In this session, Dr Carmen Morales, PhD, a Genetic Counsellor and Laboratory Coordinator at Reproclinic, Barcelona, discussed PGT-M, formerly known as PGD, preimplantation genetic testing for monogenic/single gene defects.
Pre-implantation genetic testing is performed on embryos that are created through an IVF cycle to detect genetic abnormalities; thus, this allows a selective transfer of the unaffected embryos. To do this, a biopsy is needed to be performed on those embryos to take a small sample for the genetic analysis.
There are different types of pre-implantation genetic testing, and they are classified based on the type of genetic abnormality tested or detected.
In all kinds of PGT, the doctor needs the following:
It’s usually preferred to perform ICSI (Intracytoplasmic sperm injection) where a single sperm is injected inside the egg.
The embryo biopsy can be done on day-3 or, typically, day 5 of embryo development. This just involves taking a small sample of the embryo which is the one sent to the genetic laboratory where it’ll be analyzed for the type of genetic abnormality the doctors are testing and that’s been requested.
It’s very important to know that the embryos are not leaving the IVF laboratory. The only thing that leaves the IVF, that the doctor sends to the genetic lab, it’s the sample from the embryo. Moreover, the embryos are kept in the ideal lab, and they can be cultured until they are transferred if a fresh embryo transfer is going to be done, but usually, since the result of the genetic diagnosis can take several days, these embryos are normally frozen until their genetic diagnosis is ready.
The laboratory will send the genetic report to the IVF, and based on the results of it as well as on the morphology of the embryos, even if it’s a normal embryo but has a variable morphology, the doctor isn’t going to transfer it. However, based on that, they are going to select the unaffected embryos for transfer.
There are two main types of obtaining the sample and one of them is the embryo biopsy. Dr Morales shows the following image of an embryo biopsy performed on day-3, which is the cleavage stage:
In the cleavage stage (picture b), the embryos are having eight to ten cells and doctors, with a laser or mechanically, make a hole in the area surrounding the egg which is called the zona pellucida. After the hole is done, they are aspirating one of the cells of the embryo which then will be put in a tube to be sent to the laboratory.
Moreover, doctors can also do a biopsy, usually on day 5 or day 6, when the embryo reaches the blastocyst stage (picture c). Here, the hole is also done in the zona pellucida, and the doctor will take between five to ten cells from this one cell called the trophectoderm. This sample will be sent to the laboratory for analysis. Therefore, doctors can have biopsies of a single cell or a very few number of cells.
In addition, nowadays, very few IVF labs have an in-house genetic laboratory, and usually, they are externalizing the genetic tests. Depending on the country, they like the idea of shipping the sample abroad.
In the genetic laboratory, the sample is processed and for the sample testing there are different technologies available. Dr Morales isn’t going to explain all of them in detail because they are very technical concepts, but the technology they going to use will depend on the type of PGT to be performed as well as on the availability of the different platforms in the genetic lab.
For instance, the technology most used nowadays for Aneuploidy screening is the Next Generation Sequencing; nevertheless, they can also be tested with the qPCR and Microarrays. The use of one or another technology will be based on what is available in the genetic laboratory.
We as professionals have to have in mind that when we’re talking to these couples, they usually have experienced the disease because they already have previously affected children, there are only other very close family members affected or maybe one of the members of the couple is affected of the genetic disorder. – explains Dr Morales.
In simple terms, Dr Morales defines a gene as a unit of the DNA that contains instructions for a specific function of the body, mutations or abnormalities in the gene can make this gene not work properly and this can cause a medical condition, a genetic disorder.
This monogenic or single gene disorder is when abnormalities of this specific gene it’s causing a specific disease. One gene, one disease. This are the kinds of genetic disorders that are tested with PGT-M.
Dr Morales explains that there are two copies of each gene; one inherited from the mother and the other from the father. This is the same for all the genes that are located in the autosomal chromosomes, except for the sex chromosomes that are X and Y. The rest of them are autosomal chromosomes.
Thus, for all the genes everyone inherits one from the mother and one from the father, and the ones located on chromosomics, females will have two copies: one is inherited from the mother and one from the father. On the other hand, males only have one copy of these genes located in chromosomics.
Depending on the type of gene, because a gene can have a different pattern of inheritance, there is an Autosomal Dominant inheritance, Autosomal Recessive, X-linked pattern of inheritance, or Mitochondrial inheritance.
This means that from the two copies of the gene, there’s only one copy mutated that is enough to cause the disease. For example, Huntington disease, Neurofibromatosis, Achondroplasia.
As can be seen in the picture, there’s a man who’s affected by neurofibromatosis, so one of the two copies of the NF, gene is mutated. Thus, the risk for this couple to have affected children in its pregnancy is 50%; only the ones that are inheriting this abnormal mutated gene.
There’s a need for both copies of the gene mutated, meaning abnormal, to be affected. In the example, each one of the couple only have one of the copies mutated, so they are perfectly normal, healthy, and not affected. These are called carriers, but they can transmit, both of them, the abnormal copy to the offspring.
Here, there’s a 25% that their children can be affected by this recessive disorder, all the rest will be affected, but 50% will be carriers. Even though the couple is completely healthy, they are at risk of having an affected child with this recessive disorder.
Examples of this disorder can be: Cystic Fibrosis, Beta-thalassemia, Phenylketonuria, and more.
The gene is located in chromosome X and will be inherited dominantly, meaning that one mutated copy of a gene in chromosome X is enough for you to be affected. Thus, there can be females and males affected. In the case of an affected female, she can have 50% of the offspring affected, independently if they are boys or girls.
Men can only have one mutated copy because it’s the only copy of the gene they have in chromosome X. Thus if this copy is mutated, they are affected.
Females need both copies of the gene to be affected. This event is very rare. Usually, in these kinds of diseases, it can be seen in the pedigree that only males are affected while females are the ones who are transmitting the disease through generations.
In this case, there would be a non-affected carrier model with a mutation in one chromosome X, and the risk of having affected children will depend on whether the children are boy or a girl. If they are boys, 50% of them can be affected, and if they are girls, none of them will be affected, only 50% will be carriers like their mother.
It’s important to remember that the male is transmitting to all the sons the chromosome Y, while to the daughters the chromosome X.
This is quite different from the others mentioned. The mitochondria are structures that are inside the cell, and they are the source of energy for research. They are essential for life.
The main characteristic of the mitochondria is that they have their own DNA. Also, the mitochondria are inherited from the mother only because the sperm doesn’t have mitochondria. Moreover, when there’s fertilization, all the mitochondria in the embryos come from the egg and, thus, from the mother.
With these kinds of diseases, usually, when there’s a phenomenon called Heteroplasmia (as can be seen in the picture below), the mother, for instance with mild or no symptoms. This mother can have a mix of normal mitochondria and an abnormal one, so the manifestation or symptoms of the disease will depend on the percentage of abnormal mitochondria this mother has. Usually, if the percentage is less than 18%-20%, the doctor considers that this person can be asymptomatic or have very mild symptoms. If the person doesn’t have the abnormal mitochondria, this is higher. The symptoms can be more severe.
During the formation of the eggs in the homogenesis, there’s something called the “Bottleneck effect”, meaning that the mitochondria are randomly distributed within the eggs that are formed, so some eggs can have a high percentage of abnormal mitochondria and some of them can have a very few percentages. Thus, after fertilization, there can be embryos with different loads of abnormal mitochondria and, depending on this load, these embryos will be non-affected, mildly affected or severely affected.
In this type of PGT, the doctor will be testing not only if there is the presence of the mutation or not, but also will be testing the percentage of abnormal mitochondria, the load of another mitochondria the embryos have. So, if it has been established a cutoff of 18%, and with 18% of abnormal mitochondria consider that this embryo has 95% of probability of being a non-affected, healthy.
This is the cutoff. Embryos with 18% or less of abnormal mitochondria can be considered for transfer.
I have to tell you that this mitochondrial PGT is not performed in a lot of laboratories, very few labs in the world are doing that, and it’s not very frequent. According to my experience, most of the patients who are carriers of mitochondrial disease, opt for egg donation because, for them, it’s an easier process. – states the doctor.
The main difference between the PGT-M and for example, PGT-A is that it requires a pre-examination step. Therefore, before the couple starts with IVF, the doctor needs to do the following:
This is very important because, in this kind of test, doctors are working with a very small amount of DNA, a very small sample from one cell to very few cells. Therefore, there are important issues that they need to avoid.
This can be avoided by following very strict protocols, like cleaning everything and handling the samples and embryos very carefully. Nevertheless, contamination can also come from paternal and maternal DNA. For example, to avoid paternal DNA, as Dr Morales recommended, undergo ICSI because when you are doing conventional fertilization, you can have extra sperms that are attached to the zona and they are a source of paternal DNA that can contaminate, eventually, the sample when you are doing the embryo biopsy.
Moreover, you need to minimize maternal contamination that it’s not the most frequent one by denoting very well the eggs to eliminate all the cumulus cells that surround the egg and that from maternal origin can be the source of maternal DNA.
There are also some issues that are more technical related to the PCR technique that the doctor’s going to use, the amplification thing to analyze the sample.
Something used by the doctor, called allele drop out, where you have the mutation copy of the father and the mother, but with this allele drop out only one of those is amplifying; thus, in the end, this can lead you to a misdiagnosis because maybe you think it’s affected but it’s carrier as the normal allele has not been amplified.
For this reason, to avoid all of this, it’s important to do a prototyping approach. The doctor will establish the prototype in the family, and try to search and find the combination of what they call genetic markers that are in the surrounding area of the mutation, so close to the mutation that they need to be sure that they are inherited together with the mutation. Therefore, in case there is a failure of amplification or allele dropout in the specific mutation, they are testing other areas surrounding this mutation that can help them give the diagnosis and increase the accuracy of the test because they need to reach an accuracy of the test more than 90-99%. So, they need to be sure that the diagnosis is good.
Moreover, to find the combination of these markers, that are informative and different from the mother as well as from the father, the doctors need to establish the haplotype and test the family members. They usually test the couple if they have previously affected children, but if not, sometimes they have to use the father, the mother of affected siblings, etc. In each family they also have to assess the case and see the availability of the family members to be included.
Taking everything into account, Dr Morales further explains that they need to review the case to be sure that the test can be done and it’s technically possible. They also need to do the preclinic work-up to try to establish this haploid typing and see if technically they can do it because sometimes it’s not so easy if there is a high degree of consanguinity in the couple and it’s not so easy to find these informative markers. You have to go far away from the mutation and there is more risk of misdiagnosis.
For this reason, in this setup, you are not just searching for these markers, you are setting up the protocol you are going to do in your lab, and you are going to establish the risk of misdiagnosis that this specific test will have which should be very low because if it’s high then you have to add more markers. When this preclinical workup is finished, you see that the PGT is feasible in this couple, then you issue a report and, only when the report is ready, the couple can start the IVF cycle.
In addition, after the report for the IVF cycle and the embryo analysis are ready, there is a consultation called post-test counselling, based on the results the doctors have to explain to the couple the results and the recommendations for transferring if there are normal embryos, and also, if not, the fate of the normal embryos that they are not going to transfer, and more.
For PGT-M there are different testing, strategies, and technologies that each laboratory can have or use different platforms, approach, or design the experiment in a different way.
This is something important that Dr. Morales mentions. Sometimes, this workup is laboratory-specific, meaning, if the couple decides to repeat the IVF in another clinic that is working with another genetic lab, most probably, this workup test has to be repeated because this test is lab-specific. The lab can choose different markers, and use different technology, and they need to be sure that they are going to be able to analyze this embryo with 90-99% accuracy.
It’s also important to know that this PGT-M for monogenic disorder or single gene disorder can be combined with PGT for aneuploidy screening. The same sample can be used for both technologies, but this will depend on the indication and specific for the couple. However, it can be combined and you don’t need to take a second sample from the embryo. For this, Dr. Morales provides the following example:
This approach can be very complicated, but this is an example of what the doctors have. Here is a disease. Here you can see the gene with the invitation and the doctor is going to choose some genetic markers on both sides of the mutated genes, so for the clinical workup they use the couple and an affected boy that they have. This is the haplotype, and they are searching for these markers which are different in size. They establish this haplotype and, later when they are analyzing the embryos, they are able not only to test the mutation, the one in the third position of the example shown, but also to test the rest of the markers established in the haplotype.
What’s going to happen in this embryo is that there’s an allele drop out, a failure of amplification of the original, but this is not interfering in the result because the doctors have other regions to test. They are testing several regions, so at the end they are sure that they are giving a proper diagnosis.
There is some ethical debate about this. In some countries, it’s allowed while in others is not. In general, if they’re doing only the HLA matching, they can find around 25% of the members that can be compatible. If doctors are also adding the PGT for a monogenic disorder, because the cause of the disease in the previous child is a genetic one, the chances of finding a normal embryo for the disease plus that match with the previous baby are lower, probably one in six for a recessive disorder and one innate for dominant. So, the chances are not very high and the couple has to be counselled very well because this takes time.
In most of the countries, you need a specific approval to do this test. When the test is approved, they have to do the preclinical workup, and then, they have to do the IVF. If they have one embryo that it’s normal and it’s matching, you have to transfer them and if you are pregnant you have to wait nine months. This will take a long time, so you need to be sure if your son or child is severely affected. You need to consider that you will need a lot of time to achieve a sibling that can be HLA-matching.
Considering all the points mentioned by Dr Morales, PGT is usually for serious conditions; but how serious should the condition be to do PGT-M?
The seriousness of a disease is a perception, so it can be very variable. Some patients with the same symptoms are not perceived as having the same disease, so it’s not easy to establish which is severe and which is not because, according to Dr. Morales, sometimes it’s a perception.
Moreover, with the HLA matching test, there’s a kind of rise in ethical issues; for instance, a child is born to cure another one even though the chances are low. However, what the doctor is going to do with the embryos that do not match but are completely healthy?
With regards to diseases, the first symptoms can appear after 60 years old, so this person can have a very happy and fulfilling life up to that year; thus, this can raise some ethical and controversial issues. For example, for Huntington’s disease, the doctors have the Exclusion testing and, for instance, in a couple where the male is at risk of having Huntington’s disease because his father is affected by this disease, but he is still young and he’s not having any symptoms. However, he doesn’t want to know if he’s affected or not because he is not able to handle it, but he has the right to have healthy children. Therefore, one thing that he can do is the Exclusion testing.
In this test, the doctor is going to test the embryos but not for the mutation of Huntington’s disease. The doctor is going to see if these embryos have the same haplotype as the grandfather. Then, they are only going to transfer the embryos with the haplotype of the grandmother that they know is healthy.
The haplotype of the grandfather can be affected or not, it’s not known, so these embryos that are discarded have 50% chances of being normal. This test also requires a lot of counseling and an analysis of the case.
For all these matters, it may be acceptable or not, this is different in each country. Even in Europe, each country can have their different policies and regulations for what tests can be directly done and what tests need to be assessed case by case where you need specific approval from the committee or the national commission. Therefore, the law is different in each country, and this should also be explained to the couple before they start treatment because sometimes they don’t have time to comprehend the whole process if, before starting treatment, you need specific approval from the government or law in this in this country.
Dr Morales shows some data from Europe from different sources on assisted reproduction techniques. Most of the countries that are performing assisted reproduction techniques, PGT-M is allowed and very few of them are bad.
On the other hand, PGT-A is not allowed in as many countries. Also, gender selection is not allowed in any country in Europe, for social reasons.
This data it’s for heterosexual couples and same-gender couples, the number of countries that are along this test, it’s much lower.
Furthermore, the numbers of PGT cycles done in Europe come from the data of ESHRE PGT Consortium, which is the last series and data published that collected data from 2013 to 2015.
It can be seen that around 70,000 cycles for PGT-M and, from all the cycles, they get pregnant with pregnancy-positive heartbeat around 34% for embryo transfer. Also, considering the type of genetic disorder, you can see that 50% of the cases are autosomal dominant disorders, meaning that one of the members of the couple was affected by this disease. Also, HLA matching is representing 5% of the cases.
The main disorders done in Europe for autosomal recessive are:
For autosomal dominant are:
For X-linked are:
These are the main ones, but there’s a huge list of diseases that can be tested.
To summarize, Dr Morales talks about the following points:
I don’t know what will happen in 20 years. Nowadays, it’s not a safe procedure, so I understand that maybe this is in the future gene-editing, inserting the embryos with the healthy gene, but not for now. I think maybe in the future. This is still under research because it’s not always working, we don’t know their side effects, but I think this can be the future. Not for now, maybe not in 5 years, but in the end, probably it would be the future of IVF instead of doing genetic tests.
There have been a lot of published studies and there is no difference in terms of pregnancy rates and outcomes from biopsy embryos and non-biopsied embryos. After seeing that, I have to tell you that the person who is doing the biopsy should be perfectly trained and very skilled. In that case, there is no damage in the embryo because there are a lot of cycles done not only for PGT-M but PGT-A which is more frequently done than PGT and PGT-M. In my opinion, on what is published and what I saw, if the person that is doing the biopsy and the conditions of the lab are good, there is no detrimental in the embryo development.
We don’t know the genetic cause of all diseases and not all genetic diseases are monogenic, meaning caused by defects in one single gene. I don’t have more information about your autoimmune disease, whether it can be a single gene or not. Sometimes, what we find is that the couple or the patients coming to the clinic without a clear diagnosis, or they are going because ‘I have a child with this problem’ but have not been properly diagnosed, we’ll have to report.
Firstly, before starting anything, we need to have a clear genetic diagnosis. Not all diseases, of them, have a genetic component. For example, you have diabetes type 2 or whatever, a lot of diseases, but some diseases are not monogenic. It means it’s not one gene, one disease. Multiple genes interact together, and the combination of multiple genes and sometimes related also with the environment can lead you to a disease that has a genetic component because sometimes they run in families, but it’s not so easy to find the genetic cause.
In your case, I don’t have a lot of information about your immune disease, maybe it’s not a monogenic disorder, most probably. If we cannot find the genetic cause, meaning one gene, or one disease, I think we cannot test the embryo. For this reason, we will need an assessment of your case because, in case we suspect a monogenic disorder, meaning there are some other family members affected in your family, we see the pattern of failures, and then there are some tests that can be done to screen all the genes that have been published to our knowledge and are related to this specific disease to see if we can find a mutation.
Unfortunately, there are a lot of genetic diseases that we don’t know what the cause is, and every day, the knowledge is increased, but not always possible. In this case, it’s not monogenic, not caused by a single gene, and a lot of diseases are not like that, so these diseases cannot be prevented in the embryos because they are not inheritable or they can be inherited, we don’t know exactly how they are transmitted, so in that case I will need a bit more information about your case to see if something can be done or not.
PGT-M is not fixing any disease, it’s only a genetic tool to tell you these embryos are affected of this disease or not, and then based on these results you can choose to transfer the unaffected ones. It’s not that you’re going to change the embryos in any way. You can have all the embryos abnormal and then you are not going to be able to transfer one. This can happen.
With PGT-M, we are not fixing the embryos, we’re just breaking the embryos and we are telling the couple these are unaffected and these are affected, so the ones that are unaffected can be safely transferred.
I don’t think so. The last review I found was all policies in Europe and European countries, I think at the end of 2018. I think in Germany, PGT-A, to my knowledge unless they have changed recently, it’s not allowed. PGT-M is allowed, but the case should be assessed and there’s an ethical committee that has to review the indication. It should be done case by case. PGT-A I don’t think so, for PGT-M is so strict I don’t think that PGT-A will be more permissive, usually, it’s the other way round. PGT-M, since already these couples have the option to do prenatal testing in most countries and it’s more widely accepted than PGT-A.
In some countries, it’s not considered that so many opportunities are severe enough, or that PGT-A is more controversial. To my knowledge no, but it can be easily checked.
Thousands, I mean, there are a lot. Some of them are more frequent than others for different kinds of effects, for example, cystic fibrosis is quite frequent in the European population and sometimes, because of a founder effect or we have a common ancestor that has this mutation or for any kind of reason, having been a carrier of this mutation has been preferably selected in this population because it’s beneficial for something else or on some island only one person travels with the mutation there and then it’s like the common ancestor of a lot of people.
Some genetic diseases are more frequent in some populations than in others or populations with a high level of consanguinity. You can find specific genetic disorders in this population that you cannot find in another one, but there are thousands of genes, and most of them can suffer mutations. Some mutations can happen sporadically, which means that most of the diseases are inherited, but some patients have the mutation they know about them. For example, in achondroplasia, some patients are healthy and completely normal, and they have a child with achondroplasia because, at the low of mutation, a mutation has reproduced in this embryo. However, from now on, the person can transmit the mutation to the offspring.
So, how many diseases are there? Thousands. For this reason, it’s very important to assess each case because sometimes the diseases are very rare, and you have never seen them before. You have to check, to assess the case, to see the mutation. Some of them detect a mutation, but if it’s pathogenic or not, it’s not quite clear. In that case, it’s not recommended, but there are thousands of genetic disorders.
All the functions of our bodies are regulated by a gene, so the genes will have thousands of genes, and eventually, they can cause a genetic disorder of them.
Yes, definitely. Here at Reproclinic we specialise in patients from abroad, so yes, there is no problem. If you are interested, you can email me, and we can provide you with all the information, the clinic will help you because depending on the country with these issues and these things, we are going to explain the whole process.
