Uterine leiomyoma (ULM), also referred to as uterine fibroids or myomas, represents the most prevalent benign tumor in the female reproductive system (1). Epidemiological evidence indicates that the incidence of ULM increases with age, with a particularly high prevalence among women of reproductive age. In women over 35 years, the incidence rate ranges from approximately 20% to 40% (2). Several factors, including environmental and psychological influences, contribute to ULM development. Notably, the elevation of estrogen receptor concentrations is widely accepted as a primary driver of ULM formation (3). Furthermore, familial clustering observed in numerous ULM cases suggests a substantial genetic component in its pathogenesis (4). Despite these insights, the precise molecular mechanisms underlying ULM remain incompletely understood, necessitating further investigation into potential pathogenic pathways and biomarkers to improve patient quality of life.

Oxidative stress refers to the disruption of the equilibrium between reactive oxygen species (ROS) production and the body’s endogenous antioxidant defenses (5). While basal levels of ROS are crucial for maintaining cellular homeostasis, excessive ROS can inflict structural and genetic damage, including protein and lipid peroxidation, which can impair cellular function and trigger apoptosis, contributing to disease progression (6). The uterus, within the female reproductive system, is particularly vulnerable to oxidative stress and subsequent DNA damage, which may be pivotal in ULM development (7, 8). Whole genome sequencing has elucidated the genetic foundation of ULM, revealing that over 70% of leiomyoma (LM) cases harbor mutations in the mediator complex subunit 12 (MED12), with mutation rates positively correlating with tumor quantity. ROS is implicated in both promoting MED12 mutations and facilitating tumor growth (9). Research suggests that uterine muscle contractions and vascular changes during the menstrual cycle induce hypoxic microenvironments within the uterine muscle, which subsequently trigger gene expression promoting leiomyoma proliferation under low oxygen conditions (10). Leiomyoma cells exhibit lower expression of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), compared to normal uterine muscle cells, particularly in hypoxic environments. ULM is characterized by elevated oxidative stress and insufficient antioxidant defense capacity (11). Therefore, investigating the role of oxidative stress in ULM pathogenesis is essential for advancing diagnostic and therapeutic strategies. Biomarkers of oxidative stress may emerge as valuable diagnostic tools for identifying patients with ULM. While medical intervention is often necessary for ULM treatment, antioxidants have shown potential in both prevention and therapeutic management, though further research is required. In summary, oxidative stress represents a critical and emerging aspect of ULM pathology, playing a central role in its progression. Further experimental and clinical research is needed to elucidate the cellular and molecular mechanisms linking oxidative stress to ULM.

Mendelian randomization (MR) represents a sophisticated approach in epidemiology and related fields for establishing causal relationships, utilizing genetic variation to evaluate the causal influence of potential risk factors on health outcomes. By leveraging the random assortment of alleles, MR effectively mitigates confounding factors that often obscure the exposure-outcome relationship in observational studies, enabling a more reliable and unbiased assessment of exposure effects on various outcomes compared to traditional methodologies (12, 13). MR analysis is contingent upon three fundamental assumptions: relevance (the genetic variant must be strongly associated with the exposure), independence (the genetic variant should be independent of confounders that affect the exposure-outcome relationship), and exclusion restriction (the genetic variant influences the outcome solely through its effect on the exposure, with no alternative direct causal pathways). Ensuring these assumptions are met is critical for the validity of causal inferences derived from MR studies (14). In parallel, the advent of microarray technology has revolutionized biological research by enabling high-throughput transcriptome analysis, transitioning investigations from single-gene studies to genome-wide expression profiling. Over the past two decades, microarray data have contributed substantially to advancing diverse areas of biological research, offering significant advantages for expression profiling and large-scale data analysis (15).

This study employed publicly available datasets, including GSE64763 and GSE31699, which are associated with ULM, alongside 436 oxidative stress-related genes. We chose GSE64763 because the dataset contains rich gene expression data covering multiple disease states and normal control samples, which is critical for identifying disease-associated genes and pathways. In addition, Dai Huang et al. (16) and Yumin Ke et al. (17) also used the GSE64763 dataset to conduct ULM-related research. The GSE31699 dataset, on the other hand, focuses on molecular marker studies of ULM, providing in-depth gene expression analysis, which is important for studying disease progression and treatment response mechanisms. Research by Lei Cai et al. (18) is based on this dataset. Genome-wide association study (GWAS) data for ULM and relevant candidate genes were sourced from the IEU OpenGWAS database. A comprehensive analytical framework was utilized, comprising differential expression analysis, weighted gene co-expression network analysis (WGCNA), MR, receiver operating characteristic (ROC) curves, and other statistical methodologies to identify oxidative stress-related biomarkers in ULM. Gene set enrichment analysis (GSEA) was performed to elucidate functional pathways associated with these biomarkers, while immune infiltration analysis was conducted to examine their role in the immune microenvironment. Additionally, potential therapeutic agents targeting the expression of these biomarkers were predicted ( Supplementary Figure S1 ). Investigating the mechanisms underlying oxidative stress-related biomarkers in ULM is pivotal for advancing diagnostic and therapeutic strategies, as well as for guiding future research in this domain.

ULM is the most prevalent benign tumor in the female reproductive system, often asymptomatic and incidentally detected during routine medical examinations. Although the exact etiology of ULMs remains unclear and specific tumor markers for diagnosis are lacking (36), growing evidence implicates oxidative stress as a key factor in their development and progression (11, 37). Oxidative stress can induce abnormal proliferation of ULM cells, promote DNA synthesis, and alter cell cycle dynamics, thereby driving tumor growth (38–40). Additionally, it influences cell apoptosis, inflammatory responses, and angiogenesis, further exacerbating ULM pathogenesis (10, 41–43). However, the precise roles of oxidative stress-related genes in ULMs remain insufficiently understood. In this study, transcriptional datasets from the GEO database were analyzed, identifying four biomarkers (ANXA1, CD36, MICB, and PRDX6) associated with oxidative stress in ULMs through differential analysis, WGCNA, and MR. MR analysis revealed that these biomarkers exert significant effects on ULM. ROC curve analysis further underscored their diagnostic potential for ULMs. Additionally, a predictive alignment chart incorporating these biomarkers was developed, effectively forecasting ULM risk.

In the NCBI (National Center for Biotechnology Information), there are more than 10 datasets on ULM. These data sets are used in different studies for different purposes and sample characteristics. For example, the study by José A Castro-Martínez et al. (44) used two datasets, GSE12814 and GSE23112. GSE23112 focused on microRNA expression, while GSE12814 was associated with molecular signatures of the del(7q) UL subgroup. Through comprehensive transcriptome analysis, this study provides an in-depth understanding of the molecular mechanisms of uterine diseases and provides scientific support for future diagnosis and treatment strategies. Another study (45) combined four datasets, GSE64763, GSE45188, GSE30673, and GSE593, to deepen our understanding of the pathogenesis of ULM and to identify genes involved in the development of ULM. Among them, GSE64763 is consistent with the one selected in this study; GSE45188 showed the DNA methylation profile of myometria in uterine fibroids and non-fibroids. GSE30673 focuses on MED12 mutation; GSE593 contained genome-wide expression profiles of 5 uterine fibroids and 5 normal uterine tissues. Compared with other datasets, we finally decided to use the two datasets GSE64763 and GSE31699 after comprehensively considering factors such as research design, requirement matching degree and sample size.

ANXA1, a 37-kDa protein belonging to the annexin A family, plays multifunctional roles in both innate and adaptive immunity (46). In recent years, the role of ANXA1 in tumors has garnered increased attention (47), with studies showing its expression varies by tissue type (48). For instance, ANXA1 is overexpressed in melanoma, hepatocellular carcinoma, gastric cancer, and lung cancer (49), while its expression is diminished in prostate and esophageal cancers (50). Jamaluddin et al. conducted genetic and proteomic analyses on ULM and adjacent normal muscle tissues to characterize the expression patterns of extracellular matrix (ECM) proteins in MED12 mutation-positive and -negative ULMs. Among ECM-related proteins, ANXA1 was found to be downregulated in ULMs of various sizes (51), aligning with the findings of this study and suggesting potential therapeutic targets.

MHC Class I polypeptide-related sequence B (MICB), a non-classical HLA-I gene within the human major histocompatibility complex, is highly polymorphic, with 40 polymorphic sites and 26 protein variants identified to date (52–54). MICB is a stress-inducible protein primarily expressed on cell membranes, acting as a ligand for NK cell receptors (53). Its surface expression is minimal in normal cells but markedly upregulated in tumor cells or cells infected with viruses (55). This study is the first to implicate MICB in ULM, laying the groundwork for future investigations. However, further research is needed to elucidate the underlying mechanisms of this interaction.

CD36, a transmembrane protein and member of the class B scavenger receptor family, interacts with other transmembrane proteins on the cell surface to mediate ligand binding and signal transduction (56). It is widely expressed on the surface of microvascular endothelial cells and plays a pivotal role in regulating endothelial cell function (57). In tumor microvasculature, CD36 binds to thrombospondin-1 (TSP-1), mediating endothelial cell apoptosis within tumor vasculature (58). Knapp et al., in their study on energy substrate transport proteins in ULM, discovered that CD36 expression was reduced in patients with ULM compared to matched healthy muscle layers (59). These findings align with our study results.

PRDX6, a peroxidase enzyme composed of 224 amino acids, is expressed in various tissues and organs. Recent research has highlighted its significant role in cancer. PRDX6 is notably overexpressed in cervical cancer tissues, where its overexpression promotes proliferation, migration, and invasion of cancer cells while inhibiting apoptosis (60). Our findings similarly indicate elevated PRDX6 expression in ULM. Research has demonstrated that PRDX6 knockout in HepG2 cells results in slowed cell division, mitochondrial dysfunction, and cell cycle arrest at the G2/M phase (61).

GSEA of ANXA1, MICB, CD36, and PRDX6 revealed common enrichment in signaling pathways, including chemokine signaling and cytokine-cytokine receptor interactions. These pathways are hypothesized to play a role in ULM development. Xia et al., in their comprehensive analysis of four ULM-related mRNA datasets, suggested that extracellular matrix-receptor interactions could lead to abnormal extracellular matrix formation in ULM. Additionally, focal adhesion and cell adhesion molecules were implicated in ULM pathogenesis. Xia et al. further proposed that chemokine signaling and cytokine-cytokine receptor interactions are likely involved in ULM progression (45). Prates et al. demonstrated that ANXA1 influences cervical cancer development by activating the transcriptional expression of formyl peptide receptors (FPRs) and the inhibitor of DNA binding 1 (ID1) (62). FPR plays a central role in the chemokine signaling pathway, while ID1, a DNA transcription regulator, is closely linked to DNA replication. Our findings are consistent with their results, reinforcing the involvement of these pathways in ULM development.

Through in-depth analysis of the functional and pathway information enriched by GO and KEGG, we found several clues closely related to the pathogenesis or related hypotheses of ULM. Studies have shown that ROS, as a pro-inflammatory mediator, can regulate cell proliferation and is known to activate the MAPK/ERK pathway in endometriosis (63), suggesting that ROS may also be involved in the pathological process of ULM, although this hypothesis needs to be further verified. Notably, ganirelix as a potential therapeutic agent has demonstrated significant toxicity to ULM cells, suggesting that it may induce tumor reduction in a broad patient population (64). In addition, chronic inflammation caused by visceral fat plays a key role in cell differentiation and proliferation, which is a necessary condition for the onset of ULM. Increased visceral fat deposits may alter cholesterol composition, thereby increasing the risk of subclinical atherosclerosis in patients with ULM (65). Fibromodulin (FMOD), as an important component of the extracellular matrix, not only plays a role in structure, but also is regarded as a novel tumor-associated antigen for a variety of malignant tumors, including ULM, and has the potential as a biomarker for cancer diagnosis and treatment (66). Relaxin has also been suggested to be involved in the pathogenesis of ULM, providing a new perspective for disease research (67). At the same time, TNF-α up-regulates MMP-2 expression and promotes cell migration by activating extracellular signal-regulated kinase (ERK) signaling pathway in uterine fibroid smooth muscle cells, which provides a theoretical basis for the development of ULM treatment strategies targeting TNF-α and MMPs inhibitors (68).

Immunoreactive cells, including lymphocytes, macrophages, granulocytes, and dendritic cells, are crucial participants in immune responses. Recent studies suggest that the composition and abundance of these cells may influence the development and progression of uterine ULMs (69). Using the CIBERSORT algorithm, we analyzed the immunoreactive cell infiltration characteristics in ULM versus normal tissues, revealing significant differences in five types of immunoreactive cells between the two groups. Supporting our findings, Zannotti et al. reported a greater abundance of macrophages within and around ULMs compared to distant myometrial tissue (69). The dysregulation of macrophage proliferation, infiltration, and accumulation contributes to pathological fibrosis and uncontrolled tissue repair, with key roles played by factors such as MCP-1, GM-CSF, TGF-β, and TNF-α. Similarly, Protic et al. observed a higher density of CD68-positive macrophages in ULMs and adjacent myometrium compared to distant myometrium (70). In this context, TNF-α secreted by macrophages may enhance activin A mRNA expression in ULM cells, potentially leading to excessive extracellular matrix production, tissue remodeling, and tumor growth. However, a clinical study by Liu et al. found no significant differences in MCT-positive mast cells or CD45-positive leukocytes between ULM and normal tissues in premenopausal women (71).

In another study, Wang et al. identified significant overexpression of miR-30a, miR-34, and miR-24, in addition to Let-7, in ULM. These miRNAs may inhibit ULM proliferation and differentiation by suppressing HMGA2a, acting as protective factors against the overexpression of oncogenes such as RAS and MYC ( 72). Extensive research has also explored miR-196a’s role in various cancers, showing its involvement in key biological processes related to tumorigenesis. Hu et al. demonstrated that miR-196a regulates the proliferation, invasion, and migration of esophageal squamous cell carcinoma by targeting ANXA1 (73). In breast cancer, miR-196a expression correlates with certain HOXC genes involved in tumor progression (74), while in cervical cancer, it targets netrin 4, influencing cell proliferation and migration (75). Although ULMs are benign, their abnormal cellular proliferation mirrors that of malignant tumors, suggesting that miR-196a may play a similar role in ULM pathogenesis. Furthermore, miR-155-5p exhibits dual roles, acting as an oncogene or tumor suppressor depending on the cellular environment and cancer type. Research by Navarro et al. indicated that miR-155-5p is implicated in the inflammatory processes often associated with ULMs (76).

Our research identified a strong binding affinity between ANXA1 and amcinonide, as well as between MICB and ribavirin. Previous studies have demonstrated that amcinonide, an anti-inflammatory agent with affinity for glucocorticoid receptors, can induce depigmentation in both black and albino mice. It significantly reduces the number of DOPA-positive epidermal melanocytes in these animals (77). Ribavirin, a broad-spectrum antiviral agent effective against HCV, HIV, and RSV, has been shown to decrease activin A levels while increasing follicle-stimulating hormone concentrations in the serum and liver of Wistar rats (78). The drug-binding analysis in this study suggests that amcinonide and ribavirin may serve as potential therapeutic agents for ULM, interacting with ANXA1 and MICB, respectively. While these findings hold promise for therapeutic interventions in ULM, further investigation into the precise mechanisms of action is necessary.

In conclusion, this study identified oxidative stress-related biomarkers in ULM and provided a comprehensive analysis of drug networks and immune cell infiltration, shedding light on the molecular mechanisms of these biomarkers in ULM. The findings offer potential new avenues for ULM treatment.

However, several limitations should be noted. The relatively small sample size used in this study may have introduced bias and affected both the statistical power and the biological relevance of the results. Future studies will aim to include larger sample sizes to further validate the accuracy of these biomarkers and their association with disease. At the same time, we will also consider implementing cross-validation strategies on a broader dataset to further consolidate and extend our research results. Additionally, the study focused exclusively on gene expression levels, which do not necessarily reflect direct biological effects. To validate these findings, further animal and clinical studies employing different experimental approaches will be required.