rebiopsying if the embryo is suitable for it or not.

Chrysa Karakosta, NewLife Greece: It has evolved. The bad thing is I’ve been through all these stages. We’ve been here a while. I’m looking forward to the next stage of PGT-A testing, which is going to be non-invasive testing. Then we won’t have to worry about damaging an embryo, which is the main concern embryologists have in the laboratory.

But are there misconceptions about PGT? Big time. PGT doesn’t mean that if we have a healthy embryo, you will have a definite pregnancy or a live birth. That’s the big misconception. People hear the high stats that we get following PGT-A testing, but what they forget is that, first of all, we have to find that embryo to get you pregnant. Unfortunately, when we’re discussing older reproductive ages, finding that healthy embryo means we need a lot of embryos or a lot of luck. As women age, genetic abnormalities increase. This is a fact. There are no magic lotions, potions, tablets, or supplements to change that.

So, I think this is the important point when we’re speaking to patients and deciding whether we should do it. We should focus on explaining that PGT-A, despite these advances and how much more accurate it is, our genetics laboratory gives a 97% accuracy, still, it’s 97%, and still, it doesn’t correct embryos. It’s a diagnostic tool. It tells you things about the embryo. But if the embryo is bad morphologically or abnormal genetically, there’s nothing PGT can do to assist us or improve that result.

Carolina Andrés, Clínica Tambre, Spain: With embryo arrest, I would imagine that it does not survive the thawing procedure, right? This is something difficult to predict. What we can count on is a very good vitrification system. First of all, it’s important to do a very good embryo selection, to have a very good embryo of good quality, and that’s what’s going to help us reduce the risks that the embryo does not survive after the thawing procedure.

We can never know 100%, but in the equation, we have to have a number of good-quality embryos and obviously a good vitrification procedure. All the freezing procedures – the vitrification, which is the ultra-fast freezing methodology that’s carried out at this moment – are very efficient. Actually, the survival rates are very high. They’re around 98%, 95%.

So we can say that the chances are high, but we can never assure that we are not going to have the possibility of losing that embryo. There may be something within the embryo. A day five 4AB is an embryo of great quality, at least if it’s Gardner criteria, which is the one we carry at the clinic, and that’s an excellent embryo. So, at least from the quality, because we mostly analyse that part when we take into account the survival rate, the chances would be that it has a higher chance of surviving.

Chrysa Karakosta, NewLife Greece: Obviously, no matter how many questions you ask, no one can tell you the reply for sure. But I will give you a few pointers that I think are important to know for PGT-A and better survival.

We’ve seen over the years that we have much better survival when our timing is more precise. For example, it would be a good question to ask the laboratory: Are they using time-lapse incubators to pinpoint when to do the biopsy? Do they have a laser system? Because if they’re not a PGT-A country doing a lot of PGT-A, do they have a laser, or are they going to use old techniques to make a hole in the zona of the embryo to get those cells out?

Do they have experienced laboratory staff to do it? For example, our lab may have nine embryologists, but not all of those embryologists do PGT-A testing. In order for us to allow them to do PGT-A testing, they are the most experienced. They go through a lot of training. They do a lot of things before they’re allowed to touch any of the embryos that are true PGT-A cases, because it’s a huge risk.

All of these questions have to be asked. But it’s not just the laboratory part they need to ask about. They need to know who’s going to be the genetic laboratory analysing these cells. It’s totally different to find a local clinic that doesn’t have the experience to analyse embryos through NGS, as you said, next generation sequencing — or if they have a big provider that not only uses high-tech technology, NGS sequencers, and things like that, but also sometimes AI technology in combination to get an even better result. All of these things should be asked.

Carolina Andrés, Clínica Tambre, Spain: This is what I mentioned before about PGT-M, which is the pre-implantation genetic testing for monogenic diseases. This kind of testing probably changes from one country to another, but I will say what we do here. Chrysa, if you have anything to add, please feel free.

In Spain, yes, we can test the embryo for specific diseases. There is a group of diseases that can be tested without any consultation. The genetic laboratory will require a previous genetic report and probably new samples from you and your partner, and maybe other members of the family, to analyse very well where the mutation is. This is in order to do a good study of your specific case and be able to approach the analysis of the embryos more specifically with the best technique.

There is also another group of diseases that require approval from our Spanish committee. Normally, the diseases that can be tested are those that appear early in development and have a high probability of being very severe during the first years. For other diseases, where development is later, we have to ask this committee for approval. Only if they give us approval can we go ahead with the PGT. This process takes about six months until we receive the result — approval or not.

Chrysa Karakosta, NewLife Greece: Similar. But we apply for everything. We need to get approval for everything. If it’s a condition like, for example, sickle cell disease or cystic fibrosis, because they are diseases that definitely need testing, we get the approvals faster. If it’s something that isn’t as well-known, the committee will ask us for more paperwork. Either a specialised geneticist will come in and look at the case and say: What are the chances of a live birth? What are the chances of the baby, the child, having an issue? Then they make a decision accordingly.

There are more specific cases than PGT-A, as we were talking about earlier, and all of those cases, at least in our laboratory, go through genetic counselling. They need to speak to a counsellor to understand: Is it really necessary? Is this going to solve their problem, or is the chance of finding an abnormality 99%, and we’re not going to find a healthy embryo? There are lots of things they need to speak about before we actually process it.

Carolina Andrés, Clínica Tambre, Spain: Yes. At least what we do in Spain, we can transfer mosaic embryos. It is also something that the clinic decides, because you can decide whether the PGT laboratory reports the mosaicism or not.

At least we do ask our laboratory for the PGT to report these results. From the mosaic embryos, we have the possibility of transferring, but certain guidelines only recommend a specific mosaic embryo. The PGDIS and the COGEN established these recommendations for transferring mosaic embryos. There are different types of mosaicism, so this is a huge topic. Mosaic embryos are classified based on how many chromosomes are involved, if the whole chromosome is involved or just a part of it, and then the percentage of cells affected.

Depending on the grading, it can be recommended. Mostly, low, simple mosaics are recommended. These are mosaics with a low number of cells affected with aneuploidy, and “simple” means that only one or two chromosomes are involved in the mosaicism. But this transfer will always be done with previous genetic counselling from our laboratory. It’s compulsory before considering any transfer. After the transfer, prenatal diagnosis is recommended—amniocentesis is the biggest recommendation, but at least some prenatal diagnosis is agreement.

Chrysa Karakosta, NewLife Greece: AI technology is being used everywhere. It is the future. It is coming into the genetic laboratories. One of the biggest genetics providers, Cooper Genomics, is utilising it. They have a platform that states that after starting to use this platform, they were able to have more euploid embryos because they had a better detection of the quality and quantity of the DNA they were able to detect.

Can I analyze this? I can’t, I’ll be honest. But I have to believe them because studies are backing this up.

Is AI the only technology that we should use? No. The human eye is never going to be bypassed. The embryologist’s point of view when they see an embryo in the lab should be a valid point that people take into account—the clinicians, the patients.

Even if we have an embryo that comes out after genetic testing and AI technology and everything says it’s good, but we have a very poor embryo morphologically, we still need to state this; we still need to be heard. That embryo has less chance of surviving, less chance of implanting, and less chance of developing into a live birth. So yes, AI, but to a point.

Carolina Andrés, Clínica Tambre, Spain: There are different things to consider when looking at this specific case. Of course, advanced maternal age is something that’s going to condition us very much on the genetic result. Unfortunately, we know that the genetic content is very much affected as maternal age increases. Embryonic arrest on day 3 is mainly associated with male factor, but it is also known that embryos can arrest on day three due to genetic abnormalities, large chromosomal alterations that can lead to this arrest.

If we want to optimise as much as possible, looking into the male factor is something very interesting. Given the situation, I would consider analysing that factor a bit more. Maybe analysing fragmentation, for example. We do a lot of sperm DNA fragmentation testing at our clinic, where we analyse simple and double-stranded DNA. We know that especially double-stranded DNA has a big role in embryo development. That could be something to take into consideration for future steps.

Chrysa Karakosta, NewLife Greece: What I wanted to point out is that sometimes embryo development could be considered a mini PGT-A testing without the testing. This is nature filtering out embryos. Even if we take an example of an egg donation case where eggs come from a 20-year-old woman—superwoman—still, even if we get 10 eggs and 8 fertilise, that does not mean we’re going to get a blast. That isn’t how nature works. Even if we’re using Superman’s sperm, some embryos can’t develop because they simply can’t develop into an embryo that can implant. That should always be taken into account.

Studies have shown that in women aged 42, we sometimes need up to 22 eggs to get one healthy embryo. So, unfortunately, in these older maternal ages, we do need more eggs, more embryos to get to that one healthy embryo. But it doesn’t mean that we need to test 22 embryos—it means that sometimes it takes a lot of embryos to create that one healthy embryo. I wish you all the best of luck, and I hope this one is the one.

Carolina Andrés, Clínica Tambre, Spain: Yes, actually, we have a big number of international patients, and they come from various parts of the world. A lot from European countries with different legislation, and also from the US, Australia. We see lots of patients coming from countries where legislation is more restrictive in these kinds of procedures.

Host: Sandy Christiansen, Clinical Embryologist & Fertility Educator and Advocate: It’s important to check who is performing the actual biopsy in the laboratory. Most often, professors, doctors, or your treating OB-GYNs might not be the ones trained in that biopsy procedure. They may have worked in clinics that perform these procedures and have experience in clinical genetics, which is great. They can provide genetic counselling, but they might not be the person performing the procedure in the lab.

Chrysa Karakosta, NewLife Greece:  On the day of egg collection, when we clean the eggs from the surrounding cells, we see three stages of maturity. These are:

• Metaphase II: completely mature eggs that we can perform ICSI on and hopefully fertilise.

• Metaphase I: an intermediate stage of maturity. If we culture them in the lab, they sometimes mature, and we can use them.

• Germinal vesicle (GV): remains immature no matter how much we culture it.

Metaphase Is, if they mature to MII, can be used. But we always warn patients that they have smaller chances of fertilising, developing into blastocysts, and lower chances of resulting in viable embryos. It’s a similar concept to testing embryos at day 5, 6, or even 7. When we test embryos that don’t meet the criteria by day 5, we see lower chances of having euploid embryos.

Carolina Andrés, Clínica Tambre, Spain: We do consider them for microinjection since we have to leave them some time after the denudation procedure before ICSI. It’s true, and I agree with Chrysa, that the chances they fertilise and form a blastocyst are lower compared to MII. But in order to increase the number of eggs, it’s worth considering and taking them into account. Anything we can do to help maximise those chances.