Highlights

• Identifies fertilized eggs with an abnormal number of chromosomes.
• The method will help fertility doctors prioritize the most suitable fertilized eggs and thereby improve implantation rates and successful pregnancies.
• Is "non-invasive" as it uses the liquid in which the fertilized eggs are grow, known as spent culture medium (SCM)
• At Amplexa we specialize in extracting the DNA from the culture medium and performing a test on this DNA.

Causes of implantation failure

One of the primary causes of implantation failure and spontaneous abortion is an abnormal number of chromosomes (aneuploidy). Aneuploidy is common in human fertilized eggs and becomes increasingly common with age. On average, a woman at the age of 40 will have an aneuploidy incidence rate of more than 50%, and at the age of 44 the incidence rate is almost 80% [1] Unfortunately, aneuploid eggs are indistinguishable from euploid eggs in both morphology and developmental rate, making them undetectable with conventional morphological analysis [2]. As transfer of aneuploid eggs negatively affects the reproductive outcome, it is desirable to detect aneuploidy by other means during IVF treatment.

When is niPGT-A relevant?

niPGT-A analysis becomes more effective as the patient grows older. According to data from the 2018 National Summary Report, PGT analysis is most effective in women above the age of 38 [5]. We do however recommend niPGT-A to all individuals undergoing IVF-treatment, in order to reduce the number of treatments needed for a successful pregnancy, by prioritizing chromosomally healthy fertilized eggs.

How can niPGT-A benefit an in vitro fertilization treatment?

niPGT-A increases the chance of a successful implantation.

One of the primary causes of implantation failure and spontaneous abortion is an abnormal number of chromosomes (aneuploidy). Aneuploidy is common in human-fertilized eggs and becomes increasingly common with age. niPGT-A can determine the number of chromosomes, helping doctors better select fertilized eggs for implantation in the mother's uterus.

Gender selection with niPGT-A

Gender identification of a fertilized egg using an niPGT-A analysis may be affected by maternal contamination of the sample or contamination with sample collector cells. Amplexa Genetics therefore emphasizes that gender identification of a fertilized egg using niPGT-A analysis should not be considered the best assessment and should always be confirmed by other methods.

At what stage of an IVF should niPGT-A be included?

When a clinic performs an in vitro fertilization treatment, they will keep the fertilized egg in a liquid waiting for the moment to transfer to the mother.

During the development of the fertilized egg in the liquid, the fertilized eggs naturally release DNA into the liquid.

After day 5 or 6 of development, the fertilized egg is usually ready for treatment, at this stage, the clinic collects the liquid containing free DNA and sends it to Amplexa Genetics for analysis.

The new non-invasive technique

The most common method employed by clinics for aneuploidy detection is Preimplantation Genetic Testing for Aneuploidy (PGT-A). PGT-A can increase the number of successful pregnancies by prioritizing the blastocysts with the best chromosomal profiles. In conventional PGT-A, a biopsy of the developing blastocyst (trophectoderm) is collected and embryonic DNA is extracted from the trophoblasts. However, this biopsy is potentially damaging to the blastocysts and only represents the chromosome count of the specific trophectoderm region and not the embryo itself [3]. Furthermore, the sample is prone to cell-specific bias due to mosaicism and can potentially contribute to false positive and/or false negative indications of ploidy status. Recently, a non-invasive alternative to the trophectoderm biopsy has been developed. It was discovered that blastocysts secrete cfDNA into the culture medium in which they are grown. Using this cfDNA as a template for a non-invasive PGT-A (niPGT-A) approach has proven highly effective in IVF-treatment. This cfDNA has proven as representative of the actual embryo as the DNA obtained from the trophectoderm biopsy. The origin of the cfDNA is also very likely to be healthy blastocyst tissue, representing both the inner-cell mass and the trophectoderm [4]. niPGT-A offers a risk-free alternative to traditional PGT-A with high concordance to trophectoderm biopsies, inner cell mass and the whole blastocyst. The basis for the analysis is the spent culture medium (SCM) from the routine growth of blastocysts in IVF clinics. As SCM is collected and discarded upon vitrification of the blastocysts, incorporation of niPGT-A into the existing workflow is close to seamless.

When is niPGT-A relevant?

As aneuploidy rate increases significantly with maternal age, an niPGT-A analysis becomes more effective as the patient grows older. According to data from the 2018 National Summary Report, PGT analysis is most effective in women above the age of 38 [5]. We do however recommend niPGT-A to all individuals undergoing IVF-treatment, in order to reduce the number of treatments needed for a successful pregnancy, by prioritizing chromosomally healthy embryos.

How accurate is niPGT-A compared to PGT-A?

Recent studies into the concordance rate of niPGT-A compared to PGT-A have achieved great results. Part of the improvement is due to the removal of maternal contamination in the form of cumulus cells. Additionally, it has been found that the best concentration of cDNA in the SCM is found on day 5 of blastocyst growth. Taking these factors into account, niPGT-A has been found to have higher concordance rates for embryo ploidity than PGT-A (94% vs 82%) [6].

How are the best niPGT-A results achieved?

To achieve the best results from an niPGT-A, we recommend the following steps during blastocyst growth:

Step 1. Removal of maternal cumulus cells: To avoid maternal DNA contaminating the sample, it is essential to remove any cumulus cells before the oocyte is placed into the growth medium. It can be removed chemically or with a 135 µm denudation pin.

Step 2. Cell washing: To ensure no maternal DNA is left over, we recommend washing the oocytes thoroughly the following denudation and transferring them to new growing trays in 20µl droplets.

Step 3. Sample quantity: To get sufficient cfDNA in the medium we also recommend that the blastocysts have been grown in said medium for a minimum of 24h. The minimum required SCM for niPGT-A is 6µl, is recommended to collect as much SCM as possible

References

[1] L. Navarro-Sánchez, C.García-Pascual, C. Rubio, and C. Simón, “Non-invasive preimplantation genetictesting for aneuploidies: an update,” Reproductive BioMedicine Online,Jan. 2022, doi: 10.1016/j.rbmo.2022.01.012.

[2] A. Capalbo et al.,“Correlation between standard blastocyst morphology, euploidy and implantation: An observational study in two centers involving 956 screened blastocysts,” HumanReproduction, vol. 29, no. 6, pp. 1173–1181, 2014, doi:10.1093/humrep/deu033.

[3] H. F. Chen, M. Chen, and H. N. Ho, “An overview of the current and emerging platforms for preimplantation genetic testing for aneuploidies (PGT-A) in in Vitro fertilization programs,” Taiwanese Journal of Obstetrics and Gynecology, vol. 59, no. 4. Elsevier Ltd, pp. 489–495, Jul. 01, 2020. doi:10.1016/j.tjog.2020.05.004.

[4] M. Vera-Rodriguez et al., “Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development,” Human Reproduction, vol. 33, no. 4, pp. 745–756, Apr. 2018, doi: 10.1093/humrep/dey028.

[5] “2018 data reported by National Summary Report,” https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx

[6] L. Huang, B. Bogale, Y.Tang, S. Lu, X. S. Xie, and C. Racowsky, “Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy,” Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 28, pp. 14105–14112, 2019, doi:10.1073/pnas.1907472116.