Cell-free nucleic acids and melatonin levels in human follicular fluid predict embryo quality in patients undergoing in-vitro fertilization treatment

Despite many advances in assisted reproductive technology (ART), the most viable embryo selection remains a challenge for infertility treatment. This study was designed to investigate whether intra-follicular circulating cell-free DNA (cfDNA) fragments and Melatonin levels predict embryo quality in patients undergoing IVF treatment. A total of eight hundred and ninety-five follicular fluid (ff) samples were collected from 325 infertile patients undergoing IVF treatment. Patients were enrolled from August 2017 to December 2018 in the infertility center of a tertiary care hospital. A clear non-hematic follicular fluid was aspirated after the removal of eggs from the dominant follicles (>18 mm) of each patient. Melatonin and E2 levels in each follicular sample were estimated by immune-chemiluminescence using commercially available kits. ALU-qPCR evaluated cfDNA levels in individual follicular fluid samples. Our study presented a significant and negative relationship between intra-follicular cfDNA and melatonin concentration (-0.541: P < 0.001). Each individual follicle contains measurable copy number of cfDNA [mean: 1.85 ± 2.98 ng/μl (median; 1.86 ng/μl (95% Cl: 0.96–2.87)]. In pregnant women cfDNA copy number was significantly decreased in follicular fluid samples(ff) aspirated from matured oocytes than in immature ones [p < 0.01; β = -0.42 ± 0.49; median; 1.45 ng/ml (95% Cl: 0.36–2.97) vs. 3.57 ng/μl (95% Cl: 0.37–4.01) respectively. While melatonin concentration in ff samples corresponding to mature oocytes was significantly higher than in ff samples related to immature oocytes (p < 0.001). Moreover, in pregnant women cfDNA level was significantly lower in ff samples related to oocytes which produces top-quality embryos versus low quality embryos [p < 0.001; β = 1.81 ± 0.91; median; 1.25 ng/μl (95% Cl: 0.35–1.97)] vs. [(median; 3.65 ng/ml (95% Cl: 1.23–6.36)] respectively. Likewise, in non-pregnant women melatonin levels were significantly decreased in ff samples related to embryos with high fragmentation rate (≥25%) than embryos with low fragmentation rate (<25%; p < 0.001). Conclusively, this study indicates that Intra-follicular cfDNA and melatonin concentration possibly a new supplemental tool that supports to establish an advanced non-invasive early prognostic test for the patients undergoing IVF/ICSI procedure.