Does Depression Increase the Risk of Endometriosis? A Combined Population‑Based and Laboratory Study

Endometriosis is a chronic, estrogen-dependent inflammatory disorder that causes substantial pain and infertility. Observational studies have reported higher rates of multiple psychiatric disorders among individuals with endometriosis; however, confounding and reverse causation limit causal interpretation.

We performed bidirectional two-sample Mendelian randomization (MR) analyses using genome-wide association study (GWAS) summary statistics from European-ancestry populations to test causal relationships between genetic liability to depression, anxiety, insomnia, bipolar disorder, schizophrenia, and endometriosis. Instrumental variables were selected using genome-wide significance thresholds and linkage disequilibrium clumping, and robustness was evaluated using MR Egger regression, the weighted median method, and mode-based estimators, together with heterogeneity and pleiotropy tests. To assess biological coherence, SH-SY5Y cells were differentiated into sympathetic neuron-like cells, norepinephrine was quantified by enzyme-linked immunosorbent (ELISA) assay, and paracrine effects on 12Z endometriotic epithelial cells were examined using CCK-8 assays. Transcriptomic profiling was followed by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and protein–protein interaction (PPI) network analysis.

Forward-direction MR supported an association between genetic liability to depression and increased endometriosis risk, whereas anxiety, insomnia, bipolar disorder, and schizophrenia showed no robust causal evidence. Reverse-direction MR did not support endometriosis genetic liability as a causal driver of psychiatric disorders. In vitro, sympathetic neuron-like differentiation increased norepinephrine secretion and enhanced 12Z cell proliferation, with stronger effects under conditioned-medium exposure. Enrichment analyses identified immune-related pathways, with prominent enrichment in Ras signaling, phosphatidylinositol 3 kinase and protein kinase B signaling, mitogen-activated protein kinase signaling, and cyclic adenosine monophosphate signaling.

These findings suggest that depression may contribute to endometriosis susceptibility and are supported by complementary in vitro and transcriptomic evidence. This graphical abstract illustrates a potential association between depression and endometriosis identified through Mendelian randomization analysis. ATRA-induced sympathetic-like neurons derived from SH-SY5Y cells show increased norepinephrine production and promote the viability of 12Z endometriotic cells through paracrine signaling. Several signaling pathways may contribute to this process.Infographic: depression-endometriosis link, neuron differentiation, paracrine signaling, MAPK & cAMP key pathways.

endometriosis, mendelian randomization, RNA-seq, depression, GWAS

Endometriosis is a chronic, estrogen-dependent inflammatory disorder characterized by ectopic endometrial-like tissue, chronic pelvic pain, infertility, and impaired quality of life.1,2 Diagnostic delays and limited disease-modifying therapies contribute to substantial clinical and socioeconomic burden, highlighting the need to clarify modifiable upstream determinants and biologically actionable pathways.3–5 Psychiatric disorders are frequently reported in individuals with endometriosis, including depression, anxiety, insomnia, bipolar disorder, and schizophrenia.6,7 This comorbidity has direct clinical relevance because psychiatric symptoms can exacerbate pain experience, worsen functional impairment, and complicate long-term management. However, observational associations do not establish directionality. Pain severity, infertility-related stress, healthcare utilization, medication exposure, and socioeconomic factors can confound the relationship, and endometriosis itself can contribute to psychiatric symptoms through chronic pain and inflammation. In addition, clinical depression as a diagnosed phenotype should be distinguished from genetic liability to depression, which represents inherited susceptibility captured by genome-wide association study summary statistics. Clarifying whether psychiatric liability contributes to endometriosis risk, or whether endometriosis itself increases susceptibility to psychiatric disorders, is essential for understanding the biological basis of this comorbidity. Genetic studies support endometriosis as a polygenic disease and implicate pathways related to hormone signaling, inflammation, tissue remodeling, and neuroimmune interactions.8 Similarly, depression and other psychiatric disorders have been linked to immune dysregulation and altered stress responses.9,10 This genetic architecture provides an opportunity to evaluate whether psychiatric disorder liability is causally related to endometriosis risk, rather than merely correlated through clinical consequences or shared confounders. Mendelian randomization uses genetic variants as instrumental variables to test causal hypotheses under explicit assumptions, thereby reducing bias from confounding and reverse causation.11 Bidirectional two-sample Mendelian randomization based on genome-wide association study summary statistics enables efficient evaluation of directionality between psychiatric disorders and endometriosis at population scale, while sensitivity estimators and pleiotropy diagnostics improve robustness to violations of instrument validity. Therefore, we conducted a bidirectional two-sample Mendelian randomization analysis to evaluate causal relationships between genetic liability to depression, anxiety, insomnia, bipolar disorder, schizophrenia, and endometriosis. Guided by the Mendelian randomization findings that prioritized depression as the psychiatric trait most strongly associated with endometriosis risk, we next modeled sympathetic neuron–derived signaling in vitro using a sympathetic neuron-like differentiation system and assessed its paracrine effects on endometriotic epithelial 12Z cells. Finally, we performed ribonucleic acid sequencing and downstream bioinformatics analyses to characterize transcriptional programs and pathways responsive to sympathetic neuron-conditioned signals.

Mendelian Randomization Analysis Using large-scale genome-wide association study (GWAS) summary statistics, we performed a bidirectional two-sample Mendelian randomization (MR)12 analysis to investigate the potential causal relationships between endometriosis and common psychiatric disorders, including depression, anxiety, insomnia, bipolar disorder, and schizophrenia. The overall study workflow is presented in Figure 1. To support valid causal inference, genetic variants used as instrumental variables (IVs) were selected to satisfy the three core MR assumptions: (i) relevance, genetic instruments are robustly associated with the exposure; (ii) independence, instruments are not associated with confounders of the exposure–outcome relationship; and (iii) exclusion restriction, instruments influence the outcome only through the exposure and not via alternative pathways (horizontal pleiotropy).13 | Figure 1 Outline of the study design. (1) relevance; (2) Independence; (3) exclusion restriction. | Data Sources Genetic association data for endometriosis were obtained from the GWAS Catalog (GWAS ID: ebi-a-GCST90018839).14 Summary statistics for psychiatric disorders were retrieved from the FinnGen study (Release 12), reported as the most up-to-date version (Data accessed on: 2025-07-01) available at the time of data extraction (Table 1). All datasets were restricted to individuals of European ancestry to reduce population stratification and between-study heterogeneity. All data used in this study were derived from publicly available GWAS summary statistics; therefore, no additional ethical approval or informed consent was required. | Table 1 Data Sources and Characteristics of GWAS Summary Statistics Used in MR Analyses | Selection of Instrumental Variables Single-nucleotide polymorphisms (SNPs) associated with each exposure were selected as genetic instruments using a genome-wide significance threshold of P ≤ 5 × 10−8.15 For endometriosis, which had a limited number of genome-wide significant SNPs, the significance threshold was relaxed to P <1 × 10−5 to ensure an adequate number of instrumental variables. To obtain independent instruments, we performed linkage disequilibrium (LD) clumping with thresholds of r2 < 0.001 within a 10,000 kb window,16 thereby reducing bias from correlated variants. Instrument strength was evaluated using the F-statistic, and SNPs with F ≤ 10 were considered weak instruments and excluded to minimize weak-instrument bias.17 Exposure and outcome datasets were harmonized to align the effect alleles. Ambiguous and palindromic SNPs were removed to avoid strand inconsistencies. The detailed significance threshold, SNP counts, and F-statistic for each exposure are provided in Table S1. Statistical Analyses All MR analyses were conducted using R (version 4.5.1) with the TwoSampleMR package (version 0.6.19). The inverse-variance weighted (IVW) method18 was used as the primary approach to estimate causal effects. For binary traits, causal estimates were reported as odds ratios (ORs) with 95% confidence intervals (CIs). A two-sided P < 0.05 was considered statistically significant. Sensitivity analyses were performed using MR-Egger regression,19 the weighted median method,20 and mode-based estimators (simple mode and weighted mode)21 to evaluate the robustness of IVW results under different assumptions regarding horizontal pleiotropy. Between-instrument heterogeneity was assessed using Cochran’s Q test. When heterogeneity was suggested (P < 0.05), IVW estimates were obtained under a random-effects model; otherwise, a fixed-effects IVW model was applied. Directional horizontal pleiotropy was evaluated using the MR-Egger intercept test. In addition, leave-one-out analyses were conducted to assess whether any single SNP disproportionately influenced the overall causal estimate. Establishment of a Sympathetic Neuron–Endometriotic Epithelial Cell Co-Culture System Cell Culture and Differentiation of SH-SY5Y Cells Into Sympathetic Neuron-Like Cells Human endometriotic epithelial cells (12Z) and human neuroblastoma cells (SH-SY5Y) were purchased from Wuhan Pricella (Wuhan, China).22,23 Since the experiments involved established cell lines and in vitro procedures only, no additional ethical approval was required. SH-SY5Y cells were maintained in a dedicated culture medium at 37°C in a humidified incubator with 5% CO2 (Heracell 150iGP, Thermo Fisher). Culture medium was replaced every 3 days. When cells reached the logarithmic growth phase, they were seeded into 6-well plates (51110, LABSELECT) at a density of 2×105 cells/well and allowed to adhere for 24 h. To induce differentiation toward a sympathetic neuron-like phenotype, SH-SY5Y cells in the experimental group were cultured in differentiation medium consisting of DMEM supplemented with 2% FBS, 1% penicillin–streptomycin, and 10 μM all-trans retinoic acid (ATRA) for 7 days.24 Control cells received the same medium containing 0.1% DMSO instead of ATRA. After differentiation, both groups were switched to standard growth medium (DMEM with 10% FBS and 1% penicillin–streptomycin) and maintained for an additional 48 h before sample collection. Quantification of Norepinephrine Secretion by ELISA Norepinephrine (NE) secretion was quantified using an ELISA assay to validate functional sympathetic neuron-like differentiation. After 7 days of induction, cell supernatants were collected and centrifuged at 12,000 × g for 10 min at 4°C to remove debris. NE concentration in the supernatants was measured using a Human Norepinephrine (NE) ELISA Kit (ml024646, Mlbio) according to the manufacturer’s instructions. Absorbance was read at 450 nm, and NE concentrations (ng/mL) were calculated based on a standard curve. All experiments were performed with at least three independent biological replicates. Statistical analysis was conducted using GraphPad Prism (version 10.4.1). Comparisons between two groups were performed using an unpaired two-tailed t-test, and P < 0.05 was considered statistically significant. Immunofluorescence Analysis For immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, and blocked with 3% bovine serum albumin (BSA). Cells were then incubated overnight at 4°C with primary antibodies against tyrosine hydroxylase (TH) or β-hydroxylase (DBH), followed by fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired using a fluorescence slide scanner, and fluorescence intensities were quantified via Fiji software across at least three independent experiments. Preparation of Conditioned Medium and Co-Culture with 12Z Cells 12Z cells were maintained in standard growth medium (DMEM supplemented with 10% FBS and 1% penicillin–streptomycin) at 37°C in a humidified incubator with 5% CO2. Conditioned medium (CM) was collected from differentiated SH-SY5Y cells after replacing the differentiation medium with standard growth medium and culturing for an additional 48 h. Supernatants were collected and centrifuged at 1,500 × g for 10 min at 4°C to remove cellular debris, followed by filtration through a 0.22 μm membrane filter. The filtered supernatant was designated as conditioned medium (CM) and stored at −20°C until further use. To evaluate the paracrine effects of sympathetic neuron-like cells on endometriotic epithelial cells, a direct co-culture system was established using Transwell inserts. 12Z cells were seeded in the lower chamber of a Transwell system (5% Corning, 0.4 μm pore size) and allowed to reach approximately 80% confluence. Differentiated SH-SY5Y cells were seeded into the upper chamber inserts at a 1:1 cell ratio relative to 12Z cells. The Transwell inserts had a pore size of 0.4 μm, which allows soluble factors to diffuse between chambers while preventing direct cell–cell contact. Co-cultures were maintained in standard growth medium for 48h before subsequent analyses. Control groups consisted of 12Z cells cultured alone under identical conditions without Transwell inserts. Cell Viability Assay (CCK-8) Cell viability and metabolic activity was assessed using the Cell Counting Kit-8 (CCK-8) assay under both direct co-culture and conditioned medium treatment conditions. 12Z cells were seeded in 96-well plates at 1 × 104 cells/well in 100 μL medium, with three technical replicates per condition. After cell attachment, CCK-8 assays were performed at 0 h, 24 h, and 48 h. Briefly, 10 μL of CCK-8 reagent was added to each well, followed by incubation for 2 h at 37°C. Absorbance at 450 nm was measured using a microplate reader (Multiskan FC, Thermo Fisher). All experiments were repeated at least three independent times. Statistical analyses were conducted using GraphPad Prism (version 10.4.1). To evaluate the independent and interactive effects of treatment group and time, a two-way analysis of variance (ANOVA) was applied. RNA Sequencing and Bioinformatics Analyses Total RNA from 12Z cells treated with CM or control medium (n = 3 per group) was extracted using TRIzol reagent. RNA integrity was assessed prior to library construction (RIN > 7.0). Poly(A)+ RNA was enriched using oligo(dT) beads, followed by RNA fragmentation and reverse transcription to generate strand-specific cDNA libraries. After end repair and adaptor ligation, libraries were amplified by PCR and sequenced on an Illumina NovaSeq 6000 platform with paired-end 150 bp reads. Raw sequencing reads were quality-filtered to obtain clean reads by removing adaptor sequences and low-quality reads. Clean reads were aligned to the human reference genome (Homo sapiens) using HISAT2. Transcript assembly and quantification were performed with StringTie, and gene expression levels were normalized and reported as fragments per kilobase of transcript per million mapped reads (FPKM).25,26 Differential expression analysis was conducted using DESeq2 based on gene-level read counts.27 Genes were considered differentially expressed if they met the thresholds of |log2(fold change)|≥1 and a Benjamini–Hochberg adjusted P value (q values/FDR) < 0.05. Functional enrichment analyses of differentially expressed genes were performed for Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.28 Enrichment was evaluated using a hypergeometric test by comparing the number of differentially expressed genes annotated to each term/pathway against the annotated background gene set; only genes with valid GO/KEGG annotations were included in the calculations. Terms/pathways with nominal p < 0.05 were considered significantly enriched. To investigate potential functional associations among differentially expressed genes, protein–protein interaction (PPI) analysis was performed using the STRING database. Differentially expressed genes were mapped to STRING,29 and interactions with a confidence score > 400 were retained. The resulting interaction network was visualized and further analyzed in Cytoscape.30

Selection of Genetic Instruments In the forward-direction MR analyses, we evaluated the causal effects of genetic liability to five psychiatric disorders on the risk of endometriosis. After instrument selection and LD clumping, we included 30, 23, 28, 28, and 41 independent SNPs as instrumental variables for depression, anxiety, insomnia, schizophrenia, and bipolar disorder, respectively. In the reverse-direction MR analyses, endometriosis was treated as the exposure. After harmonization and removal of ambiguous/palindromic variants, 7 SNPs were retained as genetic instruments for endometriosis across all reverse MR models. Forward MR Analyses Using the IVW method, genetically predicted liability to depression was nominally associated with a higher risk of endometriosis (OR = 1.20, 95% CI 1.00–1.43, P = 0.04) (Table 2). Sensitivity analyses using the weighted median, simple mode, and weighted mode methods yielded directionally consistent but statistically non-significant estimates. MR-Egger regression produced an imprecise estimate with a wide confidence interval (OR = 0.84, 95% CI 0.36–1.94, P = 0.69). No evidence of heterogeneity was observed (Cochran’s Q = 28.11, P = 0.51) and the MR-Egger intercept test did not indicate directional horizontal pleiotropy (intercept = 0.02, P = 0.40) (Table 3), supporting the robustness of the primary IVW finding. The nominal association between depression genetic liability and endometriosis risk should be interpreted with caution, as it does not survive correction for multiple testing across the five psychiatric traits evaluated. | Table 2 Forward-Direction Two-Sample MR Estimates for the Effects of Genetic Liability to Psychiatric Disorders on Endometriosis Risk | | Table 3 Heterogeneity and Horizontal Pleiotropy Assessments for Forward-Direction MR Analyses | For anxiety, the IVW estimate did not support a statistically significant causal association with endometriosis (OR = 1.20, 95% CI 0.97–1.47, P = 0.09) (Table 2). MR-Egger regression suggested an inverse association (OR = 0.21, 95% CI 0.06–0.77, P = 0.03); however, the MR-Egger intercept test indicated evidence of directional pleiotropy (intercept = 0.08, P = 0.01) (Table 3). Therefore, the MR-Egger estimate for anxiety was considered unreliable. Other robust methods (weighted median, simple mode, weighted mode) did not show significant associations. No significant heterogeneity was detected (Q = 22.44, P = 0.43). No causal effects on endometriosis risk were observed for genetically predicted liability to insomnia (IVW OR = 1.01, 95% CI 0.93–1.10, P = 0.80), schizophrenia (IVW OR = 1.00, 95% CI 0.93–1.07, P = 0.99), or bipolar disorder (IVW OR = 0.98, 95% CI 0.91–1.06, P = 0.61) (Table 2). Across these analyses, sensitivity methods yielded consistent null findings, and neither heterogeneity nor directional pleiotropy was evident based on Cochran’s Q tests and MR-Egger intercept tests (Table 3). We applied a Bonferroni correction to account for five independent tests (one per psychiatric trait). The adjusted significance threshold was set at P < 0.01 (0.05/5). Consequently, the nominal association observed for depression (IVW P = 0.04) did not pass multiple testing correction. The primary findings of all forward MR analyses should therefore be regarded as exploratory and suggestive rather than confirmatory. Reverse MR Analyses Using IVW as the primary analysis, there was no evidence that genetic liability to endometriosis causally influenced the risk of depression (OR = 1.04, 95% CI 0.99–1.08, P = 0.09), anxiety (OR = 1.02, 95% CI 0.95–1.08, P = 0.62), insomnia (OR = 0.94, 95% CI 0.84–1.05, P = 0.26), schizophrenia (OR = 0.91, 95% CI 0.78–1.07, P = 0.27), or bipolar disorder (OR = 1.09, 95% CI 0.99–1.21, P = 0.09) (Table 4). Estimates derived from MR-Egger, weighted median, simple mode, and weighted mode methods were broadly consistent with the IVW results (Table 4). Cochran’s Q tests provided no strong evidence of heterogeneity for most reverse-direction models; however, the endometriosis to anxiety analysis showed borderline heterogeneity (Q = 12.61, P = 0.05) (Table 5). MR-Egger intercept tests did not indicate directional horizontal pleiotropy across reverse-direction analyses (all P > 0.05). | Table 4 Reverse-Direction Two-Sample MR Estimates for the Effects of Genetic Liability to Endometriosis on Psychiatric Disorders | | Table 5 Heterogeneity and Horizontal Pleiotropy Assessments for Reverse-Direction MR Analyses | Establishment and Validation of Cell Co-Culture Model Based on the MR findings suggesting depression as a potential risk factor for endometriosis, and given prior evidence linking depression to chronic stress responses and sustained sympathetic activation, we further examined the effects of sympathetic neuronal activity on endometriotic epithelial cells. SH-SY5Y cells were differentiated with 10 μM ATRA for 7 days to establish a sympathetic neuron-like model. ELISA assays showed that NE concentrations in supernatants from the differentiated group (588.72 ± 18.60 ng/mL) were significantly higher than those of the undifferentiated control group (443.45 ± 28.98 ng/mL), indicating successful induction of NE-secreting sympathetic neuron-like properties (Figure 2A). Consistent with a sympathetic neuron-like phenotype, immunofluorescence co-staining revealed the expression of key catecholaminergic enzymes, TH and DBH, following ATRA-induced differentiation (Figure S1). TH, the rate-limiting enzyme in catecholamine biosynthesis, and DBH, which catalyzes the conversion of dopamine to norepinephrine, serve as canonical markers for sympathetic neurons. Immunofluorescence analysis revealed markedly enhanced TH and DBH signals in ATRA-treated cells relative to untreated controls. In the induction group, TH immunoreactivity localized predominantly to the soma and neurites, with DBH exhibiting a congruent distribution, collectively indicating the acquisition of a catecholaminergic phenotype. Quantitative fluorescence intensity analysis further confirmed significant upregulation of both markers following ATRA treatment. The mean fluorescence intensity of TH increased to 82.65 ± 11.40 in the ATRA group compared with 56.65 ± 9.60 in controls (*P < 0.05). Likewise, DBH expression was significantly elevated (83.99 ± 6.06 vs. 66.25 ± 7.15, *P < 0.05; Figure S1B–C). Next, direct co-culture and conditioned medium systems were established. CCK-8 assays demonstrated that, in the conditioned medium system, 12Z cells exposed to sympathetic neuron CM exhibited significantly increased cell viability at 24 h and 48 h compared with 12Z monoculture controls (Figure 2B). In the direct co-culture system, an increased metabolic activity trend was observed at 48 h (Figure 2C). Transcriptomic Profiling Suggests Signaling Pathways Involved in Sympathetic Neuron–Driven Proliferation To explore the molecular programs associated with the pro-proliferative effect of sympathetic neuron–conditioned medium on endometriotic epithelial cells, RNA sequencing was performed on 12Z cells treated with conditioned medium (CM; n = 3, CM1–CM3) and control conditions (P; n = 3, P1–P3). Principal component analysis (PCA) showed clear separation between CM-treated and control samples along PC1 (25.46%) and PC2 (20.75%), indicating a reproducible transcriptomic shift induced by conditioned medium (Figure 3A). Differential expression analysis identified 327 differentially expressed genes (DEGs), including 124 upregulated and 203 downregulated genes. The volcano plot highlighted robust changes in representative transcripts, including upregulation of RPL34, RARRES1, PEPD, and OAS3, along with downregulation of MFN2 and NFKB2 (Figure 3B). GO functional enrichment analyses indicated that DEGs were over-represented in biological processes related to signal transduction, phosphorylation, cell adhesion, and immune-associated programs (eg., innate immune response, immune response, and cellular response to lipopolysaccharide) (Figure 3C). KEGG pathway enrichment further prioritized several canonical signaling cascades implicated in proliferation and survival, including the Ras, PI3K–Akt, and MAPK signaling pathways, as well as the cAMP signaling pathway (Figure 3D). Protein–Protein Interaction Network Identifies Key Hub Proteins To further explore potential interaction modules within the CM-responsive transcriptional program, a protein–protein interaction (PPI) network was constructed based on the identified DEGs using the STRING resource and visualized in Cytoscape. Several highly connected nodes were observed in the network, including CD244, PLEK, and GNGT2, suggesting potential hub-like positions within the DEG interaction landscape. Additional connected components included immune- and signaling-associated proteins such as CD70, HAVCR2, FLT3, and EGF (Figure 4), indicating a network architecture that may integrate immune modulation with growth factor–related signaling in conditioned medium–treated 12Z cells. | Figure 4 Protein–protein interaction (PPI) network analysis of differentially expressed genes in 12Z cells treated with sympathetic neuron–derived conditioned medium. |

In this study, we performed bidirectional two-sample MR analyses to systematically evaluate the bidirectional associations between genetic liability to depression, anxiety disorders, insomnia, bipolar disorder, schizophrenia, and endometriosis. We found that genetic liability to depression was nominally associated with increased endometriosis risk in the primary inverse-variance weighted MR analysis (OR = 1.20, 95% CI 1.00–1.43, P = 0.04), but this signal was not supported after stringent Bonferroni correction (P threshold < 0.01), and sensitivity analyses yielded directionally consistent but non-significant estimates. No robust causal evidence was observed for other psychiatric traits, and reverse-direction MR analyses found no significant effect of endometriosis genetic liability on the risk of any of the included psychiatric disorders. These findings provide preliminary genetic evidence to clarify the directionality underlying the clinical comorbidity between these conditions. Globally, endometriosis has a prevalence of approximately 10% among women of reproductive age,31 and nearly half of affected patients present with comorbid psychiatric disorders including depression and anxiety in clinical settings. A meta-analysis included 9 studies confirmed that patients with endometriosis had a significantly increased risk of comorbid depression and anxiety (pooled relative risk RR = 2.93 for depression, pooled RR = 2.82 for anxiety). However, the authors also noted that limited by constraints including heterogeneity in study design and publication bias, previous observational studies have been unable to clarify the causal temporality and underlying mechanisms linking these conditions.32 Previous genetic studies have identified a significant genetic correlation between endometriosis and depression (rg = 0.27–0.36),6,33 as well as positive genetic correlations with anxiety disorders (rg = 0.33),6 eating disorders (rg = 0.616, and post-traumatic stress disorder (PTSD) (rg = 0.31).34 A study further demonstrated that genetic susceptibility loci for endometriosis are significantly enriched in pathways associated with pain and emotional regulation,8 providing a theoretical genetic basis for the comorbidity between endometriosis and psychiatric disorders. Notably, most previous MR studies on this topic have focused exclusively on depression and anxiety phenotypes, with no subsequent mechanistic validation performed.35 We verified the robustness of our findings through multidimensional sensitivity analyses, and identified that depression is an upstream causal risk factor for endometriosis. Building on the core risk factor identified via MR analyses, we proposed the scientific hypothesis that abnormal sympathetic activation serves as the key intermediate link through which depression mediates the elevated risk of endometriosis onset, and we performed preliminary validation of this hypothesis using in vitro experiments. In this study, we treated SH-SY5Y cells with 10 μM all-trans retinoic acid (ATRA) for 7 days to successfully establish a sympathetic neuron-like cell model with norepinephrine (NE) secretory function. This induction protocol has been validated in multiple studies,24,36 which enables stable differentiation of SH-SY5Y cells toward a sympathetic neuron-like phenotype, and is a well-established in vitro model for investigating sympathetic neuron function. Our cellular experimental results showed that conditioned medium from differentiated sympathetic neuron-like cells significantly promoted the increased the cell viability of 12Z endometriotic epithelial cells, which is highly consistent with the previous findings. Consistent with our results, multiple studies have confirmed that β2-adrenergic receptor (β2-AR), the primary endogenous receptor of norepinephrine, is highly expressed in endometriotic lesions, and that sympathetic activation via norepinephrine promotes the malignant biological behavior of endometriotic cells through β2-AR signaling.37 Notably, some studies have reported opposite findings, showing that β2-AR agonists can inhibit the viability and invasion of ectopic lesions via β2-AR signaling.38 This bidirectional effect of β2-AR activation may reflect context-dependent signaling outcomes under different experimental settings. Further transcriptome sequencing analysis revealed that differentially expressed genes (DEGs) were significantly enriched in the Ras, phosphatidylinositol 3 kinase and protein kinase B (PI3K-Akt), mitogen-activated protein kinase (MAPK), and cyclic adenosine monophosphate (cAMP) signaling pathways. Among these, the PI3K-Akt and MAPK pathways have been well documented as core pathways regulating the proliferation, survival, and invasion of endometriotic cells, with sustained activation observed in ectopic endometrial tissues.39,40 Meanwhile, cAMP, a key second messenger downstream of sympathetic neurotransmitters, has also been confirmed to participate in the pathological process of endometriosis. Collectively, our results provide a molecular-level explanation for sympathetic activation-mediated endometriosis progression at the transcriptomic level. Current clinical management of endometriosis remains dominated by hormonal therapy and surgical resection, with key unmet clinical challenges including high postoperative recurrence rates and a lack of curative treatment options. In addition to the sympathetic activation pathway validated in this study, previous research has uncovered other potential mechanisms underlying the comorbidity between these conditions, including dysregulation of the stress response mediated by hypothalamic-pituitary-adrenal (HPA) axis dysfunction,41 structural and functional abnormalities of the periaqueductal gray (PAG),42 and the vicious cycle of pain-depression central sensitization.43 These pathways may exert synergistic effects with sympathetic activation, collectively participating in the comorbid progression of endometriosis and mood disorders. Future studies should also explore the potential roles of burn-out and sexual dysfunction, which have been linked to both depression and endometriosis, in the comorbidity and progression of these conditions.44 Further in-depth mechanistic exploration of the crosstalk between these multiple pathways is warranted in future studies, which may identify novel targets for combined clinical intervention. Several limitations of this study should be acknowledged. First, all GWAS data included in this study were derived from individuals of European ancestry, which not only limits the generalizability of our findings to non-European populations including Asian populations, but also precludes assessment of the potential impact of ethnic differences on the genetic association between endometriosis and psychiatric disorders. Second, the in vitro functional validation in this study was performed using only the 12Z endometriotic epithelial cell line, lacking multidimensional validation using primary cells, other cell lines, and in vivo animal models. Furthermore, we did not conduct in-depth exploration of the core receptor and key regulatory genes underlying the pro-proliferative effect of sympathetic neurons on endometriotic cells. Third, our genetic analysis only covered common genetic variants, while rare variants and more complex genetic structures are increasingly recognized as contributors to psychiatric and reproductive traits.45,46 Meanwhile, the substantial clinical underdiagnosis of endometriosis may compromise the accuracy of phenotypic definition in the GWAS datasets, and we were unable to evaluate the potential roles of rare variants and complex genetic structures in subgroups with comorbid endometriosis and psychiatric disorders.

In bidirectional Mendelian randomization analyses, genetic liability to depression showed a nominal association with increased endometriosis risk that did not survive multiple testing correction; whereas reverse-direction analyses did not support endometriosis as a causal determinant of psychiatric disorders. In vitro sympathetic neuron-like models and transcriptomic analyses showed that sympathetic neuron–derived factors enhanced 12Z cell proliferation and were accompanied by enrichment of immune-related and signaling pathways. In exploratory bidirectional Mendelian randomization analyses, genetic liability to depression showed a nominal association with increased endometriosis risk that did not survive multiple testing correction. Data Sharing Statement The datasets used and analyzed in this study are available from Songwei Feng ([email protected]) upon reasonable request. Ethics Approval and Consent to Participate The present study consisted entirely of in vitro experiments using established cell lines. According to the guidelines of the Ethics Committee of The Fourth Affiliated Hospital of Soochow University, ethical approval was not required as the study did not involve human subjects, animal subjects, or primary clinical samples. Since GWAS data utilized in this study are publicly accessible, an ethical review is not necessary. Acknowledgments The authors would like to express their sincere gratitude to the undergraduate students majoring in Clinical Medicine at Suzhou Medical College, Soochow University, for their active participation and valuable contributions to this study. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Funding This work was supported by the Postdoctoral Fellowship Program of CPSF (No. GZC20251352), the China Postdoctoral Science Foundation (No. 2024M762304), the Suzhou Medical College-QiLu Medical Research Program of Soochow University (No. 24QL200112) and the National Natural Science Foundation of China (No. 82503307). Disclosure The authors declare that they have no competing interests in this work.

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