Preimplantation genetic testing for chromosomal screening is, at present, the most reliable method to assess the genetic status of preimplantation embryos. DNA isolated and amplified from trophectoderm (TE) biopsiesis the method currently used for preimplantation genetic testing, but it has significant limitations. There is increasing evidence for a ''true'' noninvasive approach consisting of the analysis of cell-free DNA (cfDNA) released by the embryo into the spent blastocyst medium (SBM) during the late stages of preimplantation development.

This study is to assess whether Noninvasive prenatal genetic diagnosis using cell-free fetal DNA in spent culture medium will substitute for trophectoderm biopsy for heritable disorders screening and if it represents the total fetal genomic DNA.

A total of 27 spent blastocyst media (SBM) from women undergoing IVF will be enrolled with agesless than44 years, whose indications for preimplantation genetic diagnosis for Euploid/Aneuploid embryo determination, were advanced maternal age, recurrent miscarriage, or recurrent implantation failure.We examined the presence of the cell free DNA in spent culture media and whether this DNA is reliable and can be representative of the chromosomal constitution of a blastocyst. So we can affirm that Noninvasive prenatal determination of fetal sex using cell-free fetal DNA (used here as a marker) provides an alternative to invasive techniques for some heritable disorders.

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.

Inclusion Criteria:

• At least two blastocysts suitable for PGD. Only embryos achieving a grade 1 (fragmentation less than 15%).

Exclusion Criteria:

• All embryos do not achieve inclusion criteria as mentioned above.

The study samples will be collected from couples attending Orient Hospital (assisted reproduction and genetics) to carry out the in vitro fertilization process and would like to perform PGD for gender selection. After obtaining the approval of the ethical committee of Damascus University, they will be informed of the research objectives and sign the approve consent.