Preeclampsia (PE) is a pregnancy-specific, multisystemic disorder characterized by clinical manifestations such as new-onset hypertension and proteinuria after 20 weeks of gestation. It stands as a significant contributor to maternal and fetal mortality rates (Mol et al., 2016). The incidence of PE varies based on geographic location, season, nutritional factors, and racial/ethnic backgrounds; however, it impacts approximately 3%–5% of women globally (Mol et al., 2016). Maternal complications associated with PE encompass eclampsia, renal failure, and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. The pathophysiological mechanisms of PE encompass endothelial damage, systemic arteriolar spasm, reduced systemic perfusion, and multiorgan dysfunction, which collectively pose a significant threat to the health of both mothers and infants (San Juan-Reyes et al., 2020; Ziganshina et al., 2020).Currently, the only effective treatment is the termination of pregnancy (Tannetta and Sargent, 2013). Unfortunately, the exact etiology and pathogenesis remain unelucidated. However, placental dysfunction, characterized by inadequate trophoblast invasion of spiral arterioles, is thought to play a central role. This dysfunction may be triggered by an imbalance between the production and deactivation of reactive oxygen species (ROS) in the placenta (Mol et al., 2016; Roberts et al., 1989; Rana et al., 2019; Brown et al., 2018).

Oxidative stress plays a significant pathogenic role in PE, with numerous oxidative stress markers showing diagnostic potential (Agrawal et al., 2018). A substantial body of evidence underscores the crucial involvement of immune responses and oxidative stress in the development of PE (Meng et al., 2021; Afrose et al., 2022; Deer et al., 2021a). The integration of immune infiltration, oxidative stress, and bioinformatics approaches provides new insights into the diagnosis and treatment of PE. Furthermore, two external datasets were utilized to validate the identified gene. Ultimately, the gene was validated through quantitative real-time polymerase chain reaction (qRT -PCR) and immunohistochemistry. The findings of this study might offer new perspectives on the role of oxidative stress in placental pathology and could also facilitate the identification of potential biomarkers and therapeutic targets, providing a novel approach to the clinical diagnosis and treatment of PE. The study route is illustrated in Figure 1.

Human PE datasets were extracted from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) in July 2023. Three PE datasets were retrieved: GSE10588 (GPL2986), consisting of 17 cases of PE and 26 control cases; GSE74341 (GPL16699), containing 12 PE cases and 10 control cases for validation; and another validation set GSE98224 (GPL6244)) containing 30 PE cases and 18 control samples. Furthermore, 1142 oxidative stress-related genes were extracted from the Genecards database. Pertinent details information is displayed in Table 1.

PE represents a multifaceted clinical syndrome that is exclusive to human pregnancy and plays a major role in causing morbidity and mortality among pregnant women and their neonates (Roberts et al., 1989). Despite its significance, the exact pathogenesis of PE remains incompletely understood, resulting from intricate interactions of various factors. PE can be triggered by a number of conditions such as systemic inflammation, placental dysfunction, hypoxia, immunological dysregulation, and oxidative stress (Jung et al., 2022; George and Granger, 2011).Oxidative stress molecular markers play a critical role in the pathogenesis of PE. During early pregnancy, abnormal invasion of trophoblasts into the maternal uterine spiral arteries leads to oxidative stress, enhancing the production of oxygen-free radicals in the placental environment (Karaşin and Çift, 2020; van Rijn et al., 2008). Certain studies have also revealed that immune response disruptions have a remarkable impact on the development of this obstetrical syndrome (Miller et al., 2022; Santana-Garrido et al., 2022; Lang et al., 2022; Deer et al., 2021b). Furthermore, advances in informatics technologies now allow the identification of diagnostic markers associated with both immune responses and oxidative stress in the context of PE.

WGCNA is a widely used method for bioinformatics analysis. In this study, We WGCNA algorithm to build a gene co-expression network, subsequently leveraging hierarchical clustering for delineating functional modules enriched with tightly interconnected genes. Analysis of the WGCNA results revealed that genes in the brown module exhibited the highest correlation with PE development. After that, a Venn diagram was used to disclose 17 genes that overlapped among the gene sets under study. GO functional analysis illustrated that these genes were involved in processes such as the positive regulation of organic hydroxy compound metabolic process, protein kinase activity, and MAPK cascade positive regulation. Similarly, KEGG analysis demonstrated that these genes were primarily associated with the positive regulation of protein kinase activity, organic hydroxy compound metabolism, positive regulation of MAPK cascade, response to inorganic substance, transition metal ion transport, and several other biological processes. Subsequently, machine-learning techniques, including LASSO, RF, and SVM-RFE, were employed to identify key disease-related genes. Furthermore, ROC curves and AUC values were utilized to assess diagnostic potential of these markers. Ultimately, a potential diagnostic marker for PE, namely, LEP, was identified.

Currently, identifying effective early prediction methods for PE remains a significant challenge. A cohort study conducted in the United States found that the maternal LEP/ceramide (cer) ratio in early pregnancy serves as a superior non-invasive serum biomarker for predicting PE, compared to the sFlt/PlGF ratio (Huang et al., 2021). This suggests that LEP may be more helpful than sFlt/PlGF in predicting PE.LEP is a protein-coding gene released by white adipocytes into the bloodstream, where it plays a crucial role in maintaining energy balance.LEP possesses various endocrine functions and isimplicated in the regulation of immune and inflammatoryresponses, hematopoiesis, angiogenesis, reproduction, anthe promotion of angiogenesis (Pérez-Pérez et al., 2018; Fasshauer and Blüher, 2015; Nieuwenhuis et al., 2020; Childs et al., 2021)^.LEP acts on variety of immune cells, influencing their development and function, and plays a key role as an immune modulator in the body. Most immune cells express LEP receptors and respond to LEP. In particular, LEP is essential for the development, function, and metabolism of T cells. Research indicates that LEP is critical for the early development of T cells, but is not required for CD8⁺ T cells (Kim et al., 2010). It has been demonstrated that leptin receptor signaling in T cells enhances cell survival, differentiation, cytokine production, and proliferation differentiatio (Fujita et al., 2002; Saucillo et al., 2014; Lord et al., 1998; Gerriets et al., 2016). Furthermore, LEP promotes B cell homeostasis by inhibiting apoptosis and promoting cell cycle progression (Lam et al., 2010). In vitro studies have demonstrated that LEP can activate B cells, inducing a pro-inflammatory phenotype (Frasca et al., 2020; Agrawal et al., 2011).LEP also binds specifically to LEP receptors on macrophages, promoting their phagocytic activity (Mancuso et al., 2018). Similar to T cells, LEP activation in monocytes induces the expression of an inflammatory phenotype (Jaedicke et al., 2013). LEP has been shown to promote the anti-apoptotic effects of dendritic cells and enhance their maturation (Mattioli et al., 2005; Moraes-Vieira et al., 2014). Studies also indicate that LEP can inhibit neutrophil apoptosis and act as a chemoattractant for neutrophils (Bruno et al., 2005; Ubags et al., 2014; Naylor et al., 2014). In addition, LEP attracts eosinophils and basophils in a manner similar to its effect on neutrophils (Suzukawa et al., 2011; Kato et al., 2011). Furthermore, LEP plays a critical role in the development of natural killer (NK) cells (Tian et al., 2002). Currently, no studies have reported LEP-induced oxidative stress activation in existing PE animal models. However, LEP has been shown to increase ROS levels in vascular smooth muscle cells and promote cell proliferation through the activating of thethe NF-κB pathway (Schroeter et al., 2012; Li et al., 2005). In the placenta of PE patients, an increase in ROS levels can be observed, potentially due to the activation of the NF-κB pathway in trophoblast cells, leading to enhanced ROS production (Wang et al., 2021). Excessively high LEP levels may disrupt glucose and lipid metabolism, leading to the excessive release of lipid peroxides and ROS, which increases the risk of PE (Kutlu et al., 2005). This may help explain our findings. The inflammatory microenvironment in PE patients promotes the infiltration of dendritic cells. However, the increased proportion of conventional dendritic cells and their over-maturation in PE patients may contribute to the increased production of pro-inflammatory cytokines and maternal damage (Zhang et al., 2017; Darmochwal-Kolarz et al., 2003). This finding aligns with our results. Decidual NK (dNK) cells actively mediate two pivotal processes in early gestation: facilitating trophoblast migration and orchestrating structural transformation of spiral arteries, potentially contributing to the development of PE (Kalkunte et al., 2009).This process may be driven by the secretion of vascular endothelial growth factor (VEGF) and placental growth factor placental growth factor (PlGF) by dNK cells, which stimulate spiral artery remodeling (Kalkunte et al., 2009; Gibson et al., 2015). Our results indicate a higher proportion of CD56^dim NK cells in preeclamptic women, whereas no significant difference was observed in the proportion of CD56^bright NK cells between the PE and control groups. T cells are classified into αβ T cells and γδ T cells based on differences in their T cell receptors. Currently, the role of γδ T cells in the pathogenesis of PE remains unclear.; however, animal studies have shown a significant elevation in γδ T cells in the placenta of PE model mice, with γδ T cell-deficient mice exhibiting improved PE symptoms (Chatterjee et al., 2017). This finding is consistent with our results. During pregnancy, type 1 T helper cell (Th1) immunity exerts pro-inflammatory effects, while type 2 T helper cell (Th2) immunity has anti-inflammatory effects. The balance between Th1 and Th2 immune responses is crucial for a healthy pregnancy. However, in PE patients, the Th1/Th2 ratio in peripheral blood is increased (El-Kabarity and Naguib, 2011). A deficiency of decidual regulatory T cells (Treg cells) is associated with poor spiral artery remodeling and insufficient trophoblast invasion, both of which exacerbate the symptoms of PE (Harmon et al., 2016).

LEP plays a critical role in placental development and function, influencing processes such as invasion, protein synthesis, cell proliferation, and apoptosis in placental cells (Pérez-Pérez et al., 2018; D'Ippolito et al., 2012; Schanton et al., 2018). Multiple studies have underscored LEP’s involvement in the pathophysiology of PE (Pérez-Pérez et al., 2018; Dos Santos et al., 2015; Hauguel-de Mouzon et al., 2006; Gutaj et al., 2020). Notably, PE patients exhibit elevated serum LEP levels compared to individuals without the condition, and elevated levels of this protein have also been reported in the placentas of PE patients. (Song et al., 2016; Kalinderis et al., 2015; Taylor et al., 2015). Multiple investigations have detected both LEP mRNA and protein expression in the placentas of PE patients (Taylor et al., 2015; Mise et al., 1998; Nishizawa et al., 2007), findings consistent with the results of the present study.

On the one hand, the elevated LEP levels may reflect a compensatory response to placental underperfusion, aiming to support neovascularization (Eleuterio et al., 2014; Lepercq et al., 2003). On the other hand, LEP is known to play a role in immune regulation, and disruptions in immune homeostasis may lead to alterations in maternal LEP expression in PE. However, further comprehensive research is needed to fully elucidate the dysregulation of LEP in the context of PE and its broader impact on maternal physiology.

PE is a complex systemic condition, with increasing evidence highlighting the immune system’s pivotal role in its development (Lu and Hu, 2019). Thus, to further investigate this, an analysis of immune infiltration was conducted to compare the immune cell profiles between normal and PE samples. The results revealed a remarkable increase in the infiltration of eight immune cell types in PE samples compared to normal samples. These immune cell types included activated dendritic cells, type 1 T helper cells,CD56^dim natural killer cells, γδT cells, macrophages, regulatory T cells, T follicular helper cells, type 2 T helper cells, and effector memory CD4⁺ T cells.

These findings indicate that LEP may serve as a promising diagnostic and therapeutic marker for PE, potentially exerting a significant role in its pathogenesis. Nevertheless, the study lacks longitudinal data, which could be valuable in understanding the temporal changes in LEP expression and its correlation with disease severity and outcomes; validating the prognostic and therapeutic implications of this model will require a larger sample size, longitudinal data, and comprehensive clinical data from multiple medical center in future research. The potential therapeutic drugs targeting LEP in PE remains to be confirmed, and future research should focus on conducting fundamental studies or clinical trials to validate the feasibility of LEP-targeting drugs in the treatment of PE. Additionally, as this study relies on publicly available data, further experimental investigations are needed to elucidate the biological functions of LEP.

In summary, through integrated bioinformatics analyses and experimental verification, we identified LEP as a key biomarker associated with immune infiltration and oxidative stress in PE.