In this session, Dr Carmen Morales, PhD, a Genetic Counsellor and Laboratory Coordinator at Reproclinic, Barcelona, has discussed PGT-A, its process, its benefits, recommendations, and results.
PGT (Pre-implantation Genetic Testing) is a genetic test performed on embryos created during an IVF cycle. It detects genetic abnormalities in embryos.
Based on this, we can selectively transfer only the unaffected embryos. This procedure requires a biopsy of the embryos to obtain samples for analysis.
There are 3 different types of PGT:
PGT-A allows the identification of aneuploidies (abnormal embryos) and the selection of a healthy and most promising embryo for transfer.
For all types of PGT, the procedure involves similar steps. An IVF cycle needs to be performed to produce embryos. The biopsy involves obtaining samples from the embryo that are sent to the genetic lab for diagnosis. Then these embryos are biopsied, which can be done on the:
The main difference is that on day 3, only 1 cell is retrieved, while on day 5, it’s possible to obtain several cells to be analysed. The embryos are kept in the IVF clinic since genetic diagnosis usually takes time. We cannot keep embryos in culture for too long, they are usually frozen. When the genetic report is ready, the unaffected embryos can be thawed and transferred.
A karyotype is a picture of a person’s chromosomes. It allows for determining the chromosome complement, including the number and structure of chromosomes. A normal karyotype has:
The difference between male and female karyotypes is that females have 2 X chromosomes, while males have 1 X and 1 Y chromosome. Abnormalities can occur in the structure of chromosomes, such as reciprocal translocations where 2 different chromosomes exchange fragments. The total amount of DNA is balanced in these patients, meaning they don’t have extra or missing DNA. There can also be abnormalities in the number of chromosomes, called aneuploidy. If a chromosome is missing, it’s called monosomy, and if there are extra chromosomes, it’s called trisomy.
Most aneuploidies are not compatible with life; very few of them are viable. Trisomy of chromosome 21 and anomalies related to sex chromosomes are among the few exceptions. Most embryos or fetuses with missing monosomy or extra trisomy chromosomes cannot survive, leading to implantation failure and spontaneous miscarriage. However, some can survive, resulting in a baby affected by a genetic syndrome.
A karyotype with trisomy 21 results in a baby having 3 copies of chromosome 21 instead of 2. It’s well known that chromosomal anomalies have a significant impact on human reproduction. In natural conception, most embryos with these anomalies are lost in the early stages, often referred to as unrecognized losses, which cannot be tested or analysed. However, a reflection of these occurrences can be seen in the detection of aneuploid embryos in IVF. The percentage of aneuploid embryos detected increases with maternal age, but even younger patients show an important percentage, never less than 20%.
If we look at chromosomal abnormalities in miscarriages, we can observe that about 50% to 70% of clinical miscarriages, depending on the study, have chromosomal abnormalities, with autosomal trisomies being the most frequent.
Preimplantation Genetic Testing for Aneuploidy (PGT-A) allows the detection of abnormalities in chromosome numbers and the selection of chromosomally normal embryos for transfer.
PGT-A aims to:
PGT-A has evolved over the last few decades, with improvements in the technologies used, the chromosomes that can be analysed, and the type of biopsy performed.
Initially, PGT-A used a technique called FISH (Fluorescent In Situ Hybridization), which allowed testing for a specific number of chromosomes, usually five to nine, from a day three embryo biopsy. Nowadays, the most commonly used technology is Next Generation Sequencing (NGS), which allows testing the entire chromosome complement at the blastocyst stage, on day 5 of embryo development.
Most aneuploidies detected originate from meiotic errors, meaning there’s a mistake during gamete formation, whether in the egg or sperm, leading to an abnormal number of chromosomes. For example, if an egg with 2 copies of chromosome 21 is fertilized by a normal sperm, it results in a trisomic embryo. Similarly, if a gamete is missing a chromosome, like chromosome 21, it can result in an embryo with monosomy 21.
Examples of profiles obtained when analysing embryos using an NGS platform include:
Certain groups of patients are at higher risk of producing gametes (eggs or sperm) with aneuploidies, leading to abnormal embryos. These groups include patients of advanced maternal age, those with recurrent pregnancy loss, recurrent implantation failure, and male factor infertility. Patients in these subgroups, who often undergo IVF, can benefit from PGT-A to select euploid embryos with a higher potential for implantation and achieving an ongoing pregnancy.
For advanced maternal age patients, typically older than 36 or 37, it is well known that ageing affects chromosome division in the eggs, making them more likely to have extra or missing chromosomes, which, after fertilization, can result in abnormal embryos.
One of the groups of patients who can benefit from PGT-A testing is patients of advanced maternal age (AMA). As maternal age increases, the percentage of abnormal embryos also increases. For patients aged 43–44 years, the percentage of abnormal embryos in an IVF cycle can be as high as 90%.
Another group of patients where PGT-A can be suggested are those with recurrent IVF failure or recurrent pregnancy loss. Different factors can cause IVF failures or miscarriages, such as uterine abnormalities, autoimmune disorders, thrombophilia, etc. The gynaecologist or physician must order the proper tests and examinations to rule out these disorders. In some patients, it is not possible to find a clear case of IVF failure or recurrent miscarriages. In these patients, we cannot rule out that these issues were related to aneuploidies in the embryos, which may have caused implantation failures or miscarriages.
Patients with severe male factors are another group that can benefit from PGT-A. These couples with abnormal sperm parameters, which correlate with a high rate of aneuploidy in the sperm. As I mentioned earlier, after fertilization, this can lead to aneuploid embryos. One of the abnormal sperm parameters that correlates with a high aneuploidy rate is oligospermia or low sperm count.
In one of the studies presented during the webinar, a control group of patients is compared with a study group of patients with severe male factors. The study shows that couples with severe male factor have around 35% abnormal embryos during the IVF cycle, while patients with male factor issues have an increased rate of 51%. When we look at patients with oligospermia, the rate of abnormal embryos is even higher, up to 64%. This proves that this group of patients has a high rate of abnormal embryos and can benefit from PGT-A.
Mosaicism is a controversial issue in PGT-A. Sometimes, intermediate profiles are found in NGS analysis, indicating something abnormal but not a complete trisomy. This pattern may be consistent with a mix of cells in the sample, where some cells have a normal karyotype, and some cells have trisomy 7. Mosaicism refers to the presence of two or more cell lines in the embryo that are genetically different.
Mosaicism occurs in natural pregnancies, detected in 2% of chorionic villus samples and around 0.5% of amniocentesis samples. The percentage of mosaicism detected in IVF embryos is much higher, ranging from 10% to 30%. The variability in percentages depends on the laboratory doing the analysis, the platform used, the cut-off values, etc. The mosaicism rate in IVF embryos is still much higher than in prenatal samples.
Intermediate profiles consistent with mosaicism can also be caused by other factors, such as:
While these profiles can be explained by mosaicism, other factors can also explain them.
Aneuploidies detected in PGT-A often originate from errors during gamete formation. However, mosaicism is usually of mitotic origin, meaning it originates from a mistake within the embryo itself. A completely normal embryo may develop a mistake during cell division, leading to daughter cells with trisomy or monosomy. This results in a mosaic embryo with normal cells (46 chromosomes) and some cells with 45 or 47 chromosomes. The different cell lines can be randomly distributed in the embryo.
The diagnosis of mosaicism can be influenced by the biopsy technique. Since the cell lines are randomly distributed, the biopsy is also performed randomly. The area chosen for biopsy may have a different distribution of abnormal cell lines, leading to varying results. The location of the sample can influence and change the diagnosis of mosaicism.
The development of sensitive technologies and the application of the day 5 biopsy, which is safer for the embryo compared to day 3, now allow for the detection of mosaicism. On day 3, mosaicism cannot be detected as only a single cell is analyzed, which may not represent the entire embryo. On day 5, a mixture of different cells is analyzed, enabling the detection of intermediate patterns.
There are 3 categories of embryos:
What can we do with mosaic embryos? Initially, the outcome of mosaic embryos was unclear, and they were typically not transferred. Over time, studies and clinics began transferring these embryos to patients without normal embryos, resulting in ongoing pregnancies and healthy babies. A comparison of potential mosaic embryos with euploid embryos shows a reduced implantation rate, ongoing pregnancy rate, and an increased miscarriage rate for mosaic embryos.
It is generally accepted that mosaic embryos can be considered for transfer in couples who do not have normal euploid embryos. This decision should be made after proper counselling of the couple, explaining all the risks involved. Various societies and groups have published guidelines on how to prioritize mosaic embryos for transfer, depending on the type of chromosome involved or the percentage of abnormal cells in the embryo.
PGT-A has been controversial from the start, with some professionals arguing that it is not beneficial and may even be damaging, citing its cost and commercial interests. Another group, likely the majority, believes that PGT-A can be beneficial, particularly for patients at higher risk of producing abnormal embryos. Given the available technology, it may be unethical not to offer PGT-A to these high-risk patients.
PGT-A is generally allowed in almost all European countries that perform assisted reproduction techniques, though regulations and policies vary.
Unlike PGT-SR and PGT-M, PGT-A is more restricted, with several countries not allowing this technology.
Data from the ESHRE PGT Consortium covering 2013-2015 shows that around 20,000 cycles were started for PGT-A, with about 80,000 embryos analyzed. Approximately 28% of these embryos were genetically normal and transferable.
The majority of PGT-A cases are indicated due to advanced maternal age. A comparison between the successfully biopsied embryos by different types of indication, and the ones that are genetically normal and can be transferred shows that most embryos are abnormal, with a low percentage being normal and transferable. Egg donation cycles, which fall under the “Others” category, have a higher percentage of normal embryos due to the younger age and better prognosis of egg donors.
In summary, PGT-A is primarily for couples at risk of producing embryos with aneuploidies and can improve clinical outcomes by reducing pregnancy loss, miscarriages, and chromosomally abnormal pregnancies in a selected group of patients. The most significant benefit is observed in patients with advanced maternal age. Despite these benefits, the use of PGT-A remains controversial, with varying acceptance across different countries.
Related reading:
If the lab conditions are not good, you can indeed have a lot of abnormal embryos, not only chromosomally abnormal but also morphologically abnormal embryos. If the conditions are not optimized, they can cause abnormalities in the embryos, and they can be a source of aneuploidy. For that reason, the protocols in an IVF lab should be checked, and they have to follow very strict protocols. So yes, the conditions can influence the quality of the embryos.
Very young women 16-17-year-olds can indeed have a higher rate of aneuploid embryos, but it’s also true there is a higher risk of Down syndrome when mothers are very young. However, this is a result of this study shows, I don’t exactly know how many patients are 28 years old, I added this as an example. Most probably, if you are using the graphics from other published papers, possibly the percentage will be lower.
There is not a very big difference between 27 or 29, so I think before the age of 30 years old, the results will be best. I cannot tell you a specific age based only on the graphic of one paper.
Mosaicism means you have different cell lines, for example, you have one cell line that it’s completely normal, four to six chromosomes, and one cell line, there is trisomy 21. If you are only analysing one cell, you cannot detect different cell lines. If you are randomly taking the normal cell, then you are going to have a normal result of this embryo. If you, by chance, are taking the cell that has trisomy 21, the diagnosis of this embryo will be trisomy 21. We are only analysing one cell that is recommended on day-3 because more can damage the embryo development. We are not able to detect mosaic by a FISH or any other technique because we are only testing one cell.
If a laboratory is doing the biopsy and all the embryos are of very poor quality, then most probably the embryos will be chromosomally abnormal, and then you will have very bad results. You would think this lab is giving me very bad results, no you are giving them very bad samples, so it’s a conjunction of both things. You need very good conditions in the IVF lab, you need to choose the embryos we are going to biopsy to have good laboratory results.
You can see a lot of variabilities even in the same genetic laboratory that is receiving samples from different IVF labs, there can be great variability between the results. It’s the same with IVF centres, they are different, they have different conditions, different patients. Some centres specialize in very difficult cases. Then the probability of abnormality in the embryos is higher than in others where they are doing IVF to everybody, and they have very good prognosis patients. It’s not only the genetic laboratory, it’s everything that it’s giving you good or bad results.
There are two tendencies. Some professionals say since we can detect abnormal embryos in all ages, even in young patients, even though the percentage is not very high, it can be 20%, let’s do PGT-A to all patients. On the other hand, we have another group of professionals that don’t see an advantage in doing that, and they decide to offer PGT-A only for a specific group of patients.
According to published data, PGT-A is improving the outcomes mainly in patients over 35 years old. In young patients, including egg donors, if there is no other factor like a severe factor, published data didn’t show any improvement in the results. In my opinion, there are different policies, different tendencies, but I would recommend PGT-A only to specific groups that we know are at higher risk to see the benefit of this test.
In our clinics (Ferticentro and Procriar), we charge €790 for the embryo biopsy and €360 for testing each embryo. This cost is in addition to the overall IVF cycle cost, which varies. It’s a substantial investment, but for many patients, the information gained is invaluable.
