Isolation of Extracellular Embryonic DNA FromIn Vitro Fertilization (IVF) Embryonic Culture Medium and Its Potential Role for Preimplantation Genetic Screening (PGS).

Noninvasiveness is a common theme across medical specialties in the 21st century. In obstetrics, noninvasive prenatal testing provoked a paradigm shift in prenatal clinical practice through the sequencing of fetal cell-free DNA (cfDNA) in maternal plasma. However, current practice for preimplantation genetic testing for aneuploidy (PGT-A) requires trophectoderm biopsy, which entails technical challenges and embryo viability concerns. The possibility to overcome these issues reached the horizon in recent years, with the discovery that cfDNA released by the embryo to the culture media during the latest stages of development offers a new strategy to assess the preimplantation embryo genetically.

Our study evaluates the possibility to isolate fetal cell-free DNA (cfDNA) from culture media and compare the results with TE biopsy samples from the same patients who underwent an IVF stimulation cycle with intracytoplasmic sperm injection at Al-Sharq Hospital between March 2022 and April 2023. During the study's time frame, all embryos were cultured until the blastocyst stage. All enrolled patients underwent niPGD for gender determination using spent culture media as well as standard PGD with TE biopsy using Polymerase chain reaction technique for SRY gene. The embryos were individually cultured in 30-mL media droplets and embryo development progressed using sequential culture media, consistent with routine laboratory practice. For niPGD analysis, the blastocyst-stage culture media on Day 5 was collected (10-20 µl) immediately before TE biopsy was performed. Only embryos achieving a grade 1 (fragmentation less than 15%), were considered eligible for biopsy and vitrification. The embryo culture media samples underwent whole-genome amplification (WGA) using the DOPlify® WGA kit (PerkinElmer) to generate representative amplification of total DNA from spent culture media. The DOP-PCR-based WGA reproducibly amplifies total DNA to produce microgram quantities of amplified and purified genomic DNA in less than 3 hours. We aim to evaluate the concordance rates between trophectoderm biopsy results and cfDNA collected from spent media with an optimized protocol to obtain an adequate quantity and quality of genomic DNA. This interesting work is an important effort to establish the feasibility of the new noninvasive approach that can be a reliable alternative for chromosomal abnormalities assessment of in vitro cultured embryos which will lead to the birth of a living and healthy baby.

Later on, we will perform the SRY gene detection for sex determination using the Polymerase Chain Reaction on the cell-free fetal DNA isolated from spent media samples and compare our results with the TB results.