Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

The aim of this study was to investigate the association between the endometrial microbiome and local immune cell composition during the implantation window. We conducted a single-center prospective observational study including endometrial samples from 58 women without endometrial pathology. All samples were obtained during the mid-secretory phase (day 5 post-ovulation). Endometrial stromal CD3⁺ T cells, CD4⁺ T helpers, CD56⁺ NK cells and CD68⁺ macrophages were identified by immunohistochemistry and quantified as a percentage of stromal cells using HALO image analysis software. Microbiome composition was assessed by 16S rRNA gene sequencing (V4–V5 region). Lactobacillus dominance (LD) was defined as the genus with the highest centered log-ratio value within each sample. Endometrial immune cell composition showed a median 2.28% CD3⁺ T cells of stromal cells, 1.75% CD56⁺ NK cells, 1.44% CD68⁺ macrophages and 0.29% CD4⁺ T helpers. Lactobacillus-dominated microbiota was identified in 51.7% (30/58) of samples. Correlation analysis showed that Lactobacillus-related taxa were negatively associated with NK/T ratios and positively with T helper-related ratios. LD samples exhibited reduced NK cell abundance (1.17% vs. 2.12%, p = 0.006, q = 0.023) and a lower NK/T ratio (0.52 vs. 1.05, p = 0.004, q = 0.023) compared to non-LD samples. This study provides evidence for a link between microbial composition and local immune regulation in the endometrium.

Successful implantation requires a finely regulated endometrial microenvironment during the window of implantation. Chronic endometritis, defined by plasma cell infiltration, and stromal senescence, indicated by p16 expression, represent separate but potentially interacting mechanisms associated with impaired endometrial receptivity. The relationship between these processes remains poorly understood. We aim to examine whether stromal senescence is associated with plasma cell density and clustering in the human endometrium during the implantation window. Forty mid-luteal (LH+7) endometrial biopsies were retrospectively analyzed and stratified into low-senescence (3.5%, n = 20) groups. Plasma cells were identified by immunohistochemistry for MUM1 and CD138 and quantified using HALO^® software (version 3.4). Group comparisons were performed using Student’s t-test and chi-squared analysis. CD138⁺ plasma cells were significantly more abundant in high-senescence endometria than in low-senescence controls (0.065 ± 0.10 vs. 0.014 ± 0.027 cells/mm², p = 0.02). Only MUM1⁺ cells formed stromal clusters, which were more frequent in high-senescence samples (67% vs. 31%, p = 0.05). High endometrial stromal senescence during the implantation window is associated with increased plasma cell infiltration and clustering. This interplay may contribute to chronic endometritis and impaired receptivity, providing new insights into potential diagnostic and therapeutic strategies for reproductive failure.

Objectives: This study aimed to determine whether chromosomal mosaicism in blastocysts is associated with a distinct morphokinetic signature. Methods: Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 182 human embryos via trophectoderm biopsy on day 5 and analyzed by next-generation sequencing. Embryos were classified as euploid (n = 55), mosaic (n = 39: 21 low-grade, 18 high-grade), or aneuploid (n = 88), of which 18 with concurrent mosaicism. Prior to biopsy, embryos were cultured in a time-lapse system (EmbryoScope), and 12 morphokinetic parameters were assessed, including pronuclei fading (tPNf), cleavage times (t2–t9), morula formation (tM), blastulation start (tSB), and full blastocyst formation (tB). These parameters were compared according to ploidy status. Results: Patients with euploid and mosaic embryos were comparable in terms of maternal age, ART indication and embryo quality (p > 0.05). In contrast, aneuploid embryos were obtained from older patients and had lower morphological grades. Mosaic embryos showed delayed tPNf (24.8 ± 6.5 vs. 22.8 ± 2.3 h, p = 0.03) and t2 (27.6 ± 6.6 vs. 25.4 ± 2.5 h, p = 0.02) compared to euploid embryos, mainly attributable to low-grade mosaic embryos. Whole-chromosome mosaicism, but not segmental mosaicism, was associated with delayed embryo development at several intermediate cleavage time points (t3, t4, t6, t7 and t9). Aneuploid embryos showed significant delays at later stages versus euploid embryos, particularly aneuploid embryos with mosaicism at t7 (56.6 ± 8.3 vs. 52 ± 5.6 h, p = 0.02), t8 (59.1 ± 9.6 vs. 54.8 ± 6.7 h, p = 0.04), tM (90.3 ± 7.7 vs. 83.6 ± 8.2 h, p = 0.006) and tB (113.0 ± 11.6 vs. 106.6 ± 8.9 h, p = 0.03). Conclusions: Mosaic embryos exhibit delays in early development (tPNf, t2) but reach later morphokinetic milestones at rates similar to euploid embryos. In contrast, aneuploid embryos, especially those with mosaicism, exhibit marked developmental delays at later stages (t7, t8, tM, tB).

Endometrial senescence and the endometrial microbiota have both been implicated in the regulation of endometrial receptivity, yet their relationship remains poorly understood. The aim of this study was to investigate associations between p16-positive endometrial cells and microbiota composition during the implantation window and to determine whether these relationships differ according to implantation outcome. Endometrial senescence was assessed by p16 immunohistochemistry and digital image analysis, whereas microbial composition was characterized by 16S rRNA gene sequencing in endometrial biopsies collected from 68 women prior to undergoing transfer of a single euploid embryo, which was performed within six months of biopsy under the same hormonal preparation protocol. No significant differences in luminal epithelial or stromal p16 abundance were observed according to subsequent implantation outcome. Although senescence was not directly associated with implantation success, stromal p16 expression demonstrated multiple associations with the endometrial microbiota. Increased stromal p16-positivity was associated with lower relative abundance of Lactobacillus and higher abundance of Delftia. Notably, in exploratory subgroup analyses more pronounced associations were observed in women who achieved pregnancy, including a negative correlation between stromal p16 expression and Lactobacillus abundance (ρ = −0.48, p = 0.005) and positive correlation with Delftia abundance (ρ = 0.42, p = 0.016). Species-level analyses revealed implantation outcome-specific associations involving Lactobacillus iners and Limosilactobacillus vaginalis. In addition, stromal p16 expression was associated with differences in microbial co-occurrence networks, indicating broader effects on microbial community organization. Together, these findings suggest that stromal, but not luminal, senescence is closely linked to endometrial microbiota composition and microbial community organization during the window of implantation. The observation that the strongest senescence–microbiota associations occurred in women who subsequently achieved successful implantation supports the hypothesis that coordinated senescence–microbiota relationships may represent a previously underrecognized feature of the receptive endometrial microenvironment.

Embryo implantation requires a finely tuned immune balance at the maternal–fetal interface. Interferon tau (IFN-τ), a key immunomodulator in ruminant implantation, may have therapeutic potential in human reproduction. This study investigated its effects on peripheral blood mononuclear cells (PBMCs) in vitro and the subsequent impact on endometrial immune composition following intrauterine administration of these cells. The work was conducted in two stages. First, in vitro assays were performed with PBMCs from 20 patients with recurrent implantation failure (RIF) cultured with or without IFN-τ for 24 h. Cytokines (IL-10, IL-4, TNF-α, IL-6) were measured by ELISA, and T cell subsets (Th, cytT, Th1, Th2, Th9, Tfh, Th17, Treg) were analyzed by flow cytometry. IFN-τ increased IL-4 and reduced TNF-α and IL-6, indicating a Th2 profile shift. T-cell analysis revealed fewer cytT, Th1, Th9, and Th17 cells, more Th2 cells, and improved Th/Tk, Th1/Th2, and Th17/Treg ratios after IFN-τ. A second clinical study included 55 RIF patients who received intrauterine IFN-τ-modulated PBMCs. Post-treatment endometrial biopsies revealed more helper T cells and macrophages, with higher Th/total T, Th/cytT, and Th/macrophage ratios, suggesting a tolerogenic environment. Overall, IFN-τ modulates PBMCs in vitro and promotes a favorable endometrial immune profile in vivo, highlighting its potential as an immunotherapy in assisted reproduction.