During this event, Dr Antonio Urbano Carrillo, Clinical Geneticist from UR Vistahermosa, Spain, explained the essentials of PGT-A (Preimplantation Genetic Testing for Aneuploidies) and the complex world of mosaic embryos.
Dr Urbano started his presentation by explaining what aneuploidy means. It refers to the number of chromosomes in a species. In humans, we have 46 chromosomes, divided into 23 pairs. Pairs 1 to 22 are called autosomal chromosomes, and the last pair is called the sexual chromosomes. This is the difference between male and female. From pair 1 to pair 22, males and females have the same chromosomes, but the difference is in the sexual chromosomes: males have one X and one Y, while females have two X chromosomes.
On the other hand, there can be a situation where individuals have an incorrect number of chromosomes. For example, a female may have 3 chromosomes of 21. This is called trisomy 21, which causes Down syndrome. Only three trisomies are compatible with life: trisomy 21 (Down syndrome), trisomy 13 (Patau syndrome), and trisomy 18 (Edward syndrome). All other trisomies or monosomies in autosomal chromosomes are lethal, and there are no individuals with one chromosome less or one chromosome more, such as three chromosomes of 4 or 8. There are no people with three chromosomes of 8. Only babies born with trisomy 21 (Down syndrome), trisomy 13 (Patau syndrome), or trisomy 18 (Edward syndrome) survive. This is important because, in the embryo stage, only embryos with this correct chromosomal composition can achieve a pregnancy and a healthy baby.
There is a close relationship between maternal age and aneuploidy. The graphic below perfectly represents how aneuploidy in embryos increases with maternal age. In this case, when a woman is around 30-32 years old, the percentage of embryos with aneuploidy is about 25%, but this increases rapidly at age 42, where about 70% of embryos are aneuploid. Luckily, there is a technique that allows us to detect aneuploidy called PGT-A.
As shown on the slide, there are three similar embryos of very good quality. However, we don’t know anything about their chromosomes, and the potential for implantation of these embryos is not based on their morphology but on their chromosomal composition.
PGT-A is a process that involves three stages. The first is the biopsy. A blastocyst biopsy is taken on day 5 or 6 after embryo formation. There are two parts in the embryo: the peripheral part called the trophectoderm, and the inner part, which will develop into the fetus and placenta. On day 5 or day 6, we can take 5 to 10 cells for analysis. It’s important to note that years ago, only one cell could be analysed. Now, 5 to 10 cells can be studied, providing more accurate information.
The second step is to send the samples to a genetic laboratory, where the analysis is performed using a platform called NGS (Next Generation Sequencing). This is the only platform that allows the detection of most chromosomal abnormalities. The bioinformatics analysis can then provide the results.
We can see that out of these 3 embryos, only 1 has the potential to achieve a pregnancy or implant in the uterus when transferred. For embryo number 2, we can see a trisomy of chromosome 22. Briefly, there is one chromosome in red, one in blue, and chromosomes 1, 2, and 3, with 2 chromosomes in this line. One line above shows a trisomy (3 chromosomes), and one line below shows a monosomy (1 chromosome). In this case, embryos 2 and 3 do not implant after transfer, but embryo 1 has the potential to result in a pregnancy and a healthy baby.
What are PGT-A benefits? Women over 30 years of age can mitigate the effect of maternal age. The implantation rate decreases with maternal age, while the average implantation rate in PGT-A cycles remains stable across all maternal age groups. Similarly, the miscarriage rate does not increase with maternal age and stays stable across all age ranges. This is the primary benefit of PGT-A.
When only one cell is analysed, only 2 possibilities are available: euploid or aneuploid. A mix of things occurs when two or more different populations with different chromosome numbers are present in the same embryo.
As shown in the picture below, we can see red and blue in the same embryo. This is mosaicism or mosaic embryos. When we detect or can detect these two or more populations in one embryo, we can do it because of the improvement in technology. We can do it because NGS (Next Generation Sequencing) allows us to detect this situation.
The biopsy of the trophectoderm in mosaic embryos is not very common, but it’s important to note that it’s similar across all age ranges—about 15% in all ranges. For this reason, this topic is important not only for maternal age but also for women under 35 years of age, because the percentage of mosaicism in this age range is about 20%.
One of the groups decided to transfer a mosaic embryo, even though they didn’t have any or more euploid embryos. This mosaic embryo transfer ended in a healthy baby being born. Then, all the groups opened the door to using mosaic embryos because 20% is a large number of embryos that are mosaic, which, as of now, are not transferred. When they showed that transferring mosaic embryos led to a healthy pregnancy and baby, all the groups began to consider it. At this point, the scientific societies made guidelines to help doctors and patients determine which mosaic embryos to transfer.
One of the latest guidelines from the Preimplantation Genetic Diagnosis International Society (PGDIS), in green, a safe option is shown, and in red, less safe or unsafe. The level of mosaicism is the number of cells in the embryo that are suspected to have mosaicism. A 50% level means that half of the embryo’s cells are euploid, and the other half are aneuploid. You can see that from 20% to about 50%, it is considered safe. This means that if we achieve a pregnancy, the baby will likely be born healthy, but there are still some risks, such as a lower implantation rate, higher miscarriage rate, and a lower live birth rate, all of which increase as the level of mosaicism increases.
As shown on the slide, a pyramid has been created by PGDIS which shows which embryo to transfer first. First, euploid embryos are transferred, which is obvious. Then, mosaic embryos with low-level segmental mosaicism can be transferred. This means that the embryo has one more or one less chromosome.
NGS (Next Generation Sequencing), allows us to detect not only the gain or deficit of one chromosome, but we can also detect if there is a loss of a segment of a chromosome. We have high resolution to detect deletions and duplications of the 46 chromosomes.
European societies have very clear recommendations about mosaic embryos, especially low-range mosaicism. When low-range mosaicism (below 50%) is detected, PGT-A should be co-evaluated with morphology. It is not recommended to discard low-range mosaic embryos or to biopsy these embryos because there is no risk for the future baby.
In summary, all the scientific organizations’ guidelines recommend transferring embryos with low-range mosaicism (less than 50%). Before transferring an embryo with partial or segmental mosaicism, it is important to evaluate whether it has a single chromosome alteration or complex mosaicism with more than three chromosomal alterations. In this case, transferring the mosaic embryo is considered safe in terms of the baby’s health.
In conclusion, we are not alone in the decision of whether or not to transfer mosaic embryos. There is a lot of research done by groups with years of experience.
Although it is important to emphasize the importance of parental testing, it is mandatory or strongly recommended to do parental testing. The main difference between transferring mosaic embryos and euploid embryos is the importance of parental testing. After the testing, if the transfer involves more than one embryo, it is recommended to transfer the highest quality embryo in terms of morphology or morphokinetics.
If the monosomy of chromosome 4 is complete, there is no possibility.
In biology or genetics, not everything is black or white. However, if the monosomy is pure—100% of the embryo’s cells missing chromosome 4—there will be no implantation or a pregnancy loss in the first stage of pregnancy.
In this case, with 60% mosaicism, in our group UR Vistahermosa group, we decided not to transfer when chromosome 18 is involved. This is because trisomy 18 can develop into Edward syndrome.
Sometimes, a monosomy detected in the technique may not reflect the fetus itself due to testing limitations. The tested cells come from the trophoblast, which is part of the placenta, and while 90-95% of the chromosomal profile matches between the placenta and fetus, there is a 5% chance they do not.
For chromosome 18 with 60% mosaicism, you can transfer, but a partial test is mandatory to ensure accuracy.
In our clinic, UR Vistahermosa, we do not recommend transferring high-grade mosaics. However, I know other clinics may transfer them. We don’t recommend transfer when the mosaicism threshold is more than 50% because the risks are higher, and there isn’t sufficient data available compared to low-grade mosaic transfers.
There is more data on low-grade mosaics, but not enough on high-grade mosaic transfers, which makes the decision more complex.
In one of the graphics I showed, about 3% of the results are inconclusive. This can happen for several reasons:
Unfortunately, if noise is present due to technical limitations, there is little we can do. Low-grade mosaic embryos are very likely to be affected by noise, and in some cases, we may overestimate mosaicism.
However, current studies and papers hypothesize that if noise is purely technical and the embryo is otherwise good, then it can be transferred, and the embryo is likely viable.
There is a hypothesis. It’s not that humans are “bad” at reproduction, but in the first stages of embryo development, the embryo divides its cells very quickly. Unlike in adults, where there are mechanisms to detect and eliminate abnormal cells, during early development, these mechanisms do not work as effectively. The embryo focuses solely on implantation and growth rather than quality control.
There is a hypothesis supported by a study where embryos were allowed to develop to day 12. All pure aneuploid embryos remained aneuploid on day 12, while mosaic embryos observed on day 5 corrected themselves by day 12, showing euploidy. This indicates that embryos can sometimes self-correct, but only if some parts of them are good. If all parts are abnormal, no correction is possible.
No, maternal age is not the only factor. While age is closely linked to chromosomal abnormalities like Down syndrome, Edward syndrome, and Patau syndrome, there are many other diseases unrelated to chromosomes. For example, neurodevelopmental problems can result from genetic predispositions unrelated to maternal age. However, maternal age is directly associated with an increased risk of chromosomal aberrations, which is the primary concern.
Segmental aneuploidy may be more likely an artefact of the testing process compared to full aneuploidy. Full aneuploidy is easier to identify and typically has a greater impact on embryo development and implantation.
Yes, the current PGT-A technology has limitations but continues to improve with each revision. Currently, it can detect deletions or duplications down to around 10 megabases (Mb) – a unit of measurement used to help designate the length of DNA, which is roughly half a chromosome arm. With advancements, this threshold may be reduced to around 4 megabases, but some diseases caused by deletions smaller than 1 megabase cannot yet be detected.
PGT-A improves IVF outcomes by allowing us to identify and avoid transferring aneuploid embryos, which cannot implant or lead to a pregnancy. While it may not necessarily increase the overall live birth rate if there are very few embryos, it does save time by identifying which embryos are euploid and transferring them first. This reduces the number of failed transfers and shortens the time to achieve pregnancy.
The risk of a false positive result in PGT-A is low. If the PGT-A result is normal, the embryo is typically normal, with a positive predictive value of about 80%. However, this can vary. When the sample and technique are good, the positive predictive value is close to 100%. However, if there is high noise, problems with the biopsy, or DNA issues, the positive predictive value could be lower.
But in general, the risk of a false positive is low. We always recommend an invasive prenatal test, such as amniocentesis, to confirm the result, although nowadays, non-invasive prenatal tests (NIPT) are available as a good solution for couples who may have concerns about invasive procedures.
