Pooled Follicular-Fluid Melatonin Under Standardized Supplementation in IVF-ICSI Cycles: Associations with Clinical Ovarian Response Phenotypes and Exploratory Embryological Outcomes: A Prospective Observational Study

Background: Melatonin concentrations in follicular fluid exceed serum levels, suggesting local ovarian accumulation and a potential role in the follicular microenvironment. Previous studies reporting associations between intrafollicular melatonin and IVF outcomes have largely relied on dominant-follicle sampling. This study evaluated pooled follicular-fluid melatonin concentrations across clinical ovarian-response phenotypes in women undergoing standardized melatonin-supplemented IVF-ICSI cycles. Methods: Seventy-one women undergoing controlled ovarian stimulation with a GnRH antagonist protocol were prospectively enrolled. All participants received oral melatonin at 3 mg/day) from gonadotropin initiation until oocyte retrieval. Participants were classified as poor (n = 24), normal (n = 22), or high responders (n = 25) using predefined clinical ovarian-response criteria. Pooled follicular fluid was collected during oocyte retrieval and melatonin was measured by ELISA. Age-adjusted and multivariable models were used to re-evaluate between-group melatonin differences, the AMH–melatonin association, and the relationship between melatonin and retrieved oocyte number. Results: In the unadjusted analysis, pooled follicular-fluid melatonin concentrations differed across ovarian -response groups (p = 0.0132), with lower levels in poor responders than in normal and high responders. This group effect remained after age adjustment (overall p = 0.0315), with higher concentrations in normal versus poor responders (β = 0.758, p = 0.0278) and high versus poor responders (β = 0.589, p = 0.0070). The age-adjusted AMH–melatonin association remained weakly positive (β = 0.174, 95% CI 0.022–0.327, p = 0.0250), but was no longer evident after conditioning on ovarian-response group (β = 0.011, 95% CI −0.350–0.372, p = 0.9534). In a negative-binomial model, melatonin was associated with retrieved oocyte number after age adjustment (IRR = 1.435, 95% CI 1.066–1.931, p = 0.0173), whereas the association was not retained in the exploratory group-conditioned model (IRR = 1.027, 95% CI 0.893–1.180, p = 0.7103). No consistent association was observed with downstream embryological outcomes. Conclusions: Under standardized melatonin supplementation, lower pooled follicular-fluid melatonin remained associated with the poor-response phenotype after age adjustment. However, the AMH–melatonin and melatonin–oocyte-yield associations were sensitive to the structure of the ovarian-response categories. These findings are observational and hypothesis-generating and do not establish a causal treatment effect, an independent ovarian-reserve biomarker relationship, or clinical utility.