Non-Invasive Methods for Detection of Genetic Abnormalities in Human Embryos Cell-Free DNA Secreted from Human Embryos as an Alternative to DNA from Cell Biopsies for Genetic Analyses

Abstract

Chromosomal abnormalities are one of the main causes of implantation failure following embryo transfer in in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidies (PGT-A) is time-consuming, expensive, and invasive. Non-invasive PGT-A (niPGT-A) is an alternative where secreted embryonic cell-free DNA (cfDNA) in spent culture medium (SCM) is investigated. This study aimed to investigate and prepare for the implementation of niPGT-A in the IVF-unit at UNN. To reach this objective 36 anonymized frozen embryos were thawed, cultured and DNA from dissociated cells and SCM was extracted. Furthermore, two different analysis methods were chosen: digital droplet PCR (ddPCR) and Oxford nanopore sequencing. The latter required amplification by Repli-G in advance. ddPCR was performed to investigate genes located on chromosomes 21 (RUNX1 and BRWD1) and 18 (TCF4 and SMAD4), to identify trisomy 21. The embryos were viable, with DNA present in every sample, having a mean of 1.289 ng/l throughout the samples. Repli-G was used to amplify five of the embryos, which gave a mean of 2099.29 ng/l, resulting in over a thousandfold increase in concentration. The amplified samples were then sequenced, which resulted in a blocked run and limited output, with the numbers of reads generated ranging from 7607 to 90 632. Yet, a consensus sequence was generated from the cfDNA found in the SCM sample of embryo 18, which was found to match the human genome. The samples run on ddPCR had a detection of genes involved in trisomy 21, with 11 out of 18 samples having positive droplets generated for the probes targeting the genes located on chromosome 21. The starting concentrations ranged from 0.599 copies/l to 4.35 copies/l in the samples with positive droplets detected. There was, however, a lack of detection on the probes targeting the genes located on chromosome 18. In conclusion, further investigation into both nanopore sequencing and ddPCR is necessary to validate their clinical relevance, however, the possibility of using cfDNA as an alternative to cell biopsies to screen the embryos is promising.