A prospective study of non-invasive preimplantation genetic testing for Rh status, sex prediction and mtDNA copy number analysis using ddPCR on spent culture media.

Presence of embryo-derived cell-free DNA (cfDNA) in spent culture medium (SCM) represents a promising non-invasive alternative to the conventional preimplantation genetic testing (PGT). The present study evaluated the feasibility of using cfDNA isolated from SCM to determine Rhesus (Rh) status through Rh blood group D antigen (RHD) gene analysis, predicting sex-linked disorders using sex determining region Y (SRY) gene detection and assessing the association between mitochondrial cfDNA (cf-mtDNA) levels and maternal age using mitochondrially encoded cytochrome b (MT-CYB) gene quantification. SCM samples collected during the IVF process were subjected to cfDNA isolation and target regions including RHD exon 7 and exon 10, SRY and MT-CYB were analyzed using droplet digital PCR. RHD gene amplification was detected in 6 of 15 samples for exon 7 and in 11 samples for exon 10, with partial concordance between exons. SRY amplification was observed in 16 of 31 samples and was largely consistent with clinical sex data. MT-CYB was detected in 19 of 31 samples, with higher cf-mtDNA/nuclear cfDNA ratios observed in women >40 years of age. A moderate positive correlation trend was noted between maternal age and MT-CYB levels (r=0.46; P=0.354). These findings demonstrate the potential applicability of SCM-derived cfDNA for non-invasive Rh and sex determination without compromising embryo integrity, while mtDNA quantification may provide additional insights into embryo quality. Overall, SCM-based cfDNA analysis exhibited promise as a complementary tool for embryo selection in IVF workflows.