In this webinar, Dr Andreas Abraham, MD, double board-certified in Obstetrics and Gynaecology/Reproductive Endocrinology and Infertility, The Head of Eugin International Clinic in Barcelona, Spain, discussed PGT-A, PGT-M, PGT-SR, how the biopsy is performed, and when PGT-A can be indicated. He also mentioned why PGT-A can be helpful for patients of advanced maternal age (AMA), and with repeated implantation failures (RIF), based on clinical studies.
PGT (Preimplantation Genetic Testing) has begun as an experimental procedure in the 1990s, it started with PCR (Polymerase Chain Reaction), everyone is now familiar with this because of the COVID-19 pandemic because the COVID-19 test is done by PCR where we try to detect a tiny fraction of a virus DNA. In terms of embryo testing, we look for a much bigger amount of DNA. There are 3 different types of PCR. The PGT-A, where A stands for aneuploidy, is formerly known as PGS. This test checks the embryos for abnormalities in the number of chromosomes. The embryos with an abnormal number of chromosomes are called aneuploidies. Depending on the type, aneuploidy may be responsible for having fewer chances of developing a healthy pregnancy that results in a baby born with genetic problems. Then there is PGT-M, where M stands for monogenic disease, which is the testing of the embryos, which is indicated if a family or one of the two partners or two partners has a disease that runs in a family caused by a specific single gene mutation. Then we test the embryos specifically for 1 gene, and then we can identify the embryos that have not inherited the mutation, so the healthy embryos. The third form of PGT of embryo testing is PGT-SR, where SR stands for structural rearrangements. That’s the embryo testing when we know that one of the partners or sometimes even both of them have structural rearrangements indicating that the normal size or arrangement of chromosomes is affected. We know that people with these structural rearrangements are more prone to have or produce embryos with the incorrect amount of genetic material, which can cause problems with implantation or recurrent pregnancy loss.
The normal set of chromosomes in humans is 46. There are 46 chromosomes XX for a woman, XY for men. There are situations where 1 chromosome is missing, which is called monosomy, so we have a total of 45 chromosomes. Sometimes there’s an extra chromosome called trisomy, where we have a total count of 47 chromosomes. The most commonly known form of trisomy is trisomy 21, also known as Down syndrome.
Dr Abraham emphasised that we cannot test embryos at whatever stage. There are some conditions for a proper early performed embryo biopsy to test embryos. We know it’s obligatory to perform the biopsy of the embryo on day-5 or 6 of the embryo development at the blastocyst stage. At this stage, 6 to 10 cells are taken from the outer layer of the embryo, the so-called trophectoderm. That’s the part that later develops into the placenta, the inner cell mass that will form the fetus. Testing of the embryos goes along with having no fresh transfer, we have to freeze all the embryos and wait for the result, which usually takes around 2 weeks. After that, the transfer will be performed as a cryotransfer in a consecutive cycle. Another important thing to remember is that the whole process will result in a reduction in the number of embryos to transfer for various reasons. The value of PGT-A, formerly known as PGS, is still under ongoing discussion.
ESHRE (European Society of Human Reproduction and Embryology) gave out best practice recommendations for PGT-A. They defined 4 main indications where PGT-A should be used:
ESHRE specifies that the exact definition (e.g., age limit, number of pregnancy losses) to qualify for repeated implantation failure should be determined by each centre based on their numbers. For any type of PGT, Karyotype testing for both partners is obligatory in the pre technique workup.
The definition of advanced maternal age varies. Some publications define advanced maternal age from 35 onwards, some others from 38 onwards. All studies talk about good prognosis patients, and that means that these patients have at least 10 MII (mature eggs) and at least 3 high-quality blastocysts. These studies state that PGT-A in these good prognosis conditions is likely to improve the live birth rate in the first transfer and is likely to reduce the number of transfers necessary. At the same time, there is no data on the cumulative live birth rates, and there’s no data on time to pregnancy. The conclusion is if you use PGT-A in patients who are not fulfilling the conditions for good prognosis, having a lot of eggs and having a lot of blastocysts, you might not do any good, you might not increase the live birth rate and time to pregnancy.
Regarding the use of PGT-A in repeated implantation failure, there is no sufficient data to support that. The studies to promote this have used patients who have had a lot of embryos, 6 high-quality blastocysts or more.
PGT-A in repeated pregnancy loss or repeated miscarriage. If you look at the clinical evidence, there’s not a lot of data, and there is conflicting data. That’s why ESHRE advised that the use of PGT-A has to be looked at on a case to case basis, and they state that doing nothing, no invasive technique, may be as effective as PGT-A. The studies that support the use of PGT-A have had selected patients with a high number of good quality blastocysts.
If you use PGT-A in women at a young age under 35, women with a good prognosis. Most young women will have a good number of eggs and therefore also a good number of embryos, and PGT-A is most likely responsible for decreasing or reducing the time to pregnancy. There’s not a lot of data to support that, and the data which is there again relies on very good prognosis patients, and there’s no data on time to pregnancy.
The first clinical case was about a single 42-year-old woman who came to perform an IVF with her own eggs and donor sperm. She explicitly asked to do PGT-A since she was of advanced maternal age. She experienced repeated miscarriages, she had 4 pregnancies and miscarried 4 times.
We did a high dosage stimulation with a high dosage of FSH, and she produced a good response at the oocyte pickup, we retrieved 15 oocytes, 13 of them were mature eggs, and then we performed ICSI. The next day, 11 of the 13 had been fertilized. This in terms of quantity for a 42-year- old woman is a very good result. In the end, 5 embryos developed till day-5, and we did a biopsy. After 2 weeks, we got the result that 2 embryos were normal and 3 were abnormal, and none of them was mosaic. We performed a single embryo transfer on day-5, and we’ve vitrified the remaining embryos on day-6 of its development, it was an ongoing pregnancy with a slightly pre-term vaginal delivery at 36 weeks.
It might be interesting to know that over 50% of our patients who come to our clinic (Eugin) for IVF with their own eggs are over 40, but a very small fraction of these patients will have such a good response to stimulation. This patient is a textbook example of a good prognosis, good response patient.
The next case was a classical indication for PGT-A. It was a heterosexual couple, and we performed IVF with PGT-A because of repeated miscarriages and a severe male factor. This patient had 5 pregnancies, and she miscarried 5 times in total. They tried to conceive naturally for 3 years. We did stimulation with FSH, we retrieved 26 cumulus cells, so a high number. After washing, we had 21 mature eggs, after injection of the partner’s sperm with ICSI, we had 17 fertilized eggs and 9 embryos developed till the blastocyst stage at day-5. After testing the embryos, we had 3 normal, 4 abnormal and 2 mosaic embryos. We performed an elective single embryo transfer on day-5 and vitrified 2 remaining embryos. This was an ongoing pregnancy, she’s currently entering the 3rd trimester. This is a classical case where we used PGT-A indication for repeated miscarriage and severe male factor according to ESHRE recommendations.
It was a good prognosis patient, under 35, with a good ovarian reserve, a good response. Good egg quantity consecutively translates into embryo quantity and quality, and that’s crucial. We started with a very high number of eggs, and every step of the way, the number declined. We finally arrived at several 3 normal embryos, which is a big reduction.
We had a young patient where male partner had Marfan syndrome, which is a monogenetic disease. The couple was trying to conceive for 2 years, 1 spontaneous pregnancy was a termination of pregnancy due to the monogenic underlying problem. They contacted the clinic to perform an IVF with PGT-M. We did a high dosage stimulation, we got a good response, 25 eggs were retrieved, 21 were mature after washing, we reached day-5 with 6 embryos, after biopsy, we found 5 normal, 1 abnormal and no mosaic embryos. We performed a single embryo transfer and vitrified the 4 remaining embryos, it is currently an ongoing pregnancy in the 2nd trimester.
It was the same concept, it was a monogenetic disease, young and good prognosis patient, the female had a good response to the stimulation, we got a high number of eggs, good fertilization, good embryo development and in the end 5 normal embryos. However, to have 5 normal embryos, we started with 25 retrieved eggs, the reduction which is important to stress out and remember about it in the context of patient counselling.
The fourth case is an example of the use of PGT-SR. The Robertsonian translocation (ROB) is one of the most common structural rearrangements in humans. It was a young heterosexual couple where the male carries the Robertsonian translocation. They had 4 pregnancies and 4 times the female miscarried, they were trying to conceive for 2 years. We used high stimulation, we got a very good response, 32 eggs were retrieved, 29 of them were mature, after ICSI, 25 were fertilized and 7 embryos developed until the blastocyst stage. After the biopsy, 4 were normal, 3 abnormal. We did a first single embryo transfer which resulted in a biochemical pregnancy. We did a second cryotransfer, which resulted in an ongoing pregnancy and delivery by C-section at 36 weeks. It was the same situation, same concept, we had a high number of eggs and embryos, which are necessary to arrive at the end of this process.
These 4 cases cover the 3 indications for PGT. These are the main indications for these techniques, and there are variations of the same theme. The main take-home message is that you need to remember there is a massive reduction in the number of transferable embryos that go along with this technique, and that can result. Sometimes, we perform PGT, and we do not have any embryos for transfer because no blastocysts develop, or all embryos were tested abnormal, and that is something that can be the case, and that’s something a patient has to be aware of before making a decision. It’s a crucial part of your decision-making process.
Depending on the authors and their publications, the definition for advanced age starts at 35 or 38. You have a good ovarian reserve reflected in a good AMH, you had a normal response in your first IVF, you had 9 eggs retrieved, which is not a lot, and the second one went better since ICSI did the trick. You had 2 blastocysts from 6 eggs, which is a very good ratio. Not knowing how you were stimulated, I suppose you have been highly stimulated with a high dosage. Sometimes you can play a little with the triggering, there’s double triggering that might help retrieve more eggs.
For the third cycle, you would be a candidate to try PGT-A. I would have to get a little more information, but as you saw during my presentation, my focus was on the number. In your case, since the second time you had a very good outcome, out of just 6 eggs, you produced 2 blastocysts, the PGT-A could give us additional information, but one has to be aware of mosaicism.
In your third cycle, I would recommend having a thorough visit with your IVF doctor to dig a bit deeper into what I try to scratch on the surface with adding the topic of mosaicism because doing PGT-A in your case can give us additional information, but it can reduce the number of transferable embryos. I would consider it as an option since you didn’t hit 40, you had a rather good response, and I would not consider conventional IVF in your case since ICSI gave a better result in terms of fertilization and blastocyst development.
The approach to do mild stimulation is perfect, that’s the common approach. In Japan, Dr Kaito invented the vitrification technique for his protocol. They believe in mild stimulation or natural cycle – no stimulation, and then they believe in egg banking, and then after 6,7 or 8 months, they inject and produce 1 or 2 blastocysts. You had 4 rounds of stimulation, and you have 3 embryos grade 1.
PGT-A should not be performed on day-2 or day-3. I’m not sure what the plan is, and whether it is to compile embryos at day-3, and then if there is a sufficient number of embryos at day-3 to try to bring them to the blastocyst stage, that’s one thing you have to bear in mind and ask the laboratory. Every lab knows its percentage of blastocyst culture, so how many embryos they need to reach day-5. In our lab (Eugin), we know that we need 4 to 5 top embryos at day-3 to reach day-5, so that’s something you have to ask the lab at the IVF clinic you are undergoing treatment.
When it comes to more rounds of IVF before proceeding, this is the whole idea of embryo banking, to kind of compile a certain number before going for transfer, but I don’t know this clinic where you’re at. We don’t usually do embryo banking, in the case of demand, if there are special patients who ask, we might do it, but again what I told you, in our lab, I know we need 4-5 top embryos on day-3 to be able to reach day-5.
The limitations of a 40-year-old onwards are that even if they have this number, in this case, one would rather need more to be on the safe side. it’s not mathematics, it’s still biology, and at 41 years, you’re more prone to have fertilization failure even if the sperm is perfect. There is more risk of embryo developmental arrest, so all that is something to be taken into account.
I’ll give you a rather complex answer. During the talk, I mentioned that egg quality is linked to maternal age. The maternal age is egg quality. The egg is more important than the sperm cell, so egg quality translates into embryo quality, and embryo quality is responsible for implantation rates and the health state of the embryo.
Most chromosomal abnormalities are linked to maternal age, and yes, mutations are also linked to the egg and embryo quality because you can imagine with age, on a cellular level, errors occur more frequently. The maternal age is responsible for these events, and yes, consecutively, it might be good to go for egg donation because that way, you kind of erase the egg quality factor using a young donor. The average age of donors is 25, in Spain from 18 to 35, so you have a young donor.
As I told you, age is egg quality, egg equality is embryo quality, so using a young donor, you erase or minimize all problems linked to maternal age. This way, you reduce this issue, bear in mind that even an 18-year-old donor could have a Down syndrome child, it’s never 0%, but it’s a very low risk.
Imagine a woman, let’s say 22 years old, no problem whatsoever in the context of reproduction, she has the perfect partner. This couple will have various intercourse around her ovulatory date per month, per ovulation, this woman has just a 20% of success rate, this is shockingly low considering that’s the best-case scenario, that’s the perfect couple, but nature evolution gave us a very ineffective reflection.
If this woman tries to get pregnant every month after 12 months, we know she’ll increase her chances to over 90%. That means 12 ovulations, 12 eggs and if there’s fertilization, in the best-case scenario, 12 embryos, so nature needs 12 months to increase chances from 20 to over 90%.
The most common cause of non-evolution is unknown, it’s like a black box still. We can kind of do thorough testing, if there’s repeated miscarriage, repeated implantation failure, we will do karyotypes of the partners, we will dig even deeper into genetics, sperm fragmentation, and maybe we’ll find something that can explain that.
However, the most common cause is exactly what I just said, we do not know, it’s something we still don’t know, and if we look at other animals, we see that other mammals do a much better job, we have a rather complicated reproduction as humans.
Once again, that’s a black box. With the biochemical pregnancy, it’s the same thing why embryos do not implant or why they implant and then result in an early loss, we don’t know. A big part of it is genetics, especially in advanced maternal age, from 40 onwards, we know that 80% of the embryos are genetically composed of abnormal cells, so the biggest number of problems apply to genetics in that age frame.
Another factor is endometrial issues, but there are tests on the market nowadays. Even if you have a perfectly tested endometrium, and even if everything is synchronized with a perfect blastocyst, even a blastocyst after PGT-A, you can have a biochemical pregnancy or non-implantation. It depends on age again, but with advanced age, the biggest part accounts for genetics rather than endometrial issues.
It depends, if the sperm is normal, then yes, a total fertilization failure can indicate poor egg quality. If it’s advanced maternal age, if you have a patient of 40 plus, yes, then a total fertilization failure with normal sperm normal for ICSI indicates an oocyte factor.
Another important part of the answer is that, even in the context of double donation, so perfect sperm plus perfect eggs, you would think that should give us 100% fertilization and 100% blastocyst development, but it does not work like this.
In the context of double donation, where everything is optimized, we have in our clinic, and that should be in line with most other clinics who do that, we have a 2% ratio risk of having no embryos. 1.2% total fertilization failure and 0.8 of the 2% there’s no embryo development, even with perfect eggs plus perfect sperm, and that kind of gives you an idea for the question you asked.