Cell-free DNA in human ovarian follicular fluid as a potential biomarker of embryo quality in IVF cycles: a pilot study

Introduction There is growing interest in evaluating cell-free DNA (cfDNA) in follicular fluid (FF) as a molecular feature of the follicular microenvironment and examining its association with subsequent embryo development. This exploratory pilot study aimed to investigate the association between FF cfDNA integrity, measured by the Arthrobacter luteus (ALU) 247/115 ratio, and morphology-based embryo quality in IVF cycles. Methods This observational study analyzed 106 follicular fluid samples from patients undergoing IVF with intracytoplasmic sperm injection (ICSI) at a single fertility center. FF samples were collected during ovum pick-up, and cfDNA concentrations were quantified using ALU-quantitative PCR. To evaluate the ALU247/115 ratio in relation to age, participants were categorized into three age groups. Results The ALU247/115 ratio showed a significant but weak positive correlation with embryo quality score across all patients (n = 106, r = 0.23, p = 0.016). Subgroup analysis revealed stronger correlations in women under 35 years (n = 31, r = 0.38, p = 0.035) and those aged 35–39 years (n =40, r = 0.32, p = 0.044). No significant correlation was observed in women aged 40 or older (n = 35, r = 0.21, p = 0.225). Conclusions In women under 40, increased cfDNA fragmentation in FF was associated with lower morphology-based embryo quality. However, the observed correlations were modest, and the absence of preimplantation genetic testing for aneuploidy (PGT-A), implantation, clinical pregnancy, and live birth outcomes limits clinical interpretation. These exploratory findings do not establish FF cfDNA as a clinically validated predictive biomarker or demonstrate that it improves embryo selection. Larger prospective studies incorporating embryo ploidy and clinically relevant reproductive outcomes are warranted.