Integrative Analysis of Differentially Expressed miRNAs and Noncoding RNA Networks Reveals Molecular Mechanisms Underlying Metritis in Postpartum Dairy Cows.

MicroRNA data from our previous study [ 18 ] were used, in which Holstein dairy cows with metritis ( n = 4) and healthy controls ( n = 4) were selected. Blood samples were collected via coccygeal venipuncture for serum miRNA profiling using RT-PCR. For clarity, we have included a brief description of the methodology employed. Briefly, serum samples were processed, small RNAs were extracted and reverse transcribed, and mature miRNA expression was profiled using the Qiagen miScript PCR array, which targets 84 bovine miRNAs. Normalization was performed using cel-miR-39-3p and the global CT mean. Data analysis included CT quality control, normalization, and calculation of ΔCT values, fold changes, and statistical significance using a web-based tool. In the current study we employed integrative bioinformatics analyses to predict target genes, protein–protein interactions, and regulatory networks involving miRNAs, circRNAs, lncRNAs, snRNAs, and mRNAs associated with metritis, which are presented below. Nucleotide sequences of DE-miRNAs were retrieved from miRBase, ( www.mirbase.org accessed on 10 January 2025) and compared for sequence conservation between human and cattle [ 19 , 20 ]. Bovine sequences were very similar to human nucleotide sequences. Therefore, human miRNA IDs were used to construct miRNA-–mRNA interaction network and functional enrichment analysis. The target genes of DE-miRNAs were predicted using miRNet ( http://www.mirnet.ca/ accessed on 10 January 2025) [ 21 ]. This tool integrated data from multiple miR databases including TarBase, miRTarBase, and miRecords. The target prediction analysis was performed separately for upregulated and downregulated DE-miRNAs. The protein–protein interaction (PPI) network for predicted target genes of DE-miRNAs’ was generated the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online database ( http://string-db.org/ accessed on 10 January 2025) [ 22 ]. Gene Ontology (GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes (KEGGs) pathway enrichment analyses were conducted on these predicted targets. A p -value < 0.05 was regarded as statistically significant. The PPI network was exported to Cytoscape (version 3.9, accessed on 10 January 2025) for visualization [ 23 ]. Hub genes were identified as the top 30 nodes in the PPI network using the Maximal Clique Centrality (MCC) method [ 24 ], which is known for high precision in identifying essential proteins. Further analysis was conducted using ClueGO [ 25 ] (accessed on 10 January 2025) to integrate GO terms and KEGG pathways, generating functionally organized term networks (k score = 3). This tool supports the analysis of single or multiple gene lists and provides comprehensive visualization of functionally related terms. To explore the biological significance of DE-miRNAs and their associated genes, biological processes were analyzed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System ( https://pantherdb.org accessed on 13 January 2025). Additional GO terms and their co-occurring terms were investigated using QuickGO ( https://www.ebi.ac.uk/QuickGO accessed on 13 January 2025). Further analysis of hub genes included their roles, human tissue expression, and protein–protein interactions (up to 6 closely related genes) using data from STRING ( http://string-db.org/ accessed on 13 January 2025) and the human protein atlas ( https://www.proteinatlas.org accessed on 13 January 2025). Interferon-stimulated genes associated with up- and downregulated miRNAs in cows with metritis were extracted from miRNet’s target prediction output ( http://www.mirnet.ca/ accessed on 13 January 2025). Interaction networks among miRNA, circRNA, lncRNA, snRNA, and mRNA were generated using miRNet ( http://www.mirnet.ca/ accessed on 29 May 2025), separately for upregulated and downregulated miRNAs. Specific interaction maps, including miRNA–circRNA–mRNA, miRNA–lncRNA–mRNA, and miRNA–snRNA–mRNA networks, were also constructed.

Postpartum uterine diseases are categorized as puerperal metritis, clinical metritis, clinical endometritis, and subclinical endometritis based on the time of occurrence after calving and presence of clinical or subclinical signs [ 1 , 2 , 3 , 4 ]. Puerperal metritis was defined by an enlarged, flaccid uterus with a foul smelling, watery red-brown discharge, along with pyrexia and systemic illness occurring within 10 days of calving [ 4 ]. Other clinical signs include anorexia, depression, and decreased milk yield and feed intake [ 4 , 5 , 6 ]. In general, postpartum uterine diseases, irrespective of the type, inflict financial losses on dairy farms, mostly due to treatment costs and disposal of milk, decreased milk production, poor reproductive performance, and increased culling [ 2 , 5 , 6 ]. Although reproductive performance is compromised in dairy cows experiencing postpartum uterine disease, the exact mechanism by which the fertility is affected is not fully understood. Gene expression of key inflammatory cytokines in circulation varied between normal cows versus cows experiencing uterine disease [ 7 , 8 ]. Similarly, gene expression of cytokines also differed in an inflamed versus normal endometrium of postpartum cows [ 9 , 10 ]. We recently reported the influence of uterine inflammation negatively affecting the length of gestational day 16 conceptus [ 11 ]. Although several studies have considered genetic components of uterine inflammation [ 12 , 13 , 14 , 15 ], few have elucidated epigenetic changes such as altered expression of regulatory RNAs and their integration with coding genes involved in bovine metritis. The potential regulatory role of microRNAs (miRNAs) in the development and progression of bovine subclinical endometritis has been investigated by studying expression of miRNAs in uterine endometrial samples [ 13 , 14 ]. Recent studies have highlighted the role of noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and small noncoding RNAs (sncRNAs), in the regulation of immune responses, inflammation, and tissue repair [ 15 ]. The altered expression of these ncRNAs has been associated with disrupted cellular processes in the uterus [ 16 , 17 ]. Understanding how specific ncRNAs are dysregulated during uterine disease provides valuable insight into the molecular mechanisms underlying infertility and may guide the development of diagnostic or therapeutic strategies. In our previous study, 84 circulating miRNAs were profiled in postpartum dairy cows, identifying 30 differentially expressed (DE) miRNAs, 16 upregulated and 14 downregulated, in cows with metritis compared to healthy controls [ 18 ]. Building on these findings, the present study employed integrative bioinformatics analyses to predict target genes, protein–protein interactions, and regulatory networks involving miRNAs, circRNAs, lncRNAs, snRNAs, and mRNAs associated with metritis.

Of 84 prioritized miRNA previously profiled, 30 miRNAs were differentially expressed ( p < 0.05; fold regulation ≥ 2), 16 upregulated and 14 downregulated ( Figure 1 ), in circulation in cows with metritis compared to normal cows. These DE-miRNAs were analyzed to predict their target genes. Among the 16 upregulated miRNAs, 10 predicted 74 genes ( Supplementary File S1 ), while 10 of the 14 downregulated miRNAs predicted 123 genes ( Supplementary File S2 ). Figure 2 A presents PPI network for target genes of upregulated miRNAS (72 nodes; 281 edges; PPI enrichment p < 1.0 × 10 −16 ) revealing 511 significantly (False Discovery Rate (FDR) p < 0.05) enriched GO biological processes and 77 significantly (FDR p < 0.05) enriched KEGG pathways ( Supplementary File S3 ). Figure 2 B shows PPI network for the downregulated miRNAs (120 nodes and 189 edges, PPI enrichment p < 1.11 × 10 −14 ) with 299 significantly (FDR p < 0.05) enriched GO biological processes and 41 significant (FDR p < 0.05) KEGG pathways ( Supplementary File S4 ). PPI networks were constructed using the STRING database and visualized in Cytoscape. To interpret the functionally nested ontology and pathway annotation networks for genes targeted by up- and downregulated DE-miRNAs in cows with metritis, ClueGO analyses were performed and visualized in Figure 3 A–C and Figure 4 A–C, respectively. The top 30 hub genes identified using Maximal Clique Centrality (MCC) method for upregulated and downregulated DE-miRNAs are presented in Figure 5 A and Figure 5 B, respectively. Functional annotation and nested network analysis of these hub genes via ClueGO are shown in Figure 6 and Figure 7 . Table 1 A,B list the hub genes, their roles, and human tissue expression PPI (up to six closely related genes) for up- and downregulated miRNAs. Table 2 details interferon-stimulated genes targeted by DE-miRNAs in cows with metritis. Network analysis of the 16 upregulated miRNAs, including circRNA, lncRNA, snRNA, and mRNA interactions, revealed connections with 6490 unique circRNAs, 135 snRNAs, 357 lncRNAs, and 2169 genes ( Supplementary File S5 ). The full interaction network is illustrated in Figure 8 . In addition, specific miRNA to RNA interactions are detailed in Figure 9 A (miRNA to circRNA), Figure 9 B (miRNA to lncRNA), and Figure 9 C (miRNA to snRNA). Similarly, analysis of the 14 downregulated miRNAs revealed interactions with 6313 unique circRNAs, 144 snRNAs, 337 lncRNAs, and 3401 genes ( Supplementary File S6 ). The network is shown in Figure 10 . In addition, miRNA to circRNA, miRNA to lncRNA, and miRNA to snRNA interactions are shown in Figure 11 A, Figure 11 B, and Figure 11 C, respectively. Table 3 summarizes the top five coding and noncoding RNAs, ranked by high degree and betweenness centrality, targeted by the DE-miRNAs along with their endometrial expressions and potential functional roles.

Recent advances in high-throughput techniques have enabled the representation of experimental data as networks, where nodes (proteins, transcripts, or metabolites) are connected by edges to illustrate interactions among them. Network analysis helps to elucidate the role of individual proteins and their interactions, the protein–protein interactions. Centrality, a network ranking of biological components, has been widely used to identify influential nodes in complex biological networks [ 26 ]. These observed nodes with higher degrees (i.e., more connections) are more likely to represent essential proteins that significantly influence the biological process. Using these network-based methods, key biological mechanisms contributing to metritis and reduced reproductive performance in dairy cows were identified. In general, abnormal metabolic profile during the periparturient period interferes with immune function and predisposes the cow to postpartum uterine disease and subsequent infertility. Analysis of upregulated miRNAs and their associated genes in cows with metritis revealed a wide range of predicted biological processes both positively and negatively. Key predicted processes included regulation of cellular activities (metabolic process, biosynthetic process, protein modification, multicellular organismal), cell death, proliferation, stress responses, mitosis, cell cycle regulation, cell cycle arrest, adhesion, communication, endogenous stimuli, differentiation, maturation, motility, angiogenesis, receptor signaling, intracellular signal transduction, cellular component organization, development, and response to hypoxia, lipids, vitamins and ions, and hormone stimuli. In addition, processes such as immune response-regulating receptor signaling and regulation of T cells and leukocytes were also implicated. These biological processes are critical for timely uterine involution and any disruptions in these biological processes may result in delayed and impaired uterine recovery after calving. Interestingly, differentially upregulated miRNAs were associated with the cellular response to vitamins A (GO:0033189 and GO:0071300) and E (GO:0071306). In cows with metritis, higher lipid peroxidation and lower plasma concentrations of vitamin A and vitamin E were observed during the first 3 weeks postpartum compared to healthy cows [ 27 ]. Beta-carotene, a precursor to vitamin A, is essential for cellular health. Improved reproductive efficiency has been associated with increased postpartum β-carotene concentration in plasma [ 28 ]. Although dietary β-carotene supplementation during the dry period did not affect ovarian activity, progesterone production, or the diameters of the cervix and uterine horns [ 29 ], its association with hydroxyproline, a key component in uterine tissue repair, suggests that it may help reduce uterine inflammation and accelerate uterine involution after calving. In contrast, prepartum supplementation with Selenium (Se), alpha-tocopherol, or both, did not improve postpartum ovarian activity, uterine involution, or reduce clinical abnormalities [ 30 ]. However, vitamin E and Se supplementation decreased the incidence of metritis, reduced the number of services per conception, and shortened days open, though they had no effect on retained fetal membranes [ 31 ]. Notably, prepartum Se treatment, administered 3 weeks before calving, significantly hastened uterine involution in cows diagnosed with metritis [ 32 ]. Collectively, vitamins A and E appear to reduce the incidence of uterine diseases, promote uterine recovery, and support earlier resumption of ovarian cyclicity in postpartum dairy cows. Additional biological processes predicted from DE-miRNAs included cellular response to lipids (GO:0033993), regulation of ovarian follicle development (GO:0001541) embryo development (GO:0009790, GO:0043009), and embryo implantation (GO:0007566). The positive effects of fatty acid supplementation around calving on reproductive performance in dairy cows have been well documented [ 33 , 34 ]. For example, dairy cows supplemented with calcium salts of safflower oil (SO) from 30 days prepartum to 35 days postpartum exhibited improved innate immunity. Neutrophil functions, phagocytosis, and oxidative burst at 4 days postpartum (dpp), and oxidative burst at 7 dpp, were significantly higher in SO-fed cows compared to those fed palm oil (PO). Neutrophil surface expression of L-selectin and cytokine production (TNF-α, IL-1β) at 35 dpp were also increased in SO-fed cows [ 34 ]. These immunomodulatory effects may enhance the cow’s ability to combat bacterial challenges during the postpartum period. Furthermore, supplementation with unsaturated fatty acids (UFAs) from flaxseed reduced pregnancy losses [ 35 ]. Diets enriched with UFAs from flaxseed or sunflower sources promoted embryo development [ 36 ], while linolenic acid supplementation led to the development of significantly larger ovarian follicles [ 35 ]. Additionally, long-chain fatty acid supplementation postpartum was shown to hasten the resumption of ovarian cyclicity and reduce the incidence of cystic ovarian degeneration [ 37 ]. These findings indicate that lipid supplementation not only supports immune function but also improves uterine health, follicular development, and embryo viability. KEGG pathway analysis of genes associated with upregulated miRNAs revealed enrichment in several key signaling pathways, including PI3K-Akt, relaxin, p53, AMPK/MAPK, ErbB, JAK-STAT, IGF receptor, T cell receptor, HIF-1, TNF, FoxO, estrogen, GnRH, NF-κB, C-type lectin receptor, and Ras pathways. These networks involve genes and their products that mediate cellular responses to intrinsic and extrinsic stimuli. They regulate essential processes such as DNA replication, chromosome segregation, cell division, metabolism, proliferation, survival, growth, and angiogenesis, processes that are critically involved in postpartum uterine recovery and reproductive function. The PI3K-Akt signaling pathway is a key intracellular signal transduction pathway that regulates metabolism, cell proliferation, survival, growth, and angiogenesis in response to extracellular stimuli. Dysregulation of this pathway has been implicated in endometriosis in women, where elevated levels of PI3K and phosphorylated AKT (Ser473) contribute to abnormal cell proliferation [ 38 , 39 , 40 ]. It should be noted that hormones could play an essential role in these regulatory pathways; for instance, estradiol (E2) promotes endometriotic cell proliferation through the reduced expression of PTEN and the subsequent activation of AKT [ 41 ]. In cows, pathogenic Escherichia coli lipopolysaccharide induces endometrial inflammation via the Toll-Like Receptor (TLR)4-IRAK-TRAF4-NF-κB signaling axis. Activation of interleukin-1 receptor-associated kinase (IRAK) and TNF receptor-associated factor 4 (TRAF4) by LPS leads to nuclear translocation of NF-κB, triggering inflammation and apoptosis in bovine endometrial cells [ 42 ]. Analysis of upregulated miRNAs and their associated genes in metritis cows also predicted enrichment of insulin receptor binding (GO:0005158), insulin resistance (KEGG pathway), and insulin-like growth factor receptor (IGFR) signaling (GO:0048009). Insulin plays a critical role in homeorhesis during the transition period. Both insulin secretion and tissue responsiveness to insulin are altered postpartum. A disrupted metabolic profile—specifically impaired PMN function—has been linked to increased susceptibility to postpartum uterine diseases and infertility [ 43 ]. Cows that developed uterine disease had lower circulating glucose and reduced glycogen concentrations in their PMNs [ 41 ]. Reduced glycogen storage likely impairs the oxidative burst capacity of PMNs, weakening immune defense mechanisms and predisposing cows to uterine infections [ 44 ]. The top five highly upregulated miRNAs in cows with metritis were bta-miR-15b, bta-miR-17-3p, bta-miR-16b, bta-miR-148a, and bta-miR-26b. MicroRNAs are involved in the regulation of several biological functions and pathways. The proposed functions of miR-15b include hypoxia, angiogenesis, and apoptosis; miR-16 is associated with hypoxia, angiogenesis, inflammation, cell growth, and apoptosis; miR-26 is involved in cell proliferation, myogenesis, cell cycle progression, BMP signaling, and cell differentiation; miR-142 is linked to immune suppression. Increased expression of miR-15b reduced activities of anti-apoptotic protein BCL2 [ 45 ], whereas miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness through regulation of cytoskeletal elements, cell adhesion molecules, and proteolytic factors [ 46 ]. Notably, bta-miR-26b, derived from intrauterine extracellular vesicles, contributes to suppression of maternal neutrophil-mediated immunity, thereby possibly promoting uterine inflammation [ 47 ]. Despite the generally pro-inflammatory profile of the upregulated miRNAs, miR-148a appears to have a compensatory anti-inflammatory role, countering uterine inflammation through the TLR4–NF-κB axis [ 48 ]. In contrast, the top five downregulated miRNAs were bta-miR-148b-5p, bta-miR-199a-3p, bta-miR-122-5p, bta-miR-200b-3p, and bta-miR-10a-5p in cows with metritis. miR-148b target genes TNFRSF10B are involved in regulation of T cell function and apoptosis and enhanced bactericidal activity [ 49 ]. MicroRNA-199a-3p suppresses high glucose-induced inflammation by regulating the IKKβ/NF-κB signaling pathway in epithelial cells [ 50 ]. Further, miR-199a-3p is significantly lower in women with endometriosis compared to normal women [ 51 ]. MicroRNA-200 family regulates cellular proliferation and migration in the endometrium, and downregulation may interfere with regulation of the postpartum immune response in women [ 52 , 53 ]. The downregulation of these miRNAs likely impairs immune function, cellular repair, and inflammatory resolution, potentially contributing to persistent uterine inflammation and delayed involution. GO/pathway terms specific for upregulated hub genes included regulation of transcription involved in G1/S transition of mitotic cell cycle to prevent mitotic catastrophe, beta-catenin binding, phosphatidylinositol phosphate kinase activity, filopodium, and negative regulation of vasculature development and poly-purine tract binding. Regulatory mechanisms including crosstalk between TLR and Wnt/β-catenin signaling pathways may elicit both pro- and anti-inflammatory functions that show involvement in both normal and pathological conditions [ 54 ]. Phosphatidylinositol phosphate kinase is essential for the activation of the signaling pathways regulating cytokine production, cell cycle progression, survival, T cell metabolism, and focal adhesions [ 55 , 56 ]. Filopodia have roles in sensing, migration, and cell–cell interaction. In macrophages, filopodia act as phagocytic tentacles and pull bound objects towards the cell for phagocytosis [ 57 ]. Both innate and adaptive immune cells are involved in the mechanisms of endothelial cell proliferation, migration, and activation. Anti-angiogenic cytokines such as IFN-gamma and IL-12 [ 58 ] may be involved in negative regulation of vasculature development and tissue repair in the uterus of postpartum cows. GO/pathway terms specific for downregulated hub genes were angiogenesis, endoderm formation, morphogenesis, regulation of cell differentiation, macrophage differentiation, and regulation of cyclin-dependent protein serine/threonine kinase activity, essential to drive cell cycle progression and transition into different phases [ 59 ]. Chemokine family CXL ligand (CXCL) mRNAs are upregulated in dairy cows with endometritis [ 9 , 10 ]. Further, upregulated expression of bta-miR-101 may be associated with TLR2. The overexpression of TLR2 is associated with the activation of the NF-κB-mitogen-activated protein kinase B (MAP3KB) complex and consequent upregulation of pro-inflammatory cytokines and chemokines [ 60 ]. This overexpression of these inflammatory cytokines and chemokines can induce severe endometrial tissue damage postpartum and adversely prolong uterine involution and negatively impact future pregnancy outcomes. We observed that interferon-stimulated genes were downregulated in cows with subclinical endometritis, which may adversely affect embryo elongation [ 9 , 10 ]. Differentially expressed (DE) miRNAs and their associated ISG targets are listed in Table 2 . Insights from cancer biology have expanded our understanding of the role of noncoding RNAs in uterine diseases of dairy cows. Among upregulated lncRNAs, KCNQ1OT1 and NEAT1 are of particular interest. KCNQ1OT1 is involved in epigenetic regulation and may influence immune-related gene expression [ 61 , 62 ]. NEAT1 is essential for paraspeckle formation and amplifies inflammatory signaling [ 63 ]. Its overexpression in uterine tissue has been linked to chronic inflammation and impaired healing. The expression of TUG1 was significantly decreased in HUVECS following lipopolysaccharide treatment in a time-dependent manner [ 64 ]. Other lncRNAs such as XIST, HELLPAR, and TUG1 are also linked to immune modulation and epithelial cell function; their dysregulation may contribute to uterine dysfunction [ 65 , 66 , 67 , 68 , 69 ]. Upregulated circRNAs such as RANBP2, RGPD4, and NBPF10 may influence intracellular transport and immune signaling, potentially exacerbating inflammation [ 70 , 71 , 72 , 73 , 74 ]. Meanwhile, sncRNAs including SNORD17 regulate RNA modification and protein synthesis, with elevated levels indicating cellular stress responses [ 75 , 76 ]. Conversely, the downregulation of lncRNAs like SNHG16, HCG18, and NEAT1 may impair immune responses and placental development [ 77 , 78 , 79 , 80 ]. Similarly, decreased expression of circRNAs (KPNA6, MACF1) and sncRNAs (SNORA66) may disrupt in cytoskeletal organization and RNA processing [ 81 , 82 , 83 ]. It should be noted that some lncRNAs such as KCNQ1OT1, NEAT1, and XIST appear to have dual roles. KCNQ1OT1 affects gene expression and several cellular functions including cell proliferation, migration, epithelial–mesenchymal transition (EMT), apoptosis, viability, autophagy, and inflammation [ 84 ]. NEAT1 functions as an miRNA sponge, regulating gene expression involved in cell growth, invasion, EMT, stemness, and resistance to chemotherapy and radiotherapy [ 85 ]. •XIST, while originally known for X-chromosome inactivation, also influences immune gene expression and participates in multiple signaling pathways, including TGF-β, PI3K/AKT, Wnt/β-catenin, FOXO, NF-κB, mTOR, MAPK, Toll-like receptor, JAK-STAT, and T/B cell receptor pathways [ 86 , 87 ]. Upregulation of XIST may lead to aberrant gene silencing and immune dysregulation, whereas its downregulation may impair chromosomal regulation and immune homeostasis, worsening uterine dysfunction. In our network analysis, KCNQ1OT1 exhibited both upregulation and downregulation in different contexts, suggesting its role in immune homeostasis. When upregulated, it may suppress immune-related gene expression and prolong inflammation. When downregulated, it may interfere with genomic imprinting and immune regulation, hindering uterine recovery and increasing infection susceptibility. NEAT1, essential for paraspeckle formation, modulates inflammatory gene expression. Its upregulation may enhance innate immune responses but also cause chronic inflammation and tissue damage. Conversely, NEAT1 downregulation may impair protective immunity and delay healing—both conditions negatively affecting uterine health and fertility. XIST is key to X-chromosome inactivation but also regulates immune gene expression. Its upregulation may cause abnormal gene silencing and immune imbalance, while downregulation can impair chromosomal function and immune regulation, further aggravating uterine disorders. We observed that the regulatory roles of noncoding RNAs vary widely across biological processes. However, these RNAs often exert their effects by interacting with miRNAs to modulate downstream gene expression. Although the role of noncoding RNAs in many signaling pathways remains underexplored, their functional significance is becoming increasingly evident. Continued research into their mechanisms will likely uncover new insights and therapeutic targets in uterine disease.

This study used high-throughput miRNA profiling and integrative network analyses to explore molecular mechanisms underlying postpartum uterine disease, particularly metritis in dairy cows. Differentially expressed miRNAs and their target genes were implicated in key biological processes and signaling pathways involved in immune regulation, inflammation, metabolism, cellular homeostasis, and reproduction. Protein–protein interaction and ClueGO analyses linked these miRNAs to numerous enriched GO terms and KEGG pathways, highlighting roles in cellular stress, angiogenesis, immune signaling, and hormonal regulation—critical for uterine involution and reproductive recovery. Centrality analyses identified hub genes associated with inflammation resolution and tissue remodeling. Interaction networks involving miRNAs and noncoding RNAs (circRNAs, lncRNAs, snRNAs) revealed complex regulatory mechanisms. Notably, NEAT1, XIST, and KCNQ1OT1 exhibited dual roles in modulating inflammatory responses, potentially influencing disease progression or recovery. The study also identified miRNA-regulated pathways tied to vitamins A and E, lipid metabolism, insulin resistance, and immune function, factors linked to postpartum health. These findings suggest that metritis involves widespread dysregulation of immune and metabolic pathways, possibly worsened by nutritional deficiencies. In summary, this integrative transcriptomic analysis provides a detailed molecular view of postpartum uterine health, highlighting miRNA, and noncoding RNA networks plays a role in disease development and recovery, offering promising biomarkers and therapeutic targets for improving fertility in dairy cows.