What is the procedure for pre-implantation genetic diagnosis?

Pre-implantation genetic diagnosis (PGD) is a genetic analysis of embryos obtained from in vitro fertilization (IVF) with the aim of selecting healthy embryos with the capacity for implantation.

Furthermore, PGD is the most indicated technique to avoid the transmission of a hereditary genetic disease, since it allows the detection of embryos with mutations in their DNA, which will be discarded for their transfer to the uterus.

PGD must be performed by experts in both embryology and genetics, as it requires very delicate techniques such as blastomere biopsy and molecular genetics techniques.

PGD step by step

IVF with PGD is one of the most laborious treatments in assisted reproduction and therefore has several steps from ovarian stimulation to embryo transfer. Below, we will discuss each of them:

IVF

consists of performing ovarian stimulation on the woman, obtaining the eggs by means of follicular puncture and, finally, fertilizing the eggs with the sperm of the male partner or of an anonymous donor.

Embryonic development

the characteristics of the embryos in culture are assessed on a daily basis, since in order to perform the biopsy it is necessary for them to have a minimum level of quality to ensure their survival.

Cellular biopsy

extraction of one or more blastomeres (cells from the embryo) for subsequent genetic analysis. It can be performed on day 3 embryos or once they have reached the blastocyst stage on day 5. The removed cell is then carefully placed in a tube, a process known as tubing.

Genetic analysis

study of the chromosomes and/or genes in the biopsied cells to identify possible alterations in them. Specifically, chromosome analysis is better known as preimplantation genetic screening and, on the other hand, the term PGD is used for the analysis of specific genetic mutations.

Embryo transfer

genetically healthy embryos are selected for transfer to the mother's uterus or cryopreservation for future attempts.

How is PGD performed step by step?

It should be noted that the transfer of embryos can be done in a fresh or frozen cycle. This will depend on the time needed to obtain the results of the genetic analysis, and it is quite common for the embryos to have to be frozen.

Embryo biopsy

Embryo biopsy is the process of extracting one or more cells, called blastomeres, in order to analyze the embryos' DNA for any genetic alteration.

The viability of the embryo may be compromised by this biopsy, since the loss of a cell may be too stressful for the embryo to survive. Therefore, it is necessary that this cell extraction be performed by highly specialized and experienced laboratory personnel.

The embryo biopsy can be done on day 3 or day 5. In both cases, it is essential that fertilization takes place through ICSI. In conventional IVF, there are sperm and granulosa cells attached to the zona pellucida of the embryo. This poses a risk of contamination of the extracted blastomeres and therefore the PGD could give an altered result.

Blastomere biopsy on day 3

On the third day of embryonic development, good quality embryos usually have 8 cells, although they may also have between 6 and 10 cells depending on their rate of division.

Therefore, if the biopsy is done at this precise moment, only one, or at most two, blastomeres will be removed from the embryo in order not to compromise its viability.

During the procedure, a hole is made in the zona pellucida of the embryo by means of laser pulses or chemical agents, such as Tyrodes acid. Once this hole has been made, the blastomere is removed by suction.

Blastomere biopsy on day 3

After the embryo biopsy, there are two possible options for the embryos:

Blastocyst culture

the embryos are left in culture to continue their evolution while waiting for the results of the genetic analysis. Therefore, the embryo transfer will be in the blastocyst stage on day 5.

Embryo vitrification

the embryos are frozen after the biopsy for transfer in a later cycle.

If you are interested in reading more about this topic, you can access the following post: Frozen Embryo Transfer Procedure.

Trophoectoderm biopsy on day 5

By the fifth day of development, the embryo has become what is known as a blastocyst. This is a larger embryo, composed of a multitude of cells and with 3 differentiated parts:

Internal cellular mass (ICM)

gives rise to the future fetus.

Blastocele

is an internal cavity filled with fluid.

Trophoectoderm

outer layer that originates the placenta and other extra-embryonic structures.

Structure of a blastocyst

Although these are different cell populations, both ICM and trophectoderm cells have the same genetic material. It is, therefore, possible to extract a few cells from this outer layer for analysis with PGD.

When doing the biopsy with a blastocyst, care should be taken to make the hole in a place away from the ICM in order to avoid possible damage.

On the other hand, it is more convenient to use the short pulses of the laser to pierce the zona pellucida than Tyrodes acid since it has greatly reduced its thickness as the blastocyst expands and the use of a chemical agent could damage it.

Then, once a sufficient hole has been made to insert the biopsy pipette, the cells are removed by aspiration. To separate the cells to be removed from those that remain inside the embryo, the laser pulses can help.

Trophoectoderm biopsy on day 5

In this case, it will be necessary to vitrify the embryos after the biopsy in order to transfer them in a future cycle, since the results of the genetic analysis take several days and it would not be viable to leave the embryos in culture.

Pre-implantation genetic screening

Pre-implantation genetic screening is a type of PGD in which possible aneuploidies in embryos are analyzed. Aneuploidies are alterations in the number or structure of chromosomes, taking into account that the normal human chromosome endowment is 46 chromosomes. Therefore, the gain or loss of a chromosome, as well as changes in its structure, are abnormalities that can result in non-viable embryos or newborns with chromosome diseases such as Down syndrome.

In some clinics, this type of PGD is also called aneuploidy screeningand the genetic tests used for their detection are those discussed in the following sections.

FISH Study

Fluorescent in-situ hybridization (FISH) is the technique that has traditionally been used in the study of chromosomal abnormalities.

It only allows the analysis of certain regions of 9 chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y). However, these chromosomes are involved in aneuploidies that can lead to repeated miscarriages or the birth of sick children.

The process consists of adding fluorescent probes for specific regions of the chromosomes to be analyzed. It is then possible to visualize the fluorescent signal through a special microscope and to detect whether any of the analyzed regions are missing or, on the contrary, if there are more copies than they should be.

FISH chromosome analysis

Since the analysis of only a certain number of chromosomes is a limitation, FISH is considered an incomplete analysis and is currently being replaced by other techniques that allow a complete genetic analysis of the embryo.

CGH Arrays

The Comparative Genomic Hybridization Array (A-CGH) is a technique that allows us to carry out a Comprehensive Chromosome Screening (CCS), that is, the 23 pairs of chromosomes are analyzed at the same time in search of regions with some chromosomal alteration.

To carry out this genetic analysis, a control DNA and the DNA sample to be studied from the extracted embryonic cell are used. Both DNA strands are marked with different fluorescent probes and then hybridized in the microarray.

With this methodology, it is possible to compare the DNA of the embryo with that of the reference sample and identify losses (deletions) or gains (duplications) of genetic material, which could lead to embryonic defects.

Genetic analysis by CGH arrays

Although this technique is much more decisive than FISH, it only makes quantitative comparisons, that is, it detects whether there is missing or excess genetic material. Therefore, it does not detect if there are chromosome fragments that are not in the right place, such as inversions or translocations.

PGD for monogenic diseases

Monogenic diseases are those caused by mutations in a single gene, such as cystic fibrosis, hemophilia, or fragile X syndrome.

Examples of monogenic diseases

In addition, they are hereditary diseases that are transmitted from parents to children. Therefore, before doing PGD on the embryos, it is necessary to identify the specific mutation in the parents through an informativity study.

Once the mutation has been located and the type of inheritance determined (dominant, recessive or sex-linked), it is possible to study the embryos with the genetic tools discussed below.

PCR

The polymerase chain reaction (PCR) involves the amplification of specific DNA sequences for further analysis. Thus, it is possible to obtain millions of copies of the gene of interest from a single copy present in the extracted cell.

Once the desired sequence has been amplified, different molecular biology techniques are used to analyze it and locate the possible mutations that give rise to monogenic diseases.

Mass sequencing

Next-generation sequencing (NGS) is the most advanced technique for genetic studies today.

The advantages it offers over other techniques are as follows:

• It allows the study of all 23 pairs of chromosomes in the same analysis, and with a higher resolution.
• It allows the simultaneous analysis of more than 500 genes associated with hereditary diseases.
• It is possible to study aneuploidies and monogenic diseases separately or simultaneously in a single analysis.
• Reduced analysis time, avoiding the need to freeze the embryos while waiting for the results.
• It allows a high number of samples to be analyzed at the same time, making the cost of analysis more affordable.

Advantages of Mass Sequencing

Nowadays, more and more clinics are opting for this latest technology both for aneuploidy screening and for the detection of monogenic diseases.

Transfer of healthy embryos

Once the results are obtained, the healthy embryos will be transferred to the mother's uterus or vitrified for future attempts. Embryos with genetic alterations are discarded, even if they apparently have good morphological quality.

If the embryos have been vitrified prior to the results, they will be transferred in a replacement cycle. To do this, the woman will have to make an endometrial preparation with the administration of estrogen and progesterone.

Destination of embryos after PGD

If the results show that there are several embryos free of abnormalities, only the embryo or embryos with the best morphogenetic quality for transfer will be thawed, which will increase the probability of implantation.

FAQs from users

In what kind of cases is PGD being used?

PGD facilitates informed decision making and is increasingly being included as an option in genetic counselling.

The current objective is that it can reach any couple, if they so freely choose, and any hereditary pathology, as long as there is a clinical indication and it is possible to address its diagnosis technically.

For couples with a clinical history of monogenic disease, the main current indications are, among others, cystic fibrosis, spinal muscular atrophy, myotonic dystrophy (Steinert), fragile X syndrome and Huntington's disease.

Regarding chromosomal analysis, the main indication is the embryo study due to the risk of aneuploidy in advanced maternal age, without forgetting the group of patients with a history of recurrent miscarriage and severe male factor.

What is the probability that there will be no embryos left after the PGT?

The Preimplantation Genetic Test (PGT) allows the detection of genetic abnormalities in the embryos, before their transfer to the woman's uterus. Identifying embryos free of chromosomal abnormalities and genetic mutations can prevent their transmission to offspring.

The more embryos available for PGT, the greater the chances of obtaining healthy embryos suitable for transfer to the woman's uterus. Advances in embryo culture and biopsy techniques have made it possible for the genetic analysis of embryos to be carried out at the blastocyst stage, allowing a greater quantity of cells to be obtained (trophoectoderm) and maintaining the integrity of the embryo and its capacity for implantation.

However, ovarian reserve and ovarian response to gonadotropin stimulation are inversely related to maternal age, since the older the mother, the lower the reserve and response. Consequently, advanced maternal age affects the number of oocytes, their quality and the number of embryos available for biopsy.

Are PGD and PGT the same?

These are the acronyms for the genetic studies that are performed on an embryo before it is transferred to the uterus. On the one hand, PGD refers to preimplantation genetic diagnosis; while PGT stands for Preimplantation Genetic Test.

Currently, the most commonly used term for this complementary technique is PGT.

What are the advantages of doing a PGD?

Even if an embryo looks good morphologically, it may be that genetically it is not normal and may lead to implantation failure when transferred to the uterus or, worse, an unviable pregnancy that ends in miscarriage.

It can also happen that an embryo with some chromosomal disease or genetic alteration compatible with life is implanted and, finally, a baby with some serious disease is born.

Thanks to PGD, those embryos that are genetically healthy can be selected before being transferred to the uterus, reducing the abortion rates and increasing those of live newborns.

Can extracting one or more cells from the embryo negatively affect its further development?

Yes, the process can compromise the viability of the embryo, since losing some cells can be too stressful for it to survive. Even so, the survival rate is very high, especially when the PGD is done in blastocysts, since these embryos have a greater number of cells than those on day 3 and, therefore, are able to recover more easily after the biopsy.

Is PGD better on day 3 or day 5?

It is preferable to perform a PGD on day 5, when the embryo is in the blastocyst stage, since it is more developed and allows a greater number of cells to be analyzed, which improves the diagnosis and reduces the possibility of error.

Furthermore, the cells biopsied in the blastocyst are from the trophoectoderm, the external part of the embryo, so all the cells that will give rise to the future fetus are preserved intact.

Is it possible to perform a polar corpuscle biopsy?

Although the most common procedure in PGD is to carry out a genetic analysis of the embryos, there is also the possibility of analysing the genetic content of the eggs obtained for IVF by means of a biopsy of the polar corpuscle.

The polar corpuscles are cells with no function that are formed during meiosis and disappear instantly after fertilization. Therefore, the polar corpuscle biopsy is performed before fertilization takes place. In this way, the eggs with the best possibilities of achieving a good development of the pregnancy are selected.

The analysis of one of these cells will not affect the future development of the embryo. However, it will not be possible to detect anomalies that occur after fertilization nor will it provide genetic information of paternal origin, only of maternal origin.

Suggested for you

Throughout this article, we have commented that PGD serves to detect genetic and chromosomal abnormalities in embryos. If you want to know what specific diseases occur as a result of these alterations, you can read on here: What genetic or chromosomal diseases can PGD detect?

For information on the success rates of this technique, we encourage you to enter the following article: Pregnancy probability with PGD.