BackgroundIntrafollicular melatonin maintains the DNA integrity of granulosa cells and protects them against apoptosis. This ubiquitous indoleamine compound serves as a potent free radical scavenger. It reduces oxidative stress and modulates DNA damage response, which improves oocyte's quality with a higher fertilization rate.Methods:This prospective study was designed to investigate the antioxidant property of intrafollicular melatonin and its impact on IVF outcome parameters by exploring the relative expression of five microRNAs (miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p) and levels of cfDNA by real-time PCR in unexplained infertile patients. We collected 425 follicular fluid (ff) samples containing mature oocytes from 295 patients undergoing IVF. Results:Patients were sub-grouped based on intrafollicular melatonin concentration (Group A; ≤ 30 pg/mL, Group B; >70 to ≤110 pg/mL), Group C; >111 to ≤ 385 pg/mL). Our results showed that patients with ≤ 30 pg/mL intrafollicular melatonin levels have a significantly higher cfDNA levels and lower relative expression of miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p compared to other subgroups (p<0.001). Similarly, they have a low fertilization rate and a reduced number of high-quality day 3 embryos. For the probability of obtaining a good quality embryo, miRNAs had AUC value of 0.89 [95% Cl; 0.71; 0.93] with 87% sensitivity and 83 % specificity (p = 0.001), whereas cfDNA have AUC value 0.76 [cut-off points, 95% Cl: 0.61, 0.67; 0.85] along with 82.4 % senstivity and 66.7 % specificity.Conclusion:Conclusively, our study provided evidence that melatonin's antioxidant capability significantly impacts cfDNA concentration and miRNAs relative expression profile in the follicular microenvironment for optimal oocyte development and embryo quality. Therefore, it may be a potent non-invasive diagnostic tool to select high-quality day 3 embryos with such promise.