The Effects of The Menstrual Cycle On Growth Factors In Platelet-Rich Fibrin: A Cross-sectional Observational Study

Platelet-rich fibrin (PRF) is widely used in dental surgery for regenerative procedures; however, variability in its biological content remains a key limitation for standardization. This study aimed to implement a tightly controlled experimental workflow to evaluate how menstrual cycle phases influence the release of growth factors from PRF. Venous blood samples were obtained from 21 healthy female volunteers at three predefined menstrual phases (menstrual: days 1-5; follicular: days 6-14; luteal: days 15-28), with all collections performed within a fixed time window (11:00-11:30 a.m.) to minimize circadian variation. For each phase, separate tubes were allocated for complete blood count, hormone analysis (estrogen, progesterone, follicle-stimulating hormone [FSH], and luteinizing hormone [LH]), and PRF preparation. PRF was produced by immediate centrifugation under standardized conditions (708 × g for 12 min; fixed-angle rotor, 40 °; radius 88 mm; no anticoagulant). Clots were macroscopically verified (homogeneous, elastic structure), carefully separated from the red blood cell layer, and individually weighed. To standardize growth factor release, each clot was incubated in RPMI-1640 medium using a weight-based ratio (1 g PRF: 1 mL medium), followed by controlled incubation at 37 °C with orbital agitation (100 rpm). Supernatants were collected and stored at -80 °C prior to analysis. Growth factors (IGF-1, PDGF, FGF-2, VEGF, and TGF-β1) were quantified using enzyme-linked immunosorbent assay (ELISA) under uniform assay conditions. Within this standardized protocol, FGF-2 levels were significantly higher during the follicular phase compared to the menstrual and luteal phases. A moderate positive correlation was observed between FGF-2 and estrogen levels in this phase, whereas no statistically significant differences were detected for other growth factors. These findings indicate that menstrual cycle phase may contribute to variability in PRF-derived growth factor profiles. The outlined protocol provides a reproducible framework for minimizing pre-analytical and analytical variability in PRF studies. The clinical implications of phase-related differences warrant further investigation in studies incorporating larger cohorts and treatment outcomes.