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This study describes the normal variations in serum and cervicovaginal fluid (CVF) cytokine levels throughout pregnancy.This multicenter, prospective study examined trimester-specific maternal serum and CVF cytokines (interleukin [IL]-1α, IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor-α, and C-reactive protein [CRP]). A two-factor linear mixed modeling approach compared cytokine distribution, while pairwise comparisons evaluated differences over time.Trimester-specific serum cytokine data were available for 288, 243, and 221 patients, whereas CVF cytokine data were available for 273, 229, and 198 patients. CVF had significantly higher concentrations of IL-1α, IL-1β, IL-6, IL-8, and matrix metalloproteinase-8 (p < 0.001), irrespective of the trimester. At all time points, IL-10 and CRP concentrations were higher in serum than CVF (p < 0.001). Serum IL-10 increased significantly throughout pregnancy (p < 0.001).Differences in cytokine distribution across different biological fluids are evident throughout pregnancy. These findings provide a framework for examining patterns of changes in cytokines throughout pregnancy.