Preimplantation Genetic Testing

I. PREVIOUS INFORMATION FOR PATIENTS

Our personal or family history indicates that there is a risk of transmitting a hereditary condition or severe chromosomal alteration to our offspring.

Therefore, the medical team attending to us has advised us that, in our specific case, one of the medical alternatives to significantly reduce these risks is to include our pre-embryos/embryos1 in the preimplantation genetic diagnosis program.

We have been informed that the risk of such a genetic condition/chromosomal alteration can be reduced by performing genetic analysis on our pre-embryos generated through in vitro fertilization. Additionally, we have been informed about the diagnostic procedures and methods that can be used to analyse the pre-embryos, the chances of success, and the limitations and risks associated with this type of testing.

WHAT DOES PREIMPLANTATION GENETIC TESTING (PGT) INVOLVE?

Preimplantation Genetic Testing (PGT) is a type of genetic analysis conducted on the pre-embryo before its implantation in the uterus. PGT is performed on patients at risk of transmitting chromosomal or genetic abnormalities to their offspring, aiming to improve the selection of only unaffected pre-embryos for transfer to the uterus.

The PGT technique involves the combination of:

• Pre-PGT studies, where applicable.
• In vitro fertilization.
• Biopsy of pre-embryonic cells through micromanipulation.
• Genetic diagnostic techniques using molecular genetics methods.
• Pre-embryo transfer.

II. WHEN IS PGT INDICATED?

This technique is indicated for the detection of pre-embryos carrying serious hereditary diseases, structural or numerical chromosomal abnormalities that could have a significant impact on the quality of life and/or life expectancy of the future newborn, as well as the successful implantation of such pre-embryo.

III. PROCEDURE

A. Pre-PGT Phase (Pre-PGT).
In this phase, genetic characterization tests for the specific genetic alterations to be diagnosed are performed in patients carrying the genetic condition. The aim is to gather maximum information before applying PGT in relevant cases.

B. Obtaining Pre-Embryos.
The objective of this step is to obtain the pre-embryos for analysis. Assisted reproductive techniques, such as in vitro fertilization (IVF), are employed for this purpose. This is necessary even if patients do not have any reproductive abnormalities, as no other method of obtaining pre-embryos is allowed at this early stage of development. In some cases, if there are cryopreserved (vitrified) untested pre-embryos from previous cycles, they may need to be used for analysis. In such cases, the pre-embryos must be thawed (de-vitrified) and their viability confirmed before biopsy.

C. Pre-Embryo Biopsy.
The biopsy is usually performed on the fifth, sixth, or seventh day after fertilization when the pre-embryo is in the blastocyst stage. The embryonic biopsy involves extracting approximately five cells from the trophoectoderm of the pre-embryo. As mentioned earlier, the biopsy can be performed on pre-embryos generated in the current IVF cycle or on cryopreserved pre-embryos from previous cycles. Regardless, once the biopsy is done, the pre-embryos will be vitrified until the analysis results are obtained.

D. Genetic Diagnosis.
The cells obtained from the biopsy will undergo genetic analysis. Depending on the clinical indication, different analysis strategies in PGT may be followed, or even a combination of them. These strategies are briefly explained below:

1. Preimplantation Genetic Diagnosis for Aneuploidies (PGT-A). In patients with an indication to analyse numerical chromosomal abnormalities, the test used will be Preimplantation Genetic Testing for Aneuploidies (PGT-A). This technique allows determining the number of copies of each of the 23 pairs of chromosomes in the pre-embryo samples and identifying both the pre-embryos negative for chromosomal aneuploidy (no alteration in the number of chromosomes) and those positive for aneuploidy (with an alteration in the number of chromosomes) (aneuploids). PGT-A has been shown to detect all whole-chromosome aneuploidies and certain segmental aneuploidies. Additionally, some abnormalities involving a complete set of 23 extra or missing chromosomes (triploidy or haploidy) can be detected. PGT-A will be performed using a method called Next Generation Sequencing (NGS) through the PGTseq platform. The embryonic biopsy will be performed at the blastocyst stage.
   
   In some cases, there may be a need for the combined analysis of a monogenic disease and aneuploidies. In such cases, the blastocyst biopsy will be conducted, and both types of analysis can be performed on the same biopsy sample.
2. Preimplantation Genetic Diagnosis for Structural Chromosomal Abnormalities (PGT-SR). In patients where the indication is a structural chromosomal abnormality, such as chromosomal translocations or inversions, Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) is used to identify pre-embryos that are negative/balanced for the chromosomal segments involved in the rearrangement. The embryonic biopsy will be performed at the blastocyst stage. Similar to PGT-A, PGT-SR will be performed using Next Generation Sequencing (NGS) with the PGTseq platform.
   
   The number of copies of the remaining chromosomes not affected by the structural rearrangement will also be analysed. In other words, in addition to the chromosomes affected by the structural alteration, the rest of the chromosomal set will be examined for the detection of aneuploidies.
3. Preimplantation Genetic Diagnosis for Monogenic Disorders (PGT-M). In patients where the indication is a monogenic disease, Preimplantation Genetic Testing for Monogenic Disorders (PGT-M) is a molecular diagnostic technique that allows the identification of pre-embryos that are genetically normal with respect to the specific variant and gene being analysed. This enables the distinction of pre-embryos that have inherited the genetic alteration associated with the monogenic disease. PGT-M can be performed using the PGTseq-M method or alternatively, Karyomapping along with the study of familial mutations when possible. The embryonic biopsy will be performed at the blastocyst stage. The chromosomal analysis PGT-A is conducted in addition to PGT-M and is intended to reveal embryos that have an incorrect number of chromosomes in their cells.

E. Pre-embryo Transfer. The medical team at the centre will decide which pre-embryos will be transferred to the patient after considering the chromosomal or genetic makeup and viability of the pre-embryos.

IV. RESULTS

The results of genetic assays and tests should be interpreted in the context of additional laboratory test results, family history, and other clinical findings. Genetic counselling is recommended to analyse the implications of these test results.

Despite the high reliability of PGT, the technique has inherent limitations. Therefore, in any pregnancy obtained after PGT, there is an indication to offer a confirmatory prenatal study as PGT testing should not be considered a substitute for prenatal testing. It is recommended to discuss this point with your maternal-fetal medicine team in the case of an on-going pregnancy.

The overall efficiency of PGT depends on factors such as the number of available pre-embryos, their developmental stage, and the effectiveness of the cytogenetic or molecular diagnostic method used. Furthermore, when PGT is employed to detect a monogenic disease, the final outcome will be influenced by the inheritance pattern of the gene (recessive or dominant) and the number of healthy pre-embryos available at the end of the process.

In general, the average pregnancy rate per embryo transfer in PGT treatments ranges between 50% and 60%. These rates largely depend on the patient’s age, embryo quality, and underlying causes that led to the treatment indication. Different PGT techniques have been used for over 25 years, and no abnormalities associated with their use have been reported in the literature, suggesting that the procedure is safe.

The possible results in PGT-A cases may include:

• Negative: No complete chromosome aneuploidies or segmental abnormalities were detected. This result indicates that no extra or missing complete chromosomes or segmental abnormalities were detected.
• Positive: Complete chromosome aneuploidies and/or segmental abnormalities were detected. This result indicates that at least one complete chromosome is present in excess or is missing, and/or segmental abnormalities were detected.
• No Result: Refers to a failure in DNA amplification or inconclusive results. In these cases, a new biopsy is recommended to obtain another sample for analysis, provided that the pre-embryo quality allows this.

The possible results in PGT-SR cases may include:

• Negative/Balanced: Pre-embryos in which a normal number of chromosomes (46,XX or 46,XY) is predicted, or a balanced chromosomal rearrangement is detected in the biopsy sample (since the technology used does not differentiate between these two states).
• Positive: Pre-embryos in which an abnormal number of chromosomes is predicted in the biopsy sample. These are pre-embryos for which a high risk of chromosomal abnormality has been determined.
• Positive/Unbalanced: Pre-embryo that has inherited the structural chromosomal alteration in an unbalanced state. These pre-embryos show gains and/or losses of chromosomal fragments related to the structural alteration carried by the patient.
• No Result: Refers to a failure in DNA amplification or inconclusive results. In these cases, a new biopsy is recommended to obtain another sample for analysis, provided that the pre-embryo quality allows this.

The possible results in PGT-M cases may include:

• Negative: Pre-embryos that are not expected to have inherited the genetic alteration associated with the monogenic condition. Additionally, no chromosomal abnormalities have been identified.
• Positive: Pre-embryos that are expected to have inherited the genetic alteration associated with the monogenic condition. This category also includes embryos with chromosomal abnormalities.
• Carrier: Pre-embryos that are expected to be healthy carriers of the monogenic disease under study. This applies to autosomal recessive and X-linked recessive diseases. Additionally, no chromosomal abnormalities have been identified.
• No Result: Refers to a failure in DNA amplification or inconclusive results. In these cases, a new biopsy is recommended to obtain another sample for analysis, provided that the pre-embryo quality allows it.
• Non-Informative: The accuracy of the test depends on the results obtained in Karyomapping or PGTseq-M, with or without analysis of the mutation site. If there is a recombination between the mutant gene and the linked polymorphisms, this can also lead to inconclusive results in PGT-M, compromising the accuracy of the test and resulting in the pre-embryo being classified as “non-informative.” The genetic status of a pre-embryo with a “non-informative” status is unknown. In such cases, a second biopsy is not recommended.

V.LIMITATIONS OF PGT. SO-CALLED “NON-INFORMATIVE” PRE-EMBRYOS. INCIDENTAL FINDINGS

Common Limitations of all PGT Tests (PGT-A, PGT-SR, PGT-M)

It is crucial to avoid unprotected sexual intercourse from 15 days prior to egg retrieval until after the pregnancy test, which is performed approximately two weeks after the embryo transfer to the uterus. Sexual intercourse during this time could lead to a natural pregnancy from an untested embryo, invalidating any PGT results.

PGT minimizes the possibility of transferring embryos carrying the chromosomal and/or genetic alteration under study. Like any diagnostic medical technique, there is a margin of error in the test, estimated to be between 1-2% theoretical possibility of diagnostic error in the genetic status of the embryo. Therefore, in any pregnancy obtained after PGT, there is an indication to offer a confirmatory prenatal study as PGT testing should not be considered a substitute for prenatal testing. It is recommended to discuss this point with your maternal-fetal medicine team in the case of an on-going pregnancy. While highly unlikely, there is a possibility that a biopsy sample may be lost or damaged at some point in the clinic, during transport, or in the laboratory. In such cases, a new embryo biopsy will be necessary, provided that the pre-embryo quality allows for this.

Like any other laboratory technique, PGT can be affected by errors that can compromise the obtained result. Common sources of these errors are associated with human errors during sample collection and processing, errors in laboratory equipment and materials, contamination of samples by other cells or external genetic material, or non-compliance with established pre-analytical conditions to ensure the validity of the results obtained.

PGT does not offer any guarantee of achieving a pregnancy or having a healthy child (free from all genetic or non-genetic defects).

Since PGT does not analyse all types of chromosomal or genetic abnormalities, it cannot exclude the possibility that an embryo may have other types of genetic abnormalities and/or birth defects. In the general population, there is a 3-5% risk of a child being born with a birth defect or intellectual disability due to genetic and/or non-genetic causes. The use of PGT does not reduce that risk.

There is a possibility of not obtaining a result from a biopsy sample, which will be classified as “no result.” This can happen if the cells extracted from the embryo contain degraded DNA, as well as due to other technical limitations. This typically affects less than 5% of the samples. If the final result is “no result,” the medical team will advise against transferring the embryos to the uterus. In such cases, a rebiopsy of the embryo may be recommended if its quality allows for it.

PGT results may indicate an intermediate number of chromosomes, also known as “mosaic embryos.” Mosaicism refers to a combination of chromosomally normal and abnormal cells in a single pre-embryo biopsy sample. Embryo biopsies in this category have at least one complete chromosome or a segment of a chromosome falling within the mosaic range. Juno Genetics does not routinely report the presence of mosaicism in a biopsy. According to current scientific evidence, these embryos have the same implantation potential and ability to generate a live newborn as embryos without mosaicism. Therefore, mosaic findings are considered secondary and of uncertain significance. Ultimately, the decision to report mosaic findings will be made by the medical team, who may request Juno Genetics to report mosaic pre-embryos.

In any case, the final clinical recommendation regarding the selection of embryos for transfer to the uterus will be the responsibility of the medical team.

Common Limitations of PGT-A and PGT-SR Tests

This test does not search for any hereditary/genetic or non-genetic conditions within a person’s family history.

In PGTseq-A and PGTseq-SR, all 23 pairs of chromosomes are analysed, and most abnormalities in the number of copies or complete loss of a set of chromosomes (complete haploidy) can be detected. However, certain types of abnormalities cannot be detected, such as some forms of polyploidies (e.g., tetraploidies like 92,XXXX).

Another class of abnormalities that may not be detected are related to losses or duplications of small fragments of chromosomes, known as segmental abnormalities. In general, segmental aneuploidies below 3Mb are not detected. However, the detection limits for segmental aneuploidies vary depending on the chromosome and the quality of the embryonic sample. The probability of a segmental aneuploidy being present in the fetus cannot be predicted.

The detection of uniparental disomies, where both sets of chromosomes come from the same parent instead of one from the father and one from the mother, cannot be guaranteed.

The PGT platform (PGTseq) was validated using embryos generated through Intracytoplasmic Sperm Injection (ICSI). The use of conventional insemination may increase the risk of contamination from maternal or paternal sources. If undetected contamination occurs, it can result in a false negative or false positive.

The PGTseq platform cannot detect all segmental aneuploidies or copy number variants (CNVs). A “Negative” result does not eliminate the risk of a segmental aneuploidy. It is recommended that patients meet with a genetic counsellor and consider the possibility of confirmatory prenatal diagnosis. Most copy number variants (CNVs) identified prenatally and postnatally will not be detected by PGT-A as they are below the detection limit.

Breakpoints of segmental aneuploidies are not precisely determined using PGTseq. The deleted/duplicated chromosome segment may be smaller or larger than indicated in the PGT-A and PGT-SR report. Given this limitation, Juno Genetics does not provide a classification of the clinical significance of segmental aneuploidies.

Occasionally, the results of PGT-A and SR may indicate a chromosomal abnormality of parental origin in one of the couple members, such as a parental chromosomal rearrangement or extra/missing chromosomal material. This type of result is considered an incidental finding. If the results suggest a chromosomal abnormality in the parents, this result will be communicated to the patients. Additional genetic testing may be required in response to such results.

Specific limitations of the PGT-SR Test

While losses and duplications of chromosome fragments can generally be detected within pre-embryos, it is not possible to distinguish pre-embryos that have a balanced form of rearrangement (the same situation as the parent carrying the rearrangement) from those with a completely normal set of chromosomes. This is because in these two situations, the amount of chromosomal material is the same.

The accuracy of PGT-SR depends on the genetic information provided to Juno Genetics in medical records and reports from previously conducted genetic tests. The information provided to Juno Genetics will be evaluated to determine if the PGTseq-SR method could detect unbalanced products derived from the rearrangement. Incorrect definition of chromosomal breakpoints and/or errors in the family history information provided to Juno Genetics may affect the ability of the PGT-SR test to detect unbalanced rearrangement products.

PGTseq-SR will only be able to detect unbalanced products of the specific chromosomal rearrangement within the records provided to Juno Genetics. The accuracy for detecting unbalanced products of the rearrangement is >98%, assuming that the karyotype information provided to Juno Genetics is accurate.

This test reduces, but does not eliminate, the risk of an unbalanced rearrangement in embryos identified as “negative/balanced”.

Specific limitations of the PGT-M Test

The procedures performed for PGT-M focus on the identification of specific inherited genetic disorders, according to the indication for the test. However, the technique used for PGT-M allows for the detection of chromosomal status information of the pre-embryo. This information will be provided to patients whenever available. Chromosomal abnormalities frequently occur in the human pre-embryo and have the potential to cause implantation failure or miscarriage. In any case, the final clinical recommendation regarding the selection of embryos to transfer to the uterus will be the responsibility of the medical team.

This test does not rule out the possibility of other variants in the studied gene, including de novo variants.

The PGT-M test used is specifically designed to analyse the indicated region/gene of interest stated in the test request. Other additional genes/regions will not be studied.

The accuracy of PGT-M for the detection of a disorder caused by a mutation in a single gene is estimated to be at least 95%, depending on the methodology used. However, it is important to note that the risk of an affected child/pregnancy after transferring a pre-embryo predicted to be “normal” or “carrier” is not zero. The intention of the test is not to guarantee an unaffected pregnancy or delivery, but to reduce the risk of transferring an affected pre-embryo to the uterus. Despite the high reliability of PGT-M test, there are inherent limitations to the technique. Therefore, there is an indication to offer a confirmatory prenatal study as PGT testing should not be considered a substitute for prenatal testing. It is recommended to discuss this point with your maternal-fetal medicine team in the case of an on-going pregnancy.

The use of intracytoplasmic sperm injection (ICSI) as a fertilization method is highly recommended for PGT-M cases, as it helps reduce the risk of DNA contamination caused by sperm. If contamination is present but goes undetected, a misdiagnosis can occur.

VI.ALTERNATIVES TO THE PGT TECHNIQUE

• Natural conception followed by prenatal diagnosis, provided that the couple is willing to consider voluntary termination of pregnancy if fetal abnormalities are detected.
• Use of donor gametes (eggs or sperm) from a non-affected individual, depending on who is the carrier of the disease.
• Legal adoption.