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.