DISCUSSION

Endometrial epithelial proteomic remodeling during uterine receptivity

Embryo implantation is a tightly regulated biological process in which the blastocyst attaches to and interacts with the uterine endometrium [21]. The endometrium is a hormonally responsive mucosal tissue lining the uterus that provides structural support, nourishment, and immunological tolerance for the developing conceptus. Transformation of the endometrium into a receptive state requires coordinated regulation of epithelial differentiation, stromal remodeling, immune modulation, and metabolic adaptation [13, 22]. Ovarian steroid hormones, particularly progesterone, regulate adhesion molecules, cytokines, and growth factors that collectively determine uterine receptivity and embryo–maternal communication [23, 24]. In mice, embryo attachment occurs on Day 4 of pregnancy following vaginal plug detection on Day 1 and coincides with luminal closure and epithelial remodeling [11].

In the present study, comparative proteomic profiling of mouse endometrial epithelial cells between Days 1 and 4 of pregnancy identified 80 differentially expressed proteins, of which 52 were successfully characterized. Functional annotation indicated that the identified proteins were primarily associated with biological processes, including cellular metabolism, biological regulation, cytoskeletal organization, protein folding, antioxidant activity, and reproductive system development. Collectively, these findings suggest that the acquisition of uterine receptivity involves coordinated metabolic activation, stress adaptation, and structural stabilization within the epithelial compartment. Notably, this study is among the relatively few 2-DE/MALDI-TOF-based proteomic analyses focused specifically on isolated mouse endometrial epithelial cells and integrates redox/antioxidant pathways, ER stress responses, and cytoskeletal components into a single epithelial-specific dataset. In contrast to whole-endometrium transcriptomic or proteomic studies and uterine fluid proteomics, which reflect composite signals from multiple cellular compartments or secreted proteins, the present epithelial-focused approach enables more precise characterization of cell type–specific molecular changes at the embryo–maternal interface.

Furthermore, building upon previous work by Ni_ et al._ [25], our study extends these findings by situating Gstm2 within a broader proteomic context and reinforcing its biological relevance through combined proteomic identification and dual validation at both protein and transcript levels. In addition, the Gene Ontology analysis in this study was descriptive and did not include formal enrichment testing; therefore, the reported functional categories should be interpreted as general annotations rather than statistically overrepresented pathways.

Among the identified proteins, PDIA3 and HSPA5 (GRP78) are central regulators of endoplasmic reticulum (ER) homeostasis. PDIA3 (ERp57) functions as a protein disulfide isomerase involved in protein folding and ER stress responses [26]. GRP78 is a key ER chaperone participating in unfolded protein response signaling and trophoblast function [26]. Controlled ER stress signaling has been implicated in implantation and placental development, facilitating cellular adaptation during periods of increased biosynthetic demand. The modulation of these proteins in our dataset suggests that epithelial cells undergo adaptive stress responses during the transition to uterine receptivity.

Antioxidant regulation and the role of Gstm2

Redox regulation emerged as another prominent functional category. Successful pregnancy requires balanced oxidative signaling; excessive reactive oxygen species may impair epithelial integrity and embryo–maternal interactions, whereas controlled redox activity supports implantation-associated remodeling [15, 27]. Gstm2, a member of the glutathione S-transferase family, plays an important role in cellular antioxidant defense by catalyzing detoxification reactions [25]. Importantly, Gstm2 was identified alongside multiple antioxidant and stress-associated proteins, including Gstm7, Prdx2, Cat, PDIA3, and HSPA5, suggesting that it functions within a coordinated progesterone-responsive antioxidant network rather than as an isolated factor.

A previous study by Ni et al. [25] demonstrated that Gstm2 expression in the mouse uterine luminal epithelium is regulated by progesterone during the preimplantation period; however, that study focused on hormone-dependent gene regulation rather than global proteomic context. In the present study, Gstm2 is not proposed as a novel implantation-related factor; rather, our findings extend this prior observation by placing Gstm2 within a broader epithelial-specific proteomic framework. In contrast to prior single-gene or transcript-level studies, the present work identifies Gstm2 through unbiased proteomic profiling and demonstrates its coordinated regulation alongside proteins involved in redox homeostasis, ER stress responses, and metabolic adaptation.

Gstm2 showed consistent upregulation at both the protein and transcript levels during the receptive phase (Day 4), with increased localization in luminal and glandular epithelial compartments. Importantly, this upregulation occurred alongside coordinated changes in proteins associated with redox regulation, ER stress, and metabolic adaptation, suggesting that Gstm2 may be associated with a broader antioxidant regulatory network during uterine receptivity. These observations are correlative, and further functional studies will be required to define its mechanistic role.

In addition to Gstm2, several well-established markers of uterine receptivity have been reported in previous transcriptomic and proteomic studies, including adhesion molecules such as integrins, mucins, and progesterone-regulated genes such as leukemia inhibitory factor (LIF) and HOXA10 [24, 28]. Although these classical markers were not identified in the present dataset, this may reflect methodological differences, including the epithelial-specific focus and the limited proteome coverage inherent to gel-based approaches. Previous omics studies have demonstrated that uterine receptivity involves coordinated regulation of adhesion, immune modulation, and metabolic adaptation pathways [3, 11, 12]. In this context, our findings complement existing literature by highlighting proteins associated with redox regulation and ER stress responses, suggesting that antioxidant defense mechanisms may represent an additional regulatory layer during the implantation window. Furthermore, although other glutathione S-transferase family members, such as Gstm7_,_ were detected, Gstm2 exhibited the most consistent and pronounced upregulation, supporting its potential relevance within this protein family.

Importantly, the temporal overlap between Gstm2 upregulation on Day 4 and key implantation-associated events, including luminal closure and epithelial remodeling, suggests its involvement in a progesterone-responsive regulatory framework during the establishment of uterine receptivity. Progesterone regulates uterine fluid absorption and lumen closure, processes essential for blastocyst immobilization and implantation [29–31]. Elevated progesterone levels during Days 3 and 4 of pregnancy have been associated with increased uterine Gstm2 expression [32–34]. Thus, Gstm2 may be associated with progesterone-regulated pathways, although this relationship was not directly examined in the present study. Together, these observations support the interpretation that Gstm2 operates within a progesterone-responsive antioxidant network that is temporally aligned with critical implantation events in the uterine epithelium.

Cytoskeletal organization and extracellular matrix remodeling

In contrast, vimentin expression did not differ significantly between Days 1 and 4 of pregnancy, although immunofluorescence confirmed its localization within stromal and decidual compartments. Vimentin is a major intermediate filament protein that maintains cytoskeletal integrity and structural resilience [35–37]. Cytoskeletal remodeling is critical during decidualization and trophoblast invasion; however, the early preimplantation window evaluated here precedes extensive trophoblast penetration. The relative stability of vimentin expression during this period likely reflects preservation of structural readiness rather than active remodeling.

Developmental studies indicate that trophoblast giant cells begin expressing vimentin around embryonic Day 7.5, coinciding with vascular remodeling and exposure to maternal blood flow [38]. Earlier work demonstrated that trophoblast expansion and formation of maternal blood sinuses occur between embryonic Days 6.5 and 7.5 [39, 40]. Therefore, the absence of significant vimentin modulation between Days 1 and 4 is consistent with the timing of implantation events. Rather than serving as a dynamically regulated marker during early receptivity, vimentin likely provides a stable cytoskeletal scaffold that maintains endometrial structural integrity prior to invasive placentation.

Additional identified proteins, including cathepsin B and HSP47, are associated with extracellular matrix remodeling and collagen processing [41–43]. Extracellular matrix turnover and collagen stabilization are essential components of implantation and placentation. Their presence among differentially expressed proteins further underscores the coordinated structural and functional adaptation occurring within the receptive endometrium. The integration of proteomic profiling with immunolocalization and quantitative gene expression analysis strengthens the reliability of these findings, as multi-level validation enhances biological confidence and reduces the likelihood of false-positive identification [44]. The concordant upregulation of Gstm2 at both protein and transcript levels supports its biological relevance during the implantation window, consistent with evidence emphasizing the importance of redox homeostasis in uterine receptivity [15, 45]. Meanwhile, stable vimentin expression confirms its role in maintaining baseline cytoskeletal organization during early pregnancy.

Study limitations, methodological considerations, and future perspectives

Several methodological considerations should be acknowledged. Pooling of epithelial samples was employed to ensure sufficient protein yield and to minimize inter-individual variability, a strategy commonly used in exploratory proteomic studies [46]. However, this approach may obscure biological variation among individual animals. To address this limitation, selected candidate proteins were independently validated using immunofluorescence and quantitative real-time PCR, thereby supporting the reliability of the observed expression patterns.

Although gel-based proteomics provides lower proteome coverage than modern high-resolution LC-MS/MS approaches, 2-DE/MALDI-TOF remains a robust and complementary strategy for resolving protein isoforms and post-translational variants, while enabling reproducible comparative profiling of relatively abundant proteins. In the present study, this approach was particularly suited to the analysis of isolated endometrial epithelial cells, where protein yield is inherently limited, and was aligned with the exploratory objective of capturing major proteomic shifts during the transition to uterine receptivity. Importantly, this platform enables direct visualization of protein expression patterns, which facilitates biological interpretation in a cell type–specific context. Nevertheless, future studies employing high-resolution quantitative LC-MS/MS will be essential for extending proteome coverage and further refining these findings. In this study, its use was further supported by the limited protein yield from isolated epithelial cells and the exploratory objective of identifying major proteomic changes during uterine receptivity.

Furthermore, antibody validation in this study was limited to negative controls and consistency with known expression patterns, and additional validation using orthogonal approaches (e.g., Western blotting or genetic models) would strengthen confidence in protein specificity. Although stringent fold change thresholds were applied to minimize false-positive identifications, future investigations employing quantitative LC-MS/MS platforms combined with targeted functional assays will be necessary to establish causal relationships between candidate proteins and implantation outcomes. In addition, the absence of a formal multiple-testing correction may increase the likelihood of false-positive identifications, as approximately 5% of detected spots are expected to reach statistical significance by chance alone.

Furthermore, several methodological and biological limitations should be considered when interpreting the present findings. First, the use of two-dimensional gel electrophoresis (2-DE) inherently introduces analytical bias, as this technique preferentially detects abundant, soluble proteins within a moderate range of isoelectric points and molecular weights, potentially underrepresenting low-abundance, membrane-associated, or extreme pI/MW proteins. Second, although endometrial epithelial cells were isolated using established protocols, the possibility of minor contamination from stromal or other uterine cell types cannot be completely excluded, which may influence the detected proteomic profile. Third, while Day 4 samples were collected based on established peri-implantation timing following vaginal plug detection, the presence of embryos was not directly confirmed by uterine flushing. Therefore, the observed proteomic changes may primarily reflect maternal hormonal and physiological regulation rather than embryo-derived signaling. Finally, this study was conducted using a single outbred mouse strain (CD-1), and thus, the generalizability of the findings to other strains or species may be limited. Future studies incorporating embryo verification, improved cell type purification, and high-resolution proteomic approaches across multiple genetic backgrounds will further refine the understanding of uterine receptivity mechanisms.

Translational relevance and integration with contemporary omics studies

Endometrial receptivity is a key determinant of implantation success in assisted reproductive technologies, and impaired receptivity is a major contributor to recurrent implantation failure (RIF) [11, 12, 24]. In recent years, considerable effort has been directed toward identifying reliable molecular biomarkers to improve assessment of the window of implantation and optimize embryo transfer strategies [23, 24]. Although the present study was conducted in a mouse model, several of the identified pathways, including progesterone-associated signaling, redox homeostasis, and epithelial remodeling, are conserved across species [3, 11]. In this context, the upregulation of Gstm2 and other proteins involved in antioxidant defense and ER stress responses may serve as candidate biomarkers relevant to human endometrial function. Notably, dysregulation of oxidative stress pathways has been associated with impaired receptivity and implantation failure in women [11, 12].

Therefore, the candidate proteins identified in this study may represent components of a broader molecular framework underlying endometrial competence. However, translating these findings into clinical application requires validation in human endometrial samples, particularly in patients with RIF or undergoing assisted reproduction, as well as integration with existing receptivity biomarkers. Although Gstm2 was identified as a significantly altered protein in this study, it should not be considered a standalone marker of endometrial receptivity. Rather, it represents a potential candidate biomarker within a complex molecular network, as evidenced by the identification of multiple proteins associated with this process. Due to the exploratory nature of this study, validation was limited to a subset of candidates; future studies should systematically validate additional key candidates (e.g., PDIA3, HSPA5/GRP78, HSP47, and antioxidant-related proteins) using targeted and functional approaches.

Recent advances in transcriptomic and multi-omics approaches, including single-cell RNA sequencing and uterine fluid proteomics, have significantly expanded our understanding of endometrial receptivity by revealing cell type–specific differentiation trajectories, immune regulation, and hormone-responsive signaling pathways during the implantation window [3, 11, 12]. In particular, single-cell transcriptomic studies have highlighted dynamic epithelial remodeling and coordinated interactions between epithelial, stromal, and immune compartments, while human studies in RIF have identified dysregulation of pathways related to cellular stress, metabolism, and hormonal responsiveness [5, 12]. However, most of these studies are based on transcript-level data or whole-tissue analyses, which may not fully capture protein-level changes or cell type–specific molecular dynamics at the epithelial interface.

In this context, the present study complements existing literature by providing an epithelial-specific proteomic perspective, revealing coordinated changes in proteins associated with redox regulation and ER stress responses during the transition to uterine receptivity. Notably, the identification of antioxidant proteins (Gstm2, Gstm7, Prdx2, and Cat) together with ER chaperones (PDIA3 and HSPA5) suggests that redox homeostasis and protein folding capacity are jointly modulated in epithelial cells, representing an additional layer of regulation that may not be fully resolved by transcriptomic approaches alone. Within this framework, Gstm2 is not interpreted as a standalone regulatory factor but rather as a candidate protein whose upregulation is associated with broader epithelial stress-adaptation processes during the receptive phase. These findings are consistent with accumulating evidence linking oxidative stress regulation to endometrial function and implantation success [11, 12], while emphasizing the importance of integrating protein-level data to refine current models of uterine receptivity.