This section is written in partnership with UR Vistahermosa experts from Spain.

Non-invasive preimplantation genetic test:

Can we forget about an embryo biopsy?

In recent years, a great deal of progress has been made in the field of reproductive medicine with new strategies emerging aimed at a common goal: to achieve the birth of a healthy baby. These emerging techniques in the biomedical field include the non-invasive preimplantation genetic test (PGT). Its purpose is to obtain genetic information from the embryo in the least harmful way possible before it is transferred to the maternal uterus.

To do this, the first step is to carry out the technique of in vitro fertilisation (IVF), which consists of combining the oocyte and the sperm in the laboratory. This treatment is indicated in various cases, such as advanced maternal age, low sperm count, ovulation problems, or when other, simpler, assisted reproduction techniques have failed.

If the fertilisation process is successful, the fertilised egg will begin to be divide and will result in an embryo. The embryos are cultivated in special incubators in the embryology laboratory. Here, embryologists regularly monitor their development, selecting the highest quality embryos for transfer to the maternal uterus or for vitrification for later use.

Until a few years ago, embryo selection was carried out exclusively on the basis of embryo morphology, which takes into account parameters such as the number of cells, their size and the percentage of fragmentation. However, morphology is not always related to the embryo’s chromosome endowment.

Human cells are made up of 46 chromosomes, structures that contain our genetic information, our DNA. Half of them are inherited from our father, and the other half f rom our mother. We say that an embryo is euploid when all its cells contain 46 chromosomes, while an aneuploid embryo is the one has too many or too few chromosome copies.

Today, we know that aneuploid embryos are common and that it depends on – among other factors – maternal age. For instance, some 25% of embryos from 30-year-old women are aneuploid. This proportion increases up to 50% at 40 years (Franasiak JM et al., Fertil Steril, 2014).

Aneuploid embryos are not compatible with life; the vast majority of them end up in miscarriages or do not implant. We can select the best embryo based on its morphology during in vitro fertilisation techniques, but it may be aneuploid. This means that there will be a greater risk of implantation failure, a miscarriage or even having a child with serious abnormalities.

Genetic study of the embryo

The only way we currently have to minimise the risk of transferring aneuploid embryos is to perform a genetic study that will determine the number of embryo chromosomes in the preimplantation phase; in other words, before it is transferred to the maternal uterus. This study is called PGT-A (Preimplantation Genetic Testing for Aneuploidy). It is used particularly in cases of advanced maternal age, repeated miscarriages and recurrent implantation failures, among others.

This genetic test is performed from three to five days after fertilisation. First, it is necessary to perform an embryo biopsy, that is, to extract one or more cells from each embryo. This material is then analysed in the laboratory to identify the embryos with correct chromosome loads. Therefore, they have a greater chance of resulting in a normal pregnancy.

Embryo biopsy

The embryo biopsy is a crucial step during the PGT. It can be performed on the third day after fertilisation, when the embryo has six to eight cells. In this case, a small opening is made in the so-called “zona pellucida” (the membrane surrounding the embryo), where a cell is extracted for analysis. However, embryo biopsy is currently usually performed on Day 5 or 6 after fertilisation. This phase is called the blastocyst phase, and the embryo is made up of more than one hundred cells. In this case, a small group of cells is extracted from the trophectoderm, the outer cell layer of the blastocyst that will become the placenta.

The blastocyst stage biopsy offers important advantages compared to the biopsy on Day 3. On the one hand, we can obtain a greater quantity of genetic material, since we extract a larger number of cells; on the other, since the embryo contains more cells, it is less harmful. It also allows embryo mosaicism (embryos made of euplid and aneuplid cell lines) to be detected.

Advantages and limitations

Like all techniques, the PGT-A has some advantages and some limitations. Compared to the IVF cycles where the PGT has not been performed, the technique has benefits that include:

  • Improving implementation rates
  • Reducing miscarriage rates
  • Improving pregnancy by transfer rates
  • Reducing the risk of having a baby with a chromosome anomaly

On the other hand, it does have some limitations:

  • It is an invasive method in which the vast majority of embryos survive the biopsy, although there is a small possibility that the embryo will not develop.
  • It is a complex technique that requires specific equipment and personnel with extensive experience.
  • There is a possibility of misdiagnosis due to embryo mosaicism as only one of the embryo cell lines is detected.

Non-invasive PGT

In 2016, the detection of free DNA in the middle of blastocyst culture was published and suggested as a tool for determining the embryo chromosome load in a non-invasive manner. This DNA is released by the embryo into the culture medium during its in vitro development by mechanisms that are not yet entirely clear. In recent years, several studies have shown the ability to detect, extract and amplify DNA from the embryo culture medium, especially in the blastocyst phase, and its potential clinical application for non-invasive PGT has been evaluated.

Currently, there is some controversy about the usefulness of the non-invasive PGT. However, the latest publications indicate that, when DNA contamination from maternal cells is eliminated and the procedures in the embryology laboratory are optimised, the DNA released by the embryo is a good indicator of its chromosome load (Huang et al., Proc Natl Acad Sci USA, 2019; Chen et al., Front Cell Dev Biol, 2021).

How is the non-invasive PGT done?

The protocol is much simpler than the traditional PGT, as there is no need to perform an embryo biopsy. The basic steps are as follows:

  • On Day 3 or 4 after fertilisation, each embryo is washed and transferred to a new culture medium, where it is incubated until Day 5 or Day 6.
  • During this period of time, the embryo releases free DNA into the medium.
  • On Day 6, the embryos are vitrified and the culture medium is collected and analysed to detect the free DNA and identify possible aneuploidy.

The non-invasive PGT has several advantages over the traditional PGT:

  • The risk of potential embryo damage after the biopsy is eliminated.
  • The protocol is much simpler.
  • The cost of specific equipment is reduced.

However, it also has some limitations:

  • The origin of the free DNA of the cells is still unclear. There is still some controversy as to whether the free DNA released to the culture medium is representative or not of the chromosome composition of the entire embryo.
  • It is necessary to minimise the risk of contamination with maternal DNA, which may lead to a misdiagnosis.
  • It should be validated in each laboratory. This entails the optimisation and standardisation of the culture conditions and the protocols for recovering it from the culture medium to obtain a sufficient quantity of free DNA and to avoid contamination with maternal DNA.

Despite the limitations of the technique, the information provided by the non-invasive PGT can be used as a system for prioritising the identification of the embryos with the greatest possibility of being euploid, thereby increasing the likelihood of achieving a normal pregnancy.

The non-invasive PGT is a technique with great potential and with some limitations that are expected to be resolved in the near future.

Article source:
This article has been published in Creando Familias magazine by UR Vistahermosa clinic from Spain.

Picture of Dra. Estefanía Montoya
Dra. Estefanía Montoya
Geneticist, UR HLA Vistahermosa

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