To do genetic testing for an embryo, we need a viable, healthy embryo with good division and development. The biopsy can only be done on embryos that reach day 5 or 6 and the hatching or fully hatched blastocyst stage. In cycles where we plan PGT-A, we do assisted hatching at day 3 by opening the membrane, the zona, to take the sample. We take about 5 to 10 cells from the outer part of the embryo, the part that will form the placenta, not the baby.
The standard analysis today is NGS, new generation sequencing, which checks for aneuploidy — a meiotic error that occurs due to the egg’s age.
NGS acts like a huge magnifying glass, allowing us to see more, including mitotic errors and mosaicism, which is found in about 5% to 20–25% of embryos in some labs.
The sample is taken from the outside cells using a tube that sucks in 5 cells, and a tiny laser is used to divide them. After the biopsy, the sample is sent to the genetic laboratory, and the embryo is cryopreserved because NGS analysis takes 2 to 3 weeks. The embryo remains frozen until results return. If good, the embryo can be used for transfer.
The inner cell mass will create the baby; the outside cells form the placenta. We take placental cells for the genetic examination.
Preimplantation Genetic Screening (PGS), now more accurately referred to as Preimplantation Genetic Testing for Aneuploidy (PGT-A), is a technique used in conjunction with in vitro fertilization (IVF) to screen embryos for chromosomal abnormalities before they are transferred into the uterus. This screening helps identify embryos with the correct number of chromosomes, increasing the chances of achieving a pregnancy and a live birth and reducing the likelihood of miscarriage or genetic disorders.
The PGS Process:
The first step in the PGS process involves stimulating the ovaries, which is typically done through hormone injections over a period of 8–12 days. Once the follicles have grown, an egg retrieval procedure is performed. This is done under mild sedation, and a thin needle is used to collect the eggs from the ovaries through the vaginal wall. The retrieved eggs are then first sorted, and the mature ones are taken for fertilization.
The next step is to fertilize the retrieved mature eggs with sperm in the laboratory, using intracytoplasmic sperm injection (ICSI). The fertilized eggs are then cultured in a controlled environment and monitored as they develop into embryos. The embryos are allowed to grow for about 5–6 days until they reach the blastocyst stage, which consists of approximately 100–150 cells. This stage is considered optimal for PGT-A because the cells have differentiated into an inner cell mass (which will form the baby) and an outer layer called the trophectoderm (which will form the placenta).
The embryo biopsy is a delicate procedure performed at the blastocyst stage. Using a specialized technique, a few cells are carefully removed from the trophectoderm layer of the embryo, ensuring that the inner cell mass remains untouched and undamaged. The biopsy typically involves extracting around 5 cells. The procedure requires highly skilled embryologists and precise tools, such as micromanipulators and lasers, to ensure minimal disruption to the embryo. The biopsied cells are then placed in a separate container for genetic analysis, while the embryo itself is frozen.
The extracted cells are analysed using an advanced genetic testing technique; next-generation sequencing (NGS). This technique provides a comprehensive view of the chromosomal makeup of the biopsied cells. The analysis can detect chromosomal aneuploidies (abnormal numbers of chromosomes), such as trisomies (e.g., Down syndrome, which is caused by an extra copy of chromosome 21) or monosomies (e.g., Turner syndrome, where there is a missing X chromosome). The testing process usually takes around 3 weeks.
Once the genetic analysis is complete, the results are reviewed to determine which embryos are chromosomally normal (euploid) and which have abnormalities (aneuploid). The results will categorize the embryos into “normal,” “abnormal,” or “mosaic” (where some cells are normal and others are not). Only euploid embryos are selected for transfer to maximize the chances of a healthy pregnancy.
After the results are obtained and the embryos have been selected, one (or occasionally two) euploid embryos are thawed and transferred into the patient’s uterus. This is done in a subsequent frozen-thawed embryo transfer cycle. The timing of the transfer is coordinated with the patient’s menstrual cycle or hormonal preparation to ensure the endometrium is in an optimal receptive state for implantation.
Benefits of PGS
Improved Embryo Selection: One of the primary advantages of PGT-A is its ability to enhance embryo selection. With traditional IVF, selecting the best embryo for transfer is often based on morphological assessment alone, which only evaluates the appearance and growth patterns of the embryo. This method cannot reveal chromosomal status, leading to the risk of transferring embryos that look healthy but have chromosomal abnormalities. PGT-A provides an additional layer of information, allowing for the selection of embryos with the correct chromosomal number. This can lead to higher implantation rates, especially in patient populations at higher risk of producing aneuploid embryos, such as older women or those with recurrent pregnancy loss.
Reduced Risk of Miscarriage: Since chromosomal abnormalities are a major cause of miscarriage, transferring only chromosomally normal embryos can reduce the risk of early pregnancy loss.
Decreased Chance of Genetic Disorders: PGS allows for the detection of specific chromosomal abnormalities that could lead to genetic disorders, ensuring that only healthy embryos are transferred.
Optimized Embryo Selection: For patients with multiple embryos, PGS provides a scientific basis for selecting the most viable ones, thereby reducing the number of embryos transferred and minimizing the risk of multiple pregnancies.
Considerations and Limitations of PGS
Risk of Biopsy-Related Embryo Damage: There is a risk that the biopsy procedure could damage the embryo, affecting its development and reducing the chances of successful implantation.
Cost and Accessibility: PGS is an additional cost on top of standard IVF treatment, which may not be accessible for all patients. It is generally recommended for those with recurrent implantation failure, advanced maternal age, or previous miscarriages.
Mosaic Embryos: Some embryos may be classified as mosaic, meaning that some cells are normal while others are abnormal. The decision to transfer mosaic embryos can be complex and requires careful consideration and counselling.
False Result Rate: Depending on the indication of the test, there is an estimated 3% false result rate which could lead to the transfer of embryos with chromosomal abnormalities, increasing the risk of miscarriage or birth defects.
Not Comprehensive for All Genetic Disorders: PGT-A does not identify single-gene disorders (e.g., cystic fibrosis, sickle cell anaemia) or structural chromosomal rearrangements (e.g., translocations or deletions). For these specific genetic conditions, other specialized tests like Preimplantation Genetic Testing for Monogenic Disorders (PGT-M) or Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) are required. Therefore, PGT-A provides a limited but important scope of genetic screening.
In conclusion, PGT-A is a sophisticated tool that enhances the success rates of IVF by allowing for the selection of chromosomally normal embryos. The process, though complex, can significantly improve the chances of achieving a healthy pregnancy, making it a valuable option for some patients undergoing fertility treatments in certain indications.
This is a special process, when compared to conventional IVF. People going through this process are having their hormones and drugs through IVF, in the same process. However the time of the embryo transfer changes. Most of the PGTA procedures, in most labs world wide, are being carried out on embryos which are developed from day 5 or 6. In this stage of development the embryos are called blastocysts, so when we have good looking blastocysts then through a method called biopsy, we aspire some cells from the embryo, it is then cryopreserved, put back in the freezer and then we put the cells in a tube. They are then analysed in the tube so as to check on the chromosome number and structure in the biopsied sample. This is highly important as we need the right number of chromosomes otherwise we have chromosomal abnormalities that we don’t recommend to go back into the uterus. This is a screening process so we discriminate between those embryos that have the right number of chromosomes and those who don’t. Those we recommend to get back to the uterus are the ones with the right number of chromosomes.
From the patient’s point of view, it applies the same technique used for an IVF or an ICSI cycle, so induction of ovulation, oocyte collection, insemination. Once the embryos are there, some cells are removed mechanically from the trophectoderm at the blastocyst stage or the removal of the blastodermic fluid that is inside the blastocyst.
It is implied that then the embryo is frozen, is vitrified, and the analysis is done in the second phase. Once the diagnosis is made, those embryos suitable for transfer in a different cycle can be thawed and transferred at the right time into the patient’s womb.
So the difference between a normal ICSI cycle is that the cycle for PGT for aneuploidy or PGS is segmented and carried out in two different months.
PGS is Pre-Implantation Genetic Screening. Nowadays, it’s more commonly known as PGT-A – Pre-implantation Genetic Testing for Aneuploidies. This means each embryo is tested to ensure that the chromosomes for the embryo in question are correct both in number and appearance. Currently, embryologists use two moments in an embryo’s development during which the chromosomal load can be assessed – day 3, and/or day 5 of the embryo’s life.
On day 3, the embryo consists of eight cells. What embryologists do is use a laser to create an opening within the embryo’s shell in order to extract one of those cells. This biopsy doesn’t hurt the embryo and doesn’t impair its further development. The chromosomes are removed from the cell and analyzed, with results being available within 48 hours. If we find that the embryo is genetically normal and is continuing to develop within the lab, it can be transferred.
The day 5 biopsy takes place when the embryo is at the blastocyst stage. In this case, embryologists only weaken the embryo’s shell – as it grows, it will push through that weakness, allowing some outer cells to come out through the opening. That part of the embryo is called the trophectoderm. On day 5, embryologists can take four or five cells without actually touching the inner mass of the embryo, which later becomes the fetus. Following the biopsy, the embryo is frozen, chromosomes are extracted and analyzed; the process takes up to two weeks. If the embryo is chromosomally normal, we can perform a frozen embryo transfer at a later date.
What are the pros and cons of both approaches? The day three biopsy gives us very quick results, allowing for a fresh embryo transfer, which – in egg donation scenarios – still gives us a slightly higher chance of success.
However, some evidence says that some embryos found to be abnormal on day three may self-correct before day 5 and become chromosomally normal; which means we could potentially be discarding a small portion of viable embryos. The day five biopsy gives us a more reliable result, as four or five cells have been analyzed. There is still, however, a question of whether the cells that we have analyzed are representative of the rest of the embryo. We also aren’t certain whether an abnormality detected in the trophectoderm is a bad thing because we know of the existence of mosaic embryos – both normal and abnormal chromosomally – that can result in healthy live births. So, essentially, the process is the same – a biopsy is performed and the chromosomes analyzed. However, it’s difficult to say which of the two ways is currently better.
The process of PGS testing begins with creating embryos and letting them develop in a laboratory. Once they reach the blastocyst stage, we perform a biopsy of the trophectoderm, which is the part of the embryo that becomes the placenta. We take a couple of cells from the trophectoderm and send the sample to a genetics laboratory. The cells have their karyotype tested, which tells us whether the embryo itself is chromosomally normal or not.
Once we have the results, we will schedule another consultation with your doctor to discuss the results and further options. These options will depend on the test results – whether your embryos are normal, abnormal, or mosaic. Mosaic embryos contain both genetically normal and abnormal cell lines – a geneticist will be able to tell us if the degree of mosaicism allows for a safe transfer of such an embryo. In some cases, the results may not be conclusive – if that happens, we simply perform the test again.
PGS (or, as it is currently known, PGT-A) is usually performed on day five or six of embryo development, in the so-called blastocyst stage. Previously, it used to be done on day three; this approach, however, is now considered to be inferior. The reason for that has to do with what the embryo consists of in each stage: on day three, embryos usually are made up of around eight cells, while a blastocyst-stage embryo has hundreds, clearly divided into two types, the inner cell mass (which will eventually become the child), and the trophectoderm (which will develop into the placenta). As the blastocyst has a greater number of cells, performing the test is safer.
The test consists of a biopsy – embryologists take a couple of cells from the trophectoderm and sent them off to a genetics laboratory for analysis. Note that we don’t touch the part of the embryo that becomes the actual child. The laboratory analyses the cell sample and determines whether the embryo has the correct number of chromosomes or if any abnormalities are present.
Once the sample has been taken, we freeze the embryos using a technique called vitrification, which allows us to safely store specimens in nitrogen tanks.
Once the results from the genetic laboratory come back, we schedule the transfer of one healthy embryo. If we have more than one candidate for implantation, we can base our choice on the embryo’s morphological quality before the freezing process – we do this by reviewing the parameters recorded during embryo development. The rest of the healthy embryos will remain in storage until the patient decides to have them transferred. Although it may sound like a complicated process, it’s an established technique performed often.
