Spatial Distribution of Senescent Cells and Their Proximity to Immune Subsets in the Human Endometrium During the Implantation Window

<b>Background/Objectives</b>: Senescent cells contribute to endometrial remodeling during the implantation window, but their spatial organization within the stroma remains poorly understood. This study aimed to characterize the distribution of senescent (p16-positive) cells in the functional layer of the endometrium and to evaluate their spatial relationships with immune cell subsets. <b>Methods</b>: Endometrial biopsies from 68 women undergoing IVF were collected during the mid-luteal phase (LH+7, corresponding to the implantation window). Samples were analyzed by immunohistochemistry for p16 and immune markers (CD3, CD4, CD8, CD14, CD68, CD56, CD79α). Images from adjacent serial sections were digitally aligned, and senescent cell density, clustering, and nearest-neighbor distances to immune cells were quantified using HALO Image Analysis software (v3.4). Ratios of senescent-to-immune cell abundance were also calculated to account for stromal variability. <b>Results</b>: Senescent cells were heterogeneously dispersed within the stroma, with occasional high-density clusters. Quantitative analysis revealed that their abundance was lower than that of monocytes, macrophages, and total T cells, but higher than that of T-helper and B cells. Across patients, median senescent-to-immune cell ratios were approximately 1, indicating comparable abundances, except for CD4⁺ and CD79α⁺ subsets, where ratios were significantly elevated. Nearest-neighbor analysis showed that macrophages and monocytes localized in closest proximity to senescent cells (45 ± 20 μm and 45 ± 25 μm), while T-helper and NK cells were positioned at greater distances from senescent cells (102 ± 42 μm and 53 ± 23 μm, respectively). B cells showed the greatest separation (211 ± 66 μm). Correlation analysis confirmed density-driven proximity for most immune subsets, with CD4⁺ and CD56⁺ cells as exceptions, displaying limited spatial association with senescent cells. <b>Conclusions</b>: Senescent cells in the endometrium during the implantation window display heterogeneous distribution and selective spatial associations with immune subsets. Their preferential distancing from T-helper and NK cells suggests impaired local immune-senescence crosstalk, highlighting spatial profiling of senescent cells as a potential diagnostic marker of endometrial receptivity.