Molecular Cytogenetic Methods

What are molecular cytogenetic methods?

Molecular cytogenetics is the numerical and structural examination of chromosomes.

Its advantage over classical cytogenetic methods is that microdeletion, duplication or translocations that cannot be distinguished by the classical method can also be distinguished by molecular methods.

This main heading includes methods such as FISH (Fluorescent In Situ Hybridization), ACGH (Array Comparative Genomic Hybridization) and NGS (Next Generation Sequencing).

With the application of molecular cytogenetic methods in the embryo, the embryos are tested chromosomally before they are transferred into the uterus.

For this reason, the application of these tests in the embryo is called preimplantation genetic test (PGT) – genetic test performed before implantation.

PGT-A in the subheadings of this test means PGT for aneuploidy.

In other words, whether the chromosome number 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.

With PGT-M, which is another topic in the PGT methods, monogenic, that is, single gene diseases such as SMA, thalassemia and neurofibromatosis, can be distinguished, while embryos are tested before transfer in couples with chromosomal translocations by PGT-SR method. It is possible to achieve healthy pregnancies and live births.

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

In IVF procedures, the main goal is not only to achieve pregnancy, but also 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 the selection of embryos.

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

advanced maternal age

History of recurrent pregnancy loss

Recurrent IVF failure

Severe male factor

A known single gene disease carrier

A known translocation carrier

A history of fetal structural anomaly or chromosomal disease in previous pregnancies

Of course, all couples other than this group can shorten the period of reaching pregnancy or optionally any of these methods can be applied.

What are molecular cytogenetic methods?

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What is FISH?

Fluorescent's Situ hybridization (FISH) method is a molecular technique used to detect specific DNA fragments in cells.

In this method, fluorescent dyes are used to make the DNA present inside the cells visible.

In this way, it can be determined where certain DNA fragments in the cells are located and how dense they are.

The FISH method is used to control the chromosome number and structure of embryos.

With the FISH method, it is possible to evaluate 3 or 5 pairs of chromosomes from 23 pairs of chromosomes, including sex chromosomes.

Thus, it is possible to diagnose the sex of the embryo and the diagnosis of special syndromes such as monosomy or trisomy 13, 18, 21.

In addition, in order to evaluate the genetic structure of the embryo and to ensure the selection of embryos with a healthy chromosome structure in individuals with translocation carriers, it is possible to choose embryos with the use of region-specific probes and to choose embryos with the FISH method.

It takes approximately 24 hours to obtain the results of biopsies taken with the FISH method.

Therefore, it is possible to make fresh transfers in the same cycle after the results are obtained.

What is the CGH method? / What is comparative genomic hybridization?

The ACGH (Array Comparative Genomic Hybridization – Comparative Genomic Hybridization) method is a technique used to examine the structure and order 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 known DNA structure.

In IVF treatment, the ACGH method is used to examine the structure and order of the chromosomes of the embryos.

The structure and order of the chromosomes of the embryos contribute to the development and health of the embryo, as well as the health progress of the pregnancies that will occur.

For this reason, the healthiest and best developmental embryos are selected by using the ACGH method during the selection of embryos in IVF treatment.

Thus, the success rate of IVF treatment is tried to be increased.

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

What is NGS?

NGS (Next Generation Sequencing) is the newest known technique among preimplantation genetic testing (PGT) methods today.

Thanks to the new 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 breaking down 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 recognition of chromosomal mosaicism in embryos has also increased.

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

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 cells sampled from the embryo, cells with normal and abnormal chromosome numbers coexist. Depending on the mosaicity rate and type detected in the embryo, the couple can be transferred if they do not have another normal embryo.

In embryo transfers carried out without molecular cytogenetic methods, if the embryo is structurally aneuploid, that is, abnormal, three potential results can be encountered if this situation is not known.

The embryo does not attach to the uterus at all, that is, pregnancy cannot be achieved.

End of pregnancy in early gestational weeks, despite the embryo's attachment to the uterus

Pregnancy of pregnancy, but detection of anomalies in the fetus in screening or diagnostic tests in later gestational weeks.

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, but cannot be completely ignored.

For this reason, apart from the above-mentioned indications, couples have the chance to have their embryos done by their own will.

However, it should not be forgotten that these tests do not distinguish between chromosomal diseases and additional tests should be done to screen these diseases.

When the NGS technique is applied, the results are obtained in about 14 days.

For this reason, after the biopsy procedure, the embryos are frozen and the results are expected to be obtained.

When the result is achieved, a preparation period that takes about 20 days is entered for the transfer of frozen embryos into the uterus.

When the intrauterine preparation is completed, the embryos known to have a healthy chromosome structure are dissolved and transferred into the uterus.