Macrophage Density Patterns in Uterine Leiomyomas Across Patient Age Groups: Evaluation of Separately Quantified CD68-Positive and CD163-Positive Cells

Tumor-associated macrophages (TAMs), key effectors of the innate immune system, actively participate in extracellular matrix remodeling, angiogenesis, and tumor growth. However, their age-associated quantitative and phenotypic distribution in uterine leiomyoma microenvironment remains insufficiently understood. This study aims to evaluate the density and spatial distribution of CD68+ and CD163+ macrophage immunophenotypes in uterine leiomyoma stroma across distinct patient age groups. Methods: A total of 318 patients with histologically confirmed uterine leiomyoma were stratified into three chronological age cohorts: Group I (≤30 years, n = 42), Group II (31–45 years, n = 216), and Group III (≥46 years, n = 60). Archival paraffin-embedded tumor tissue sections were analyzed histologically with hematoxylin and eosin, and immunohistochemically using antibodies against CD68 and CD163. Digital morphometric analysis was performed using QuPath software on whole-slide images, evaluating median immunopositive cell densities (cells/mm2) across five standardized regions of interest per case. Results: Histological examination confirmed typical benign leiomyoma morphology, with secondary degenerative alterations (hyalinosis, sclerosis, calcification, and aseptic necrosis) being most pronounced in Group III. Immunohistochemical profiling demonstrated an age-associated increase in macrophage infiltration within the tumor stroma. Median CD68+ macrophage density was higher in older cohorts, ranging from 11.4 cells/mm2 [IQR: 8.6–13.8] in Group I to 16.7 cells/mm2 [IQR: 13.9–19.8] in Group II, and 22.4 cells/mm2 [IQR: 18.8–26.2] in Group III (p < 0.001). Concurrently, CD163+ macrophage density was likewise higher across older cohorts, measured at 9.8 cells/mm2 [IQR: 7.4–12.1] in Group I, 19.4 cells/mm2 [IQR: 16.1–22.9] in Group II, and 28.6 cells/mm2 [IQR: 23.8–33.4] in Group III (p < 0.001). The observed numerical predominance of CD163+ cells over CD68+ cells in Group III topographically correlated with areas of secondary tissue remodeling and ischemic injury. Conclusions: This study reveals significant unadjusted differences in separately quantified CD68-positive and CD163-positive cell densities within uterine leiomyoma stroma across distinct patient age groups. Higher CD163+ cell densities in older cohorts topographically co-distribute with areas of secondary tissue remodeling and ischemic alteration. Because markers were evaluated on separate sequential sections and multivariable adjustments were unfeasible due to subgroup collinearity, these parameters reflect descriptive, marker-specific stromal variations among real-world clinical cohorts rather than independent causal effects of biological aging.