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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

Year: 2026

Type: article

Abstract: 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.

Source: Experimental and Therapeutic Medicine

DOI: 10.3892/etm.2026.13245