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Challenges of Preimplantation Genetic Testing

Posted by Agnes Caruso,PhD on Dec 15, 2020 12:00:00 AM

Getting pregnant is not always simple and with an increase in the age of first-time mothers, there is an increased need for in vitro fertilization (IVF). IVF allows the generation of embryos in the lab and subsequent embryo transfer to the patient. Successful implantation of the embryo depends on many factors with chromosomal aneuploidy being one of the important factors in a failed IVF cycle. There are several ways the odds of success can be increased and risks of miscarriage are reduced.

What is Preimplantation Genetic screening or testing (PGS or PGT) and why is it used?

Improving the outcomes of IVF treatment means being able to better predict the risk of miscarriage. One of the factors affecting the success of pregnancy is embryonic aneuploidy. PGT is a way to screen or test embryos generated in the lab for a presence or absence of genetic alterations that can reduce the embryo’s viability or affect the health of the fetus.

The most common and most widely used method for embryo screening is PGT-A, which is testing for aneuploidies in an embryo. This test is mainly offered to couples with advanced maternal age or recurrent problems such as failures to implant or recurrent miscarriages. While there is a disagreement if the PGT tests increase rates of successful pregnancies, some studies indicate that euploid embryos have improved implantation odds1. PGT tests rely on sampling cells from the developing pre-implantation embryos. Most commonly these are trophectoderm cells, but blastomeres are also used (Figure 1).

embryonic development (003)

Figure 1. Early embryonic development and how the embryos can be sampled for PGT.

The first method used for PGT was the fluorescence in situ hybridization (FISH) technique. Its limitations are the requirement for selecting the chromosomal probes before testing and the ability to combine only a certain number of fluorophores in one test. Therefore aneuploidy results return only data for probes used in testing. The two methods which replaced it to improve the ability to screen for all chromosomal aneuploidies are qPCR and array CGH. Further technological developments introduced genome-wide haplotyping and low-coverage NGS sequencing.

However, the two newest methods come with their own limitations. The most important factor being that manipulation of extremely low DNA concentrations originating from one to ten biopsied cells is challenging:

  • It is impossible to perform standard QC on those samples
  • Allele and locus drop-out
  • Allele and locus drop in
  • Whole genome amplification (WGA) is an essential part of the workflow as no analysis can be performed without it

The preferred method for WGA is the use of Multiple Displacement Amplification (MDA) as it reduces amplification bias and has low error frequency2.

Using dedicated reference standards allows to evaluate method reproducibility and performance of the lab analysts. Repeated testing of the same material should deliver the same answer. Limited amount of the biopsy material, makes it impossible to use it repeatedly. Seraseq® PGT-A reference materials are formulated at low but stable concentration and are derived from cells with a confirmed aneuploidy. Moreover, the same source is used in our NIPT reference materials making

Normal, aneuploid or mosaic embryo and what does that mean?

Testing embryos by PGT-A is not as simple as we may expect. There can be three different outcomes:

    • Euploid or normal with no alterations in the chromosome number
    • Aneuploid – with an altered number of chromosomes
    • Mosaic – embryo consists of cells with a normal and abnormal number of chromosomes

While the first two outcomes are easy to interpret, mosaicism is not as simple. The reason for the difficulty is that level of mosaicism is usually variable and identifying the level in the NGS data requires skill or special software tools well trained in identifying mosaic samples.

Challenges around PGT-A reflect the fact that the method, in the majority of cases, relies on sampling trophectoderm cells. These cells will form the placenta during pregnancy. Therefore aneuploidy or mosaicism in those cells does not necessarily reflect anomalies in the embryo itself. However, the level of mosaicism appears to affect the IVF treatment outcomes and mosaic embryos are frequently not used for the transfer3.

The Future of PGT

Current rapid technology development can change the way PGT testing is done in the future. One of the methods has been adopted by some labs is the non-invasive preimplantation testing (ni-PGT-A). The method samples the embryo growth medium rather than the embryo itself. Presentations at this year ESHG showed good concordance between the ni-PGT-A and traditional method.

An expansion of single-cell testing by haplotyping and sequencing can revolutionize PGT. However, genomics is not everything. Metabolic testing of the growth media is also being assessed as an indicator for successful embryo transfer. So far those methods are in development and it is not certain if and when they can be introduced into clinical practice.

How does LGC SeraCare help in making reporting simpler?

As the PGT methods are changing to include more complex techniques, such as NGS, their development and validation are also more complicated. LGC SeraCare now offers reference materials that can be used to develop, change or validate the PGT-A assays. They can also help you to train staff and ensure consistent performance of the assays.


  1. Spinella, F. et al. Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments. Fertil. Steril. 109(1):77-83, (2018). PMID: 29191449
  2. Dean, F. B. et al. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 99(8):5261-6(2002). PMID: 11959976
  3. Munné, S. et al. Clinical outcomes after the transfer of blastocysts characterized as mosaic by high resolution Next Generation Sequencing- further insights. Eur. J. Med. Genet. 63(2): 103741 (2020). PMID: 31445143

Topics: New Reference Material, Reproductive Health, PGT, Women's health, NGS, PGS, PGT-A