Molecular Cytogemetic Methods

What is Molecular Cytogenetics?

What are molecular cytogenetic methods? Molecular cytogenetics is the numerical and structural study of chromosomes. The advantage over classical cytogenetic methods is that microdeletion, duplication or translocations, which cannot be distinguished by the classical method, can also be distinguished by molecular methods. Within this main heading, there are methods such as FISH (Fluorescent In Situ Hybridization), aCGH (arrray Comparative Genomic Hybridization) and NGS (Next Generation Sequencing). With the application of molecular cytogenetic methods in the embryo, the embryos are tested chromosomally before being transferred into the uterus. For this reason, the application of these tests in the embryo is called preimplantation genetic test (PGD) – the genetic test performed before implantation.

PGT-A, which is included in the subheadings of this test, means PGD for aneuploidy. In other words, whether the number of chromosomes in the embryo is normal or not is determined by the PGT-A method. Thus, many chromosomal problems such as trisomy 21 (Down Syndrome) are recognized before the embryo is transferred into the uterus. While monogenic, i.e. single gene diseases such as SMA, thalassemia and neurofibramotosis can be distinguished with PGT-M, which are the other two headings in PGD methods, it is possible to reach healthy pregnancies and live births by testing embryos before transfer in couples with chromosomal translocations with PGT-SR method.

For whom are molecular cytogenetic methods and genetic tests applied in the embryo suitable?

In IVF procedures, not only pregnancy is achieved, but also the main goal is to continue the pregnancy with health and to reach a healthy live birth. For this, the selection of the right embryo before embryo transfer is of great importance. In addition to techniques such as blastocyst culture, embryoscope use, artificial intelligence, preimplantation genetic tests and molecular cytogenetic methods can be used in embryo selection.

All of these methods are recommended especially for the following groups after taking a detailed history from the couples:

Advanced maternal age
A history of recurrent pregnancy loss
Recurrent IVF failure
Severe male factor
Carrier of a known single gene disease
A known translocation carrier
Fetal structural anomaly or a history of chromosomal disease in previous pregnancies

Of course, all couples except this group can be applied to shorten the time to reach pregnancy or optionally any of these methods.

What is NPP?

NGS (Next Generation Sequencing) is the newest technique known among preimplantation genetic testing (PGD) methods today. Thanks to the NGS – Next Generation Sequencing technique, it is possible to detect structural and numerical abnormalities in embryos.

In this technique, firstly, the DNA of the obtained sample is amplified, then the DNA sequences obtained by enzymatically disintegrating are read. Thus, the total number of readings for each embryo is determined. This method is a more precise measurement technique than the methods used before and the accuracy rate is much higher. Thanks to this method, the chance of recognizing chromosomal mosaicism in embryos has also increased.

What are molecular cytogenetic methods?

What is FISH?

The Fluorescent In Situ Hybridization (FISH) method is a molecular technique used to detect specific pieces of DNA in cells. In this method, fluorescent dyes are used to make the DNA inside the cells visible. Thanks to this, it can be determined where certain pieces of DNA in cells are located and how dense they are.

In IVF treatment, the FISH method is used to control the number and structure of chromosomes of embryos. With the FISH method, it is possible to evaluate 3 or 5 pairs of chromosomes, including sex chromosomes, from 23 pairs of chromosomes. Thus, it is possible to determine the sex of the embryo and to diagnose special syndromes such as monosomy or trisomy 13, 18, 21.
In addition, in order to evaluate the genetic structure of the embryo in individuals with translocation carrier and to ensure the selection of embryos with healthy chromosome structure, it is possible to use region-specific probes and to make embryo selection with FISH method.

It takes about 24 hours to obtain the results of biopsies taken by the FISH method. Therefore, after the results are obtained, it is possible to make a fresh transfer in the same cycle.

What is the CGH method? / What is Comparative Genomic Hybridization?

The aCGH (Array Comparative Genomic Hybridization) method is a technique used to study the structure and arrangement of DNA in the chromosomes of cells. This method can provide numerical and structural examination of all 23 pairs of chromosomes in the embryo. The basis of the method is based on the comparison of the DNA structure of the embryo with the DNA structure known to be normal.

In IVF treatment, the aCGH method is used to examine the structure and arrangement of the chromosomes of embryos. The structure and order of the chromosomes of the embryos contribute to the development and health of the embryo as well as to the health of the pregnancies that will occur. Therefore, during the selection of embryos in IVF treatment, the embryos with the healthiest and best development potential are selected with the use of the aCGH method. Thus, the success rate of IVF treatment is tried to be increased.

Today, instead of aCGH, the use of NGS (Next Generation Sequencing) has come to the fore as the latest technique in the examination of the chromosomal structure of embryos.

As a result of the NGS test, embryos are given 3 different results.

Euploid (normal) - There are 46 chromosomes in the embryo and the chromosomes are structurally normal. These embryos can be transferred.
Aneuploid (abnormal): The number of chromosomes in the embryo is different from 46. These embryos cannot be transferred.
Mosaic: In the cells sampled from the embryo, there are cells with normal and abnormal chromosome counts. Depending on the mosaicism rate and type detected in the embryo, it can be transferred if the couple does not have any other normal embryos.

In embryo transfers performed without molecular cytogenetic methods, if the embryo is structurally aneuploid, that is, abnormal, but this situation is unknown because the test is not performed, 3 potential results may be encountered. These results can be listed in order of frequency as follows:

The embryo does not attach to the uterus at all, that is, pregnancy cannot be achieved
Termination of pregnancy in early gestational weeks despite the attachment of the embryo to the uterus (Biochemical pregnancy formation, anembryonic – empty pregnancy, miscarriages, fetal heartbeat arrest)
Formation of pregnancy, but detection of anomalies in the fetus in screening or diagnostic tests in the following gestational weeks

In order to avoid all these negative consequences, many couples today often turn to preimplantation genetic diagnosis methods. In cases where the maternal age is young and donor oocytes are used, the chromosomal abnormality rate is extremely low and cannot be completely ignored. For this reason, in addition to the indications mentioned above, couples have the opportunity to have the tests listed above on their embryos with their own will.

However, it should be kept in mind that these tests do not distinguish gene diseases other than chromosomal diseases (such as SMA, Thalassemia, Cystic Fibrosis) and additional tests should be performed to screen for these diseases.

When the NPP technique is applied, the results are concluded in approximately 14 days. Therefore, after the biopsy procedure, the embryos are frozen and the results are expected to be obtained. When the result is obtained, a preparation period of about 20 days is entered for the frozen embryos to be thawed and transferred into the uterus. When the intrauterine preparation is completed, the embryos known to have a healthy chromosome structure are thawed and transferred into the uterus.

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