Endometriosis (EMS) is characterized by the abnormal growth, infiltration, and recurrent bleeding of endometrial tissue (glands and stroma) outside the uterine cavity, leading to pain, infertility, and the formation of nodules or masses (1, 2). It is a prevalent condition among women of reproductive age (3). The development of EMS is influenced by various factors, including sex hormones, immune response, inflammation, and genetics, though its exact pathogenesis remains elusive. Current theories attempting to explain the origins of EMS include Sampson's Theory, immune dysregulation, Mullerianosis, vascular and lymphatic metastasis, the eutopic endometrium theory, and genetic and environmental contributions (4–7). Despite the prominence of these theories, none fully account for all cases (8), suggesting that EMS pathogenesis may be a multifactorial and stepwise process. Epidemiological studies have identified several risk factors for EMS, including family history, early menarche, short menstrual cycles, low fertility, obesity, chemical exposure, and prior abdominal surgery (9–12). A significant challenge in EMS management is diagnostic delay, which often leads to disease progression, complicates treatment, increases the likelihood of recurrence, and diminishes the patient's quality of life. Notably, no definitive cure exists for EMS, and current treatments primarily focus on symptom management. Early diagnosis is critical, yet no reliable biomarkers are available in peripheral blood or the endometrium for accurate diagnosis (13). Although elevated CA125 levels are often observed in patients with severe EMS, significant pelvic inflammation, or associated conditions like endometriotic cyst rupture or adenomyosis (14, 15), it is not a definitive diagnostic marker. Consequently, the identification of novel, effective biomarkers for early EMS diagnosis remains a pressing need to facilitate clinical management and improve treatment outcomes.

Programmed cell death (PCD) is a fundamental process in the development of multicellular organisms and plays a pivotal role in the pathogenesis of degenerative diseases. Key forms of PCD, including apoptosis, ferroptosis, and pyroptosis, involve mitochondrial participation (16). Mitochondria serve as critical regulators of PCD by acting as signal amplifiers. In response to both internal and external stimuli, mitochondria alter membrane permeability, release proteins such as cytochrome C, and initiate apoptotic pathways. Under normal conditions, mitochondrially produced reactive oxygen species (ROS) activate apoptosis; however, excessive ROS can damage mitochondrial integrity and accelerate apoptosis (17). Additionally, mitochondria interact with the endoplasmic reticulum and nucleus to propagate apoptotic signals (18). Mitochondrial dysfunction impairs cellular oxidative phosphorylation, leading to insufficient ATP production. To compensate, cells shift to anaerobic glycolysis, even in the presence of adequate oxygen, which produces lactate. This accumulation of lactic acid promotes cell migration, invasion, and angiogenesis, thus exacerbating the progression of EMS (19). Studies have underscored the pivotal role of PCD in the pathophysiology of EMS, suggesting potential therapeutic avenues. For instance, abnormal apoptosis in ectopic endometrial tissue is associated with disease progression and clinical manifestations (20). Additionally, spontaneous apoptosis is diminished in the endometrial glands of patients with EMS, facilitating immune evasion and promoting ectopic growth (21). Research has also shown that creatine confers resistance to ferroptosis in ectopic endometrial stromal cells by inhibiting prion protein, thus supporting EMS development (22). Furthermore, women with mild EMS exhibit a significantly higher number of dysfunctional mitochondria in their oocytes (23). Mitochondrial energy production and metabolism are impaired in EMS-affected tissues, potentially due to oxidative stress-induced mitochondrial DNA or membrane damage, metabolic shifts, or reduced availability of energy substrates. However, the combined impact of PCD and mitochondrial dysfunction on EMS pathogenesis remains unexplored. Thus, further investigation into the roles of mitochondria and PCD in EMS is essential.

Using a comprehensive bioinformatics approach, including machine learning techniques, this study identified biomarkers in EMS linked to both PCD and mitochondrial activity. Through expression validation and diagnostic performance analysis, the study not only assessed their potential clinical utility but also explored their expression dynamics. Integrated functional, immunological, and regulatory network analyses revealed the underlying mechanisms of these biomarkers in EMS. Additionally, single-cell level analysis provided new insights into their functional roles, offering a deeper understanding of EMS pathology and laying the groundwork for advancing diagnostic and therapeutic strategies.

EMS, a condition affecting many women of reproductive age, remains a global health challenge with an unclear pathogenesis (41). It is hypothesized that the dynamic changes in mitochondrial function, which regulate energy metabolism and redox balance, may be linked to the development of EMS (42, 43). Additionally, ferroptosis, a form of PCD, has been suggested to contribute to EMS progression through the secretion of vascular endothelial growth factors and modulation of immune mechanisms (44). While PCD is a natural cellular process, mitochondrial dysfunction can affect apoptosis and may therefore be implicated in the pathogenesis of EMS. However, the specific mechanisms linking mitochondrial dysfunction, PCD, and EMS remain to be fully elucidated, requiring further investigation.

Through a series of bioinformatics analyses—including differential expression analysis, WGCNA, machine learning, and expression validation—two potential biomarkers, AIFM1 and PDK4, were identified. Both biomarkers demonstrated strong diagnostic performance for EMS. PDK4 was upregulated, and AIFM1 was downregulated in the disease group. PDK4 encodes a mitochondrial protein that belongs to the PDK/BCKDK protein kinase family and contains a histidine kinase domain (45). It plays a pivotal role in ferroptosis resistance by inhibiting pyruvate oxidation through the pyruvate dehydrogenase pathway (46). Increased expression of PDK4 has been associated with colitis, with its substrate accumulating in CD4⁺ T cells of patients with inflammatory bowel disease. Loss of PDK4 function can delay colitis development and reduce T cell activation and aerobic glycolysis (47). Inflammation also enhances PDK4 expression in C2C12 myoblasts via the Jun N-terminal kinase (JNK) pathway (48). Since EMS is often accompanied by inflammation, PDK4 may mediate this pathological process through its effect on inflammatory signaling. However, the precise mechanism remains to be explored. On the other hand, AIFM1, which encodes a flavoprotein critical for nuclear breakdown during apoptosis, is located in the mitochondrial intermembrane space of healthy cells. Upon apoptosis, AIFM1 translocates to the nucleus, where it participates in chromosome condensation and fragmentation, and promotes the release of cytochrome c and caspase-9 from mitochondria (49). Previous studies have shown that silencing CCAAT/enhancer-binding protein α (C/EBPα) can promote the proliferation and anti-apoptosis of normal eutopic endometrial stromal cells, and lead to the downregulation of AIFM1 expression (50). Therefore, AIFM1 plays a pivotal role in human development and disease by serving as a key mediator of extra-mitochondrial signals.

AIFM1 and PDK4 were notably enriched in pathways related to the cell cycle, complement and coagulation cascades, as well as systemic lupus erythematosus (SLE). These pathways play pivotal roles in the pathogenesis of EMS and its associated disorders. For instance, cyclin B1 is implicated in promoting ectopic endometrial cell proliferation under ovarian hormone regulation (51). Dysregulation of cell cycle regulators interacting with p27 (Kip1) is essential in driving ovarian clear cell carcinoma progression linked to EMS, enhancing cellular proliferative activity (52). EMS is also closely associated with immune dysfunction and chronic inflammation, with the complement system playing a critical role in its pathophysiology. Studies have demonstrated the upregulation of complement components (C1S, C1QA, C1R, C3) in patients with EMS, which correlate positively with tissue factor, suggesting an interaction between the complement and coagulation pathways in EMS progression (53). Moreover, EMS and SLE, both prevalent in females, share significant immunological overlap, as SLE is a severe autoimmune condition affecting multiple systems, while EMS is a chronic inflammatory disorder involving hormonal and immune dysregulation (54, 55). Thus, AIFM1 and PDK4 may influence EMS onset and progression by modulating the cell cycle, complement and coagulation pathways, and autoimmune mechanisms.

This study underscores the pivotal role of the immune system in EMS. Immune cell infiltration levels across 22 types were analyzed, revealing a significant upregulation in M0 macrophages, resting mast cells, naive B cells, and M2 macrophages in EMS. In contrast, infiltration levels of resting dendritic cells, activated NK cells, and follicular helper T cells were downregulated. These findings suggest that the suppression of activated NK cells in endometriotic lesions may aid ectopic endometrial cells in evading immune surveillance, thereby promoting their survival and implantation. This observation aligns with previous research (56). In healthy women, peritoneal NK cells exhibit enhanced cytotoxicity; however, in patients with EMS, elevated local estrogen levels alter the number and phenotype of NK cells, impairing their ability to effectively eliminate ectopic cells and fostering lesion development (57). Correlation analysis of immune cells revealed a strong positive correlation between resting mast cells and M2 macrophages, indicating a potential synergistic role in immune regulation and inflammation resolution. Previous research has shown that the local microenvironment of EMS lesions can trigger mast cell activation, with these activated mast cells releasing various inflammatory mediators that contribute to EMS-related pain via inflammatory pain pathways (58). Conversely, the negative correlation between T follicular helper cells and M2 macrophages suggests a mutually restrictive relationship during immune responses. Furthermore, a strong positive correlation was observed between resting dendritic cells and AIFM1, while a negative correlation was found between M2 macrophages and AIFM1. M2 macrophages are central to the immune microenvironment in EMS (59), and blocking macrophage-associated immune checkpoint CD47 can effectively alleviate EMS (60). These findings indicate that AIFM1 may play differential regulatory roles across immune cell types, modulating the immune balance. Additionally, the expression of AIFM1 and PDK4 was correlated with immune cell infiltration levels, further suggesting that these biomarkers could serve as promising therapeutic targets for EMS.

In this study, 10 distinct cell types were identified, including endothelial cells, epithelial cells, gametocytes, smooth muscle cells, fibroblasts, MSCs, neutrophils, monocytes, NK cells, and T cells, all of which have been previously associated with EMS. Endothelial cells, for instance, play critical roles in promoting inflammation, angiogenesis, and permeability in EMS, particularly in deep infiltrating cases (61). This suggests their vital involvement in angiogenesis and the sustenance of ectopic tissues. Ectopic epithelial cells, on the other hand, resist apoptosis via the NNMT-FOXO1-BIM pathway and can stimulate CD4⁺ T cells through the HLA II complex, fostering chronic inflammation (62). Lymphocytes in EMS show signs of immune activation and exhaustion, with NK cells exhibiting increased cytotoxicity and T cells displaying elevated expression of checkpoint genes (63). These alterations likely contribute to the immune evasion of ectopic tissues. Fibroblasts and smooth muscle cells are also integral to pathological fibrosis and angiogenesis in EMS, leading to persistent fibrotic phenomena that contribute to classic EMS symptoms such as pain and infertility (64, 65). Monocytes and macrophages play essential roles in the progression, vascularization, and painful manifestations of EMS. Macrophages, in particular, are multifaceted cells crucial for embryonic development and tissue homeostasis in healthy conditions. However, under inflammatory stress, monocytes migrate from the bloodstream and differentiate into macrophages, which then perform functions such as infection defense and wound healing. The role of macrophages in inflammation has garnered significant attention in recent research (66). Neutrophil levels are notably elevated in the blood, peritoneal fluid, and ectopic endometrium of patients with EMS. Ectopic endometrial lesions actively recruit neutrophils, which, in turn, promote early-stage angiogenesis within the inflammatory microenvironment, thereby creating a positive feedback loop (67, 68).

In EMS samples, smooth muscle cells and endothelial cells exhibited a higher likelihood of interaction, suggesting abnormal intercellular communication and signaling in the pathological context of EMS. Such dysregulated interactions may contribute to inflammation, tissue adhesion, and lesion formation in EMS. Consequently, targeting the crosstalk between these two cell types could offer a novel therapeutic approach for EMS treatment. The distinct expression patterns of AIFM1 and PDK4 in various cell types highlight their specific roles in the disease, underscoring their potential as biomarkers for understanding the immune microenvironment of EMS. This understanding is crucial for developing more targeted and precise treatment strategies.

This study identified potential biomarkers, AIFM1 and PDK4, in the pathogenesis of EMS through comprehensive bioinformatics analysis. These biomarkers effectively distinguished EMS from healthy controls. Additionally, by examining the communication between cells and the signaling pathways involved, this research provides valuable insights for diagnosing and treating EMS. However, the study has some limitations, including the absence of experimental validation of the biomarkers' functional roles and a relatively small sample size. Future experimental studies are needed to validate the conclusions derived from this bioinformatics analysis.