What is Preimplantation Genetic Testing (PGT)?

Preimplantation genetic testing involves procedures that detect chromosomal abnormalities or genes causing diseases before pregnancy, allowing the transfer of healthy embryos into the uterus of the prospective mother. These procedures are applied using IVF methods to identify potential chromosomal anomalies in embryos. Preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) are encompassed within PGT.

Who can benefit from PGT?

– Advanced maternal age (38 years and older)

– Known carrier of translocations

– Severe male factor infertility (cases where the sperm count is less than 5 million per ml or no sperm in the semen – cases of azoospermia)

– Recurrent IVF failures

– History of single-gene defects and/or the presence of children requiring HLA-matched transplantation

– Recurrent early pregnancy losses

– Mitochondrial disorders such as MtDNA mutations

– Selection of embryos to shorten the time to achieve pregnancy in couples with no risk factors: Embryo Ranking

– Investigation of epigenetic modifications

When is embryo biopsy performed?

In preimplantation genetic diagnosis, genetic material from the embryo is required for genetic analysis. Biopsy is an invasive technique to obtain the embryo’s DNA. The PGT procedure can be applied at three different stages of embryo development:

1. Pre- and post-fertilization period polar body biopsy from oocytes
2. Blastomere biopsy at the embryo cleavage stage
3. Trophoectoderm tissue biopsy at the blastocyst stage (chromosomal content of the embryo obtained from 5-10 cell trophoectoderm on days 5, 6, or 7 depending on the embryo development stage)

What are the advantages of trophoectoderm biopsy?

– It has the highest diagnostic power.

– It allows analysis of trophoectoderm cells without including the inner cell mass (ICM) in the biopsy.

– It is cost-effective.

– It has no impact on the developmental competence of the embryo.

– It provides a high rate of DNA amplification.

What are the disadvantages of trophoectoderm biopsy?

– The IVF center must be qualified in long-term embryo culture and vitrification.

– Global experiences in this regard are still inadequate.

– There is limited time for fresh embryo transfer. Therefore, a frozen-thawed embryo transfer cycle will be required if PGT is to be performed.

Does trophoectoderm biopsy harm the embryo?

When performed properly by trained hands, surgical removal of 5-10 trophoblast cells does not significantly affect blastocyst viability. The best evidence of this is the double embryo transfer experiment with similar blastocysts. It has been shown that when biopsy is performed on only one blastocyst and not the other, the embryos implant equally. Recently, it has been reported that even repeated embryo biopsies with two consecutive freeze-thaw cycles did not significantly affect embryo viability.

Preimplantation Genetic Diagnosis – Aneuploidy (PGT-A)

Aneuploidy is the most common genetic abnormality in humans, and its high incidence in embryos is the main cause of failed implantation, pregnancy loss, and congenital birth defects. Diploid cells normally contain 46 chromosomes, a condition known as euploidy. Aneuploidy is a modified condition involving deviations from multiples of 23 in copy numbers. Typical examples include monosomy or trisomy, resulting in 45 or 47 chromosomes, respectively.

What is Preimplantation Genetic Testing (PGT)?

Preimplantation aneuploidy in embryos is primarily a result of chromosomal/chromatid segregation errors that occur during meiosis (in sperm or egg) and eventually affect all cells in the resulting embryo equally. These mechanisms can generally be grouped as follows: (1) Errors in segregation (failure of homologous chromosomes or sister chromatids to separate) and (2) Premature separation (early separation of homologous chromosomes or sister chromatids).

The majority of meiotic errors occur in the mother’s meiosis (90-99%); recent studies estimate that ~50-70% originate from meiosis I and ~30-50% from meiosis II. The likelihood of aneuploidy originating from paternal meiotic events is much lower, ranging from 1% to 10%. Embryonic aneuploidy is believed to be influenced by environmental factors. Some parental lifestyle conditions, such as obesity, smoking, exposure to radiation, and contraceptive pill use, can increase meiotic errors.

Preimplantation genetic testing (PGT-A) reduces the risk of aneuploidy, improves IVF success, shortens pregnancy duration, and reduces the rate of multiple pregnancies.

PGT-A, previously expressed simple grouping of embryos into ‘normal’ or ‘abnormal’ based on chromosomal profiles; however, recent data advocate for a much more refined classification, encompassing “euploid,” “aneuploid” (e.g., monosomy/trisomy), “mosaic,” and “segmental abnormal” embryos. Additionally, mosaic and segmental abnormal groups can be further refined based on their characteristics. The purpose of such classification is to achieve an advanced ranking system allowing the selection of embryos with the highest likelihood of resulting in a clinical pregnancy.

Various PGT-A methods include predicting the presence of mosaicism from a single trophectoderm biopsy. These methods are primarily based on classifying the number of inter-chromosomal copies within a ‘mosaic range’. Mosaicism can arise primarily during embryonic mitotic cell division and non-disjunction, leading to sister cells with different chromosomal structures (trisomy, disomy, and/or monosomy). Contemporary methods for determining the quantity of chromosomes from a multicellular sample (i.e., trophectoderm) can identify mosaicism by observing an intermediate copy number showing a mixture of euploid and aneuploid cells. Modern PGT-A methods can detect mixtures of euploid and aneuploid cells with high accuracy, but there are differences among clinics regarding the reported frequency of embryos classified as mosaic with PGT-A at the blastocyst stage. The latest estimates range from 4% to 22%. The reasons for this discrepancy are biological (conditions affecting the rate of mosaicism in laboratories).

Another relatively new category of PGT-A classification involves detecting segmental imbalances. Typically, the probability of detecting imbalances of 10 Mb or larger is considered. Unlike embryos classified as ‘mosaic’, there is very little data on the clinical outcomes of embryos assumed to have segmental imbalances.

Preimplantation Genetic Testing – Structural Rearrangements (PGT-SR)

Balanced translocations, Robertsonian translocations, insertions, and inversions are abnormalities that alter the natural sequence of chromosomal segments but leave copy numbers unchanged. Carriers of such anomalies typically show no symptoms, but recombination and segregation during meiosis can lead to chromosomal copy number abnormalities in eggs and sperm. This condition can lead to infertility, an increased risk of pregnancy loss, and an increased chance of having children with physical and mental disabilities. Therefore, while PGT-A serves as a screening tool for spontaneously occurring chromosomal abnormalities, PGT-SR is a targeted test performed when known chromosomal abnormalities are present in parental genomes. PGT-SR requires a personalized examination of parental karyotypes because embryos obtained using IVF are tested for imbalanced chromosomal structures.

Preimplantation Genetic Testing – Single Gene Disorders (PGT-M)

Since the rate of consanguineous marriage in our country is quite high (23.5%), combating severe debilitating single gene disorders should be one of the most important health policies. PGT applications for single gene disorders have become one of the most effective technologies in preventive medicine. PGT can be applied to any type of single gene disorder, but it is most commonly used for diseases that cause severe morbidity. The main diseases include Spinal Muscular Atrophy (SMA), Cystic Fibrosis, Beta-Thalassemia, Duchenne Muscular Dystrophy (DMD), Huntington’s disease, and Fragile X syndrome.

In couples carrying familial genetic diseases transmitted by a single gene, the process of selecting healthy embryos that do not carry the mutation causing the genetic disease through examining embryos is called PGT-M. Before this test is performed, a detailed map of the gene region carrying the mutation causing the disease is drawn up, and the mutation region and its surroundings are identified by a genetic specialist. Then, the process of DNA amplification is performed on biopsy samples taken from embryos obtained through the IVF method. Subsequently, healthy embryos that do not contain the mutation are selected through mutation analysis of single genes. In the second step, the selected healthy embryos can undergo the PGT-A process to determine their chromosome numbers. Thus, embryos carrying the correct number of chromosomes both in terms of single genes and chromosome numbers are selected.

The International Preimplantation Genetic Diagnosis Society (PGDIS) recommends PGT-M for the following situations:

1) Carriers of Mendelian diseases who want to have an unaffected child,

2) HLA typing for finding an HLA-matched stem cell for a sibling requiring stem cell therapy,

3) Structural chromosomal abnormalities and different translocation carriers in infertile carrier couples to increase the chance of obtaining unaffected pregnancies,

4) Unexplained recurrent pregnancy losses (RPL) aiming to reduce complications and side effects caused by recurrent miscarriages.

RPL is defined as two or more pregnancy losses and can have many causes. More than 50% of recurrent pregnancy losses have no known cause.

Preimplantation Genetic Diagnosis – HLA Compatible Embryo Selection (PGT-HLA)

We call the necessary genetic procedures performed to bring a fully HLA-matched sibling to families with a child requiring tissue-matched transplantation PGT-HLA. This genetic procedure can also be applied to prevent genetic diseases that the family can be a carrier of, as well as used in the treatment of childhood cancers, especially hematological cancers. Diseases with HLA typing in embryos include Acute Lymphoblastic Leukemia (ALL), Chronic Myeloid Leukemia (CML), Aplastic Anemia, X-linked Adrenoleukodystrophy (X-ALD), Blackfan-Diamond Anemia (DBA), K-ras, Burkitt’s Lymphoma, Congenital Neutropenia, Histiocytosis, Hurler Syndrome, JMML, Hyper Immunoglobulin M Syndrome, Myelodysplastic Syndrome (MDS), Hemophagocytic Syndrome, Non-Hodgkin Lymphoma (NHL), Glanzmann Disease, Neuroblastoma, FERMT3 (Leukocyte Adhesion Deficiency), Beta Thalassemia, CD3 deficiency, Fanconi Anemia, Alpha Mannosidosis, Blackfan-Diamond Anemia (DBA), Wiskott-Aldrich Syndrome (WAS).

Non-invasive PGT

Since any embryo biopsy performed at any time with any available method is an invasive procedure, we call obtaining embryonic samples with different methods without performing embryo biopsy non-invasive PGT. Non-invasive tests not only prevent embryo damage but also provide the opportunity for genetic research that does not require an experienced specialist. Non-invasive PGT methods: 1) Blastocyst Aspiration: Minimal invasive aspiration of blastocyst fluid, 2) Analysis of depleted culture medium. The disadvantage of this method is its weak diagnostic power because DNA amplification may be insufficient. The advantage is that it is not an invasive procedure.

What factors affect the efficiency of PGT?

1) Ovarian stimulation in IVF treatments

2) DNA source for PGT and biopsy methods

3) Embryo selection

How should the follow-up of pregnancies be after the transfer of embryos selected with PGT method?

In pregnancies obtained with embryos found to be healthy after the PGT method, prospective parents should be informed about prenatal tests. Because even if PGT is performed, prenatal tests and the follow-up of pregnancy with 2nd level ultrasonography are recommended for these pregnancies. Amniocentesis should be recommended in cases of PGT-M (Single Gene Disorders) and cases where mosaic embryo transfer is performed.

Whole Exome Sequencing (WES) is performed for whom?

– Couples experiencing mature egg problems, fertilization issues, or embryo development problems in repeated IVF attempts,

– Families with inherited diseased children, for the purpose of identifying the genetic mutation causing this disease,

– In multifactorial diseases that can arise due to various genetic factors such as cardiac hypertrophy, Brainstem Ataxia (cerebellar ataxia), for the purpose of identifying genetic mutations,

– To determine the genetic mutation causing undiagnosed inherited diseases.

Who is Whole Genome Sequencing (WGS) performed for?

– It is applied to patients for whom diagnosis cannot be made with the WES test. The difference between WGS and WES is that WGS also analyzes the intronic regions of genes and the DNA located in mitochondria.

What is a Mosaic Embryo?

The definition of chromosomal mosaicism is the presence of cells with two (or more) different chromosomal structures together. Mosaicism arises from mitotic events during postzygotic development.

How often is mosaicism seen in embryos?

Mosaicism is the most common abnormality in pre-implantation embryos. Mosaicism arises due to non-separation, as most mosaic embryos have monosomic and trisomic blastomeres among normal cells. This finding leads to speculation about the accuracy of embryo biopsy because taking a biopsy from only one cell that gives a normal result does not provide information about the rest of the embryo. Due to the increase in false positive/false negative results in approximately 40-60% of mosaic embryos, some clinicians are not inclined to perform biopsy, especially in cases of division at the biopsy stage for PGT-A. Clearly, more research is needed in this area to fully understand the importance of mosaic embryos in PGT.

What is Genetic Screening?

Genetic screening is a screening method that helps determine disease risks by analyzing genetic material.

What methods are used for genetic screening?

Genetic screening methods include examining genetic material using various genetic tests and analyses. Next-Generation Sequencing (NGS) is the most commonly used method for the genetic diagnosis of both chromosomal and single gene disorders.

What are the advantages of genetic screening?

Genetic screening offers many advantages in terms of determining disease risks and planning treatment. These advantages include early diagnosis, determining treatment options, and providing genetic counseling services.

What is the purpose of Preimplantation Genetic Testing (PGT)?

PGT is used for many purposes. It is possible to increase pregnancy rates and minimize the risk of fetal aneuploidy by selecting genetically healthy embryos in couples undergoing IVF treatment due to infertility. Additionally, with PGT, the risk of transmitting inherited diseases to their children is eliminated for couples carrying a hereditary disease.

How often is mosaicism seen in embryos?

Mosaicism is quite common during early pre-implantation embryo development; it is estimated to occur in approximately 10-30% of embryos, and according to some sources, it can be present in up to 50% of all embryos. However, the frequency of mosaicism decreases in later stages of pregnancy. It is estimated that the frequency of mosaicism in live births is less than 0.2%. It is predicted that maternal age does not affect mosaicism.