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.

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.

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).

Objective: The aim of this study was to evaluate whether intrauterine administration of autologous peripheral blood mononuclear cells (PBMCs) activated with interferon tau (IFN-τ) before embryo transfer improves implantation and pregnancy outcomes in IVF patients. Methods: This single-center, prospective, randomized, controlled trial was conducted at Nadezhda Women’s Health Hospital (Approval No.: 6/28022023). The study was registered at ClinicalTrials.gov (NCT05775198). Randomization was computer-generated with allocation concealed via sealed envelopes. Participants and statisticians were blinded to group assignment; clinicians were not. Women aged 21–50 undergoing frozen–thawed embryo transfer with euploid embryos were included. Exclusion criteria included uterine anomalies, autoimmune, oncologic conditions, infections, or use of immunosuppressants. Participants (n = 340) were randomized 1:1 to receive either intrauterine infusion of autologous PBMCs activated in vitro with IFN-τ or standard IVF care without PBMC treatment. PBMCs were cultured with recombinant IFN-τ, washed, and infused 24 h prior to single euploid blastocyst transfer. A total of 14 patients were excluded from analysis because of early dropout, leaving 326 (n = 167; n = 159) patients for modified intention-to-treat analysis. Primary outcomes included implantation rate (elevated urinary or blood hCG), clinical pregnancy (fetal heartbeat at 6–8 weeks), and live birth rates. Miscarriage rate and safety were secondary objectives. Patients were followed up until 6 weeks post pregnancy resolution. Results: In the intervention group, 38.3% of patients achieved implantation, compared to 27.7% in the controls (OR 1.6, 95% CI: 1.0–2.6, p = 0.04). Live birth rates were also significantly higher in the IFN-τ-modulated PBMC group (28.7% vs. 17.6%, OR 1.9, 95% CI: 1.1–3.2; p = 0.02). While the clinical pregnancy rate was higher, it did not reach statistical significance (34.7% vs. 25.8%, p = 0.08). There was no difference between the groups in terms of miscarriage (p = 0.4). No serious adverse events were reported after treatment, during pregnancy or in the postnatal period. Conclusions: Intrauterine treatment with IFN-τ-activated PBMCs before ET significantly improves implantation and live birth rates in IVF patients.

Platelet-rich plasma (PRP) has emerged as a regenerative therapy for improving endometrial receptivity, although its effects on the endometrial immune microenvironment remain poorly understood. This prospective paired-sample study investigated changes in the endometrial immune microenvironment following intrauterine PRP administration during the window of implantation. Twenty-seven women undergoing assisted reproductive treatment provided paired endometrial biopsies before and after PRP administration. Stromal CD3+ T lymphocytes, CD4+ T-helper lymphocytes, CD56+ natural killer cells, and CD68+ macrophages were quantified by immunohistochemistry and digital image analysis. No significant overall changes in CD3+, CD4+, CD56+, or CD68+ cell abundance were observed after PRP administration (all p > 0.05). Nevertheless, exploratory analysis of individual paired trajectories showed that 77.8% of patients met the study-specific criterion for a coordinated immune remodeling pattern, defined as a concordant increase or decrease in at least three of the four analyzed immune cell populations. Patients with a coordinated-increase pattern had lower baseline CD68+ macrophage abundance than those with a coordinated-decrease pattern, with a large standardized effect size (Hedges’ g = 1.28) and nominal statistical significance (p = 0.04). However, this association did not remain significant after FDR correction (q = 0.165). These findings indicate heterogeneous, patient-specific patterns of endometrial immune-cell change following intrauterine PRP administration and suggest that baseline macrophage abundance may represent a candidate factor associated with subsequent immune remodeling patterns. The coordinated-remodeling classification and its association with baseline macrophage abundance should be considered exploratory and hypothesis-generating.

The role of the endometrial microbiome in embryo implantation remains incompletely understood. While most studies have focused on taxonomic composition and Lactobacillus dominance, the ecological organization of microbial communities associated with implantation success has received limited attention. The aim of this study was to investigate whether implantation outcome following euploid embryo transfer is associated with differences in endometrial microbial network structure and community organization. Endometrial biopsies collected during the window of implantation from 95 women undergoing subsequent euploid embryo transfer were analyzed using 16S rRNA gene sequencing. Microbial diversity, co-occurrence networks, community structure, hub taxa, network robustness, and microbial association patterns were evaluated. The Pregnant group demonstrated a more interconnected microbial network with a greater number of significant associations, larger ecological modules, and increased network integration than the Non-pregnant group. Seventeen microbial associations were unique to the Pregnant network, whereas eleven were unique to the Non-pregnant network, indicating implantation-associated ecological rewiring. Lactobacillus-centered association patterns also differed between groups. These findings suggest that successful implantation is associated with coordinated endometrial microbial community organization rather than changes in individual taxa. Microbial network structure may represent a novel dimension of endometrial receptivity assessment.