{"paper_id":"88e39e2a-6d2e-4c71-901a-edffbec09fc6","body_text":"Endometriosis is a chronic, oestrogen-dependent inflammatory disease characterised by the presence of endometrial-like tissue outside the uterine cavity, affecting approximately 10% of women of reproductive age worldwide, corresponding to an estimated 190 million individuals globally [ 1 , 2 ]. Clinically, the disease manifests as chronic pelvic pain, dysmenorrhoea, dyspareunia, and infertility, with a diagnostic delay averaging 7–10 years due to symptom normalisation and the lack of non-invasive diagnostic tools [ 1 ]. According to the most widely accepted retrograde menstruation theory, menstrual debris implants onto peritoneal surfaces; however, although retrograde menstruation occurs in the majority of women, endometriosis develops in only a subset [ 3 , 4 ]. This observation underscores the multifactorial nature of the disease, emphasising the complex interplay between genetic predisposition, epigenetic modifications, immune dysfunction, and environmental influences [ 3 ]. While genome-wide association studies have identified numerous genetic loci associated with endometriosis, these variants explain only a small proportion of disease heritability [ 5 , 6 ], and functional studies are needed to clarify the specific molecular pathways contributing to disease development.\nOxidative stress plays a central role in the pathogenesis of endometriosis [ 7 , 8 ]. Iron released from erythrocytes reaching the pelvic cavity during retrograde menstruation leads to excessive production of reactive oxygen species (ROS) through the Fenton reaction [ 8 ]. This oxidative environment contributes to genomic instability by causing lipid peroxidation, protein oxidation, and DNA damage [ 7 , 8 ]. The accumulation of DNA double-strand breaks and base damage in endometriotic cells has been associated with defective DNA repair mechanisms [ 9 , 10 ]. The homologous recombination (HR) pathway is critical for the error-free repair of DNA double-strand breaks, and variants in genes involved in this pathway threaten genome integrity [ 11 ]. Polymorphisms in DNA repair genes such as  XRCC1 ,  XRCC3 , and  BLHX  have been reported to be associated with endometriosis risk and chromosome instability [ 12 , 13 ]. These findings suggest that deficiencies in DNA repair mechanisms play an important role in the molecular pathogenesis of endometriosis [ 9 , 10 , 14 ].\nGEN1  (Gap Endonuclease 1) is a Holliday junction resolvase enzyme that plays a critical role in homologous recombination [ 15 ]. Resolution of four-way Holliday junction structures formed during the repair of DNA double-strand breaks is essential for maintaining genome integrity and chromosome segregation [ 15 ].  GEN1  is a member of the Rad2/XPG nuclease family and specifically binds to Holliday junctions, resolving them by introducing symmetric nicks in the non-crossing strands [ 11 , 16 ]. Unlike other Rad2/XPG nucleases, human  GEN1  protein contains a chromodomain for DNA recognition and binding [ 16 ]. Nuclear exclusion and activation of  GEN1  are regulated throughout the cell cycle, and this spatial control is critical for preventing genomic instability by restricting  GEN1  activity to mitosis [ 15 , 17 ]. Furthermore,  GEN1  has been shown to play a role in maintaining centrosome integrity, and  GEN1  deficiency has been associated with aberrant centrosome numbers, multiple mitotic spindle poles, and spontaneous DNA damage [ 18 ]. A recent whole-exome sequencing study identified a  GEN1  gene variant (c.1574C>T, p.Ser525Leu) associated with endometriosis in a Turkish family, highlighting the potential role of DNA repair mechanisms in endometriosis pathogenesis [ 19 ].\nThree-dimensional organoid culture systems have become powerful tools for disease modelling by recapitulating the physiological features of human tissues in vitro [ 20 , 21 ]. Human endometrial organoids were first successfully established by two independent groups in 2017, demonstrating long-term expansion capacity, genetic stability, and hormonal responsiveness [ 22 , 23 ]. Endometrial organoids preserve the cellular heterogeneity, transcriptomic profile, and functional characteristics of the original tissue, providing a unique platform for investigating endometrial physiology and pathology [ 23 ]. In endometriosis research, patient-derived organoids have been shown to capture clinical heterogeneity of the disease, reflect molecular features of ectopic lesions, and be suitable for drug screening studies [ 24 ]. These organoid models maintain disease-specific characteristics including aberrant activation in integrin, PI3K-AKT, and WNT signalling pathways, enabling identification of potential therapeutic targets [ 24 ]. The recent development of internationally harmonised protocols for endometrial organoid culture further supports the standardisation and reproducibility of this model system [ 25 , 26 ]. Furthermore, the application of functional genomic approaches, including gene silencing and gene editing technologies, to patient-derived organoids offers new opportunities for investigating the biological consequences of specific genetic variants implicated in endometriosis [ 25 ].\nAlthough the critical role of the  GEN1  gene in DNA repair mechanisms and its potential association with endometriosis have been suggested, including the identification of a missense variant (c.1574C>T, p.Ser525Leu) in a Turkish family [ 19 ], the functional consequences of  GEN1  deficiency in endometrial cells remain unexplored. Patient-derived endometrial organoids provide an ideal platform for investigating the functional effects of gene deficiencies at the cellular level [ 24 ]. In this study, we aimed to establish and characterise endometrial organoids derived from endometriosis patients and controls, compare  GEN1  expression between groups, and evaluate the effects of  GEN1  deficiency on cell proliferation and DNA damage accumulation. Our study represents the first organoid-based investigation exploring the functional role of  GEN1  in endometriosis pathogenesis, and the findings may contribute to understanding the molecular basis of the disease and elucidating the role of DNA repair dysregulation in endometriosis pathogenesis.\n\nA total of six women were enrolled in this study: three patients with laparoscopically and histopathologically confirmed endometriosis and three control subjects without laparoscopic evidence of endometriosis. The demographic and clinical characteristics of the participants are summarised in  Table 1 . No statistically significant differences were observed between groups for age, body mass index, menstrual cycle day, or parity (all  p  > 0.05, exact Mann–Whitney U test). All endometrial samples were collected during the proliferative phase of the menstrual cycle (cycle days 8–12).\nTo investigate  GEN1  expression in human endometrial tissue, immunofluorescence analysis was performed on freshly isolated primary endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) from control subjects ( n  = 3). Confocal microscopy revealed that  GEN1  protein was expressed in both cell types, with prominent nuclear localisation consistent with its known function as a Holliday junction resolvase ( Figure 1 A). E-cadherin staining in epithelial cells confirmed the preservation of cell–cell junctions and epithelial architecture during the isolation procedure ( Figure 1 A). RT-qPCR analysis demonstrated that  GEN1  mRNA was expressed in both EECs and ESCs, with approximately 1.8-fold higher expression levels detected in epithelial cells compared with stromal cells ( Figure 1 B).\nGEN1  mRNA and protein levels were compared in primary endometrial cells obtained from patients with endometriosis ( n  = 3) and control subjects ( n  = 3). RT-qPCR analysis revealed that  GEN1  mRNA expression was reduced in the endometriosis group compared with controls (0.52 ± 0.14 vs. 1.00 ± 0.19,  p  = 0.05) ( Figure 1 C). Immunofluorescence intensity analysis demonstrated a similar reduction in  GEN1  protein levels in endometriosis samples compared with the control group (0.54 ± 0.18 vs. 1.00 ± 0.22,  p  = 0.05) ( Figure 1 D). This reduction was observed in both epithelial and stromal cell populations.\nIsolated single cells and small epithelial fragments obtained from both control subjects and endometriosis patients were embedded in Matrigel and allowed to form EEOs over 7–10 days. The resulting EEOs recapitulated the epithelial organisation of the endometrium, exhibiting well-defined luminal structures and E-cadherin-positive cell–cell junctions ( Figure 2 A).\nEEO formation efficiency was lower in the endometriosis group compared with controls (18.4 ± 5.6% vs. 25.2 ± 6.8%,  p  = 0.10), representing a trend toward reduced EEO-forming capacity ( Figure 2 B). The mean EEO diameter was also reduced in endometriosis-derived EEOs compared with control EEOs (124.6 ± 34.2 µm vs. 155.8 ± 32.6 µm,  p  = 0.10), and a lower proportion of endometriosis EEOs displayed cystic morphology with luminal structures (72.4 ± 10.2% vs. 83.6 ± 10.5%,  p  = 0.10) ( Figure 2 B).\nImmunofluorescence analysis demonstrated that  GEN1  was expressed in the EEO epithelial compartments in both groups, with nuclear enrichment co-localising with E-cadherin-positive regions ( Figure 2 A).  GEN1  immunofluorescence intensity was reduced in endometriosis-derived EEOs compared with control EEOs (0.46 ± 0.15 vs. 1.00 ± 0.21 AU,  p  = 0.05) ( Figure 2 C).\nTo assess whether EEOs maintained endometrial epithelial characteristics, hormone receptor expression was evaluated. RT-qPCR analysis showed that ESR1 mRNA levels were comparable between groups (control: 1.00 ± 0.20 vs. endometriosis: 0.94 ± 0.18,  p  > 0.05), as were PGR mRNA levels (control: 1.00 ± 0.24 vs. endometriosis: 0.91 ± 0.22,  p  > 0.05), indicating preserved hormonal receptor status in EEOs from both groups ( Figure 2 D).\nRNAi-mediated gene silencing was applied to organoids derived from both control subjects and endometriosis patients. Seven-day EEO cultures were transfected with  GEN1  -specific siRNA duplexes or non-targeting control siRNA at a final concentration of 20 nM ( Figure 3 A). Transfection efficiency, assessed by siGLO Red co-transfection, was 87.3 ± 5.4% in control EEOs and 85.8 ± 6.2% in endometriosis EEOs ( p  > 0.05).\nRT-qPCR analysis confirmed efficient  GEN1  knockdown in both groups: 72 ± 8% reduction in control EEOs and 68 ± 10% reduction in endometriosis EEOs relative to respective non-targeting siRNA controls ( Figure 3 C). At the protein level, Western blot densitometry indicated a 60 ± 11% reduction in control EEOs and 53 ± 13% reduction in endometriosis EEOs ( Figure 3 D). Immunofluorescence imaging showed reduced  GEN1  signal in siRNA-treated EEOs from both groups, while EEO morphology was preserved following transfection.\nCell proliferation and epithelial integrity were examined in siRNA-treated EEOs from both control and endometriosis groups. BrdU incorporation analysis revealed a reduction in the proportion of proliferating cells following  GEN1  knockdown in both groups. In control EEOs, the percentage of BrdU-positive cells decreased from 38.2 ± 6.2% to 23.1 ± 5.6% following  GEN1  siRNA treatment, corresponding to a 39.5% reduction ( p  = 0.05) ( Figure 3 E). Endometriosis-derived EEOs exhibited a more pronounced response to  GEN1  knockdown, with BrdU-positive cells decreasing from 32.6 ± 5.8% to 16.4 ± 4.4%, corresponding to a 49.7% reduction ( p  = 0.05) ( Figure 3 E).\nE-cadherin immunofluorescence staining demonstrated that cell–cell junctions and epithelial polarisation were preserved in  GEN1  knockdown EEOs from both groups. No apparent difference in E-cadherin expression intensity or localisation pattern was observed between control siRNA and  GEN1  siRNA groups in either control or endometriosis-derived EEOs.\nGEN1  knockdown increased DNA damage in both groups. In control EEOs, γH2AX immunofluorescence intensity increased from 1.00 ± 0.22 to 1.72 ± 0.40 following si GEN1  treatment ( p  = 0.10). Endometriosis-derived EEOs exhibited a more pronounced increase, from 2.32 ± 0.44 to 3.58 ± 0.52 ( p  = 0.05). In absolute terms, endometriosis-derived EEOs showed a greater increase in γH2AX intensity (Δ1.26) than control EEOs (Δ0.72), although the relative increase was lower (54% vs. 72%) owing to the already elevated baseline in the endometriosis group ( Figure 4 ). Although the increase in γH2AX intensity in control EEOs did not reach statistical significance, the consistent directionality across groups supports a biological effect of  GEN1  depletion.\nEndometriosis EEOs exhibited elevated baseline DNA damage compared with controls, as indicated by increased γH2AX immunofluorescence intensity (2.32 ± 0.44 vs. 1.00 ± 0.28,  p  = 0.05) ( Figure 5 ).\n\nThis study is the first to investigate the function of the DNA repair enzyme  GEN1  in endometrial EEOs and its potential role in endometriosis pathogenesis. Three key findings emerged: (1)  GEN1  mRNA and protein expressions were reduced by approximately half in eutopic endometrium of endometriosis patients compared with controls; (2) patient-derived EEOs maintained consistently low  GEN1  expression, and EEO formation efficiency showed a non-significant trend toward reduction in the endometriosis group ( p  = 0.10); and (3) RNAi-mediated  GEN1  knockdown reduced cell proliferation more pronouncedly in endometriosis-derived EEOs than in controls (49.7% vs. 39.5% reduction). These findings support the notion that defects in DNA repair mechanisms play an important role in endometriosis pathogenesis and provide the first organoid-based evidence for the potential functional significance of  GEN1  in this disease [ 8 , 19 ]. Furthermore, endometriosis-derived EEOs exhibited elevated baseline γH2AX immunofluorescence compared with controls, indicating increased DNA double-strand break accumulation in the disease state. This observation, combined with the heightened sensitivity to  GEN1  knockdown, supports the notion that endometriotic cells possess an underlying genomic fragility that may be further exacerbated by additional disruption of the homologous recombination pathway. Indeed,  GEN1  knockdown directly increased γH2AX immunofluorescence in both groups, with endometriosis-derived EEOs showing a greater absolute increase in γH2AX intensity (Δ1.26 vs. Δ0.72), consistent with a role for  GEN1  deficiency in exacerbating DNA damage accumulation.\nThe reduction in  GEN1  expression observed in the present study is consistent with previous studies demonstrating altered DNA repair gene expression in eutopic endometrium of women with endometriosis. Govatati et al. demonstrated significantly decreased  PTEN  protein expression in eutopic endometrium of endometriosis patients [ 27 ]. A transcriptome meta-analysis by Poli-Neto et al. revealed downregulation of genes related to DNA repair and cell cycle control in eutopic endometrium of women with endometriosis [ 28 ]. The  GEN1  expression reduction (~48%) observed in our study aligns with this general trend and suggests that the homologous recombination pathway may be compromised in endometriosis [ 10 ]. Notably, the association of a  GEN1  genetic variant with endometriosis risk reported by Kina et al. further supports the potential importance of  GEN1  in this disease [ 19 ]. Broader genetic evidence from GWAS studies has identified multiple loci associated with endometriosis susceptibility, though the specific contribution of DNA repair gene variants remains incompletely characterised [ 5 , 6 ]. Polymorphisms in DNA repair genes, including  XRCC1 ,  XRCC3 , and  BLHX , have been associated with endometriosis risk and chromosomal instability in lymphocytes from affected women [ 12 , 13 ]. Collectively, these genetic findings converge with our functional data to suggest that impaired homologous recombination, exemplified here by reduced  GEN1  expression, may represent one of the molecular mechanisms underlying the genomic instability observed in endometriosis.\nThe patient-derived EEO model employed in our study offers significant advantages over traditional two-dimensional cell culture and animal models for investigating endometriosis pathogenesis. EEOs allow long-term expansion while preserving the genetic and phenotypic characteristics of the original tissue and reflecting disease diversity [ 21 , 24 ]. As demonstrated by Boretto et al., endometriosis-derived EEOs exhibit disease-specific features and can recapitulate the original lesion following in vivo transplantation [ 24 ]. Gu et al. emphasised that EEOs demonstrate high stability and patient specificity compared with cell lines and animal models [ 29 ]. The non-significant trends toward lower EEO formation efficiency (18.4% vs. 25.2%,  p  = 0.10) and smaller EEO size (124.6 vs. 155.8 µm,  p  = 0.10) observed in our study are consistent with disease-specific morphological differences, though these should be interpreted cautiously given the small sample size. Similar morphological differences have been reported by Boretto et al. [ 22 ], demonstrating that ectopic EEOs grow more slowly and exhibit different morphology compared with those derived from healthy tissues [ 23 ].\nThe greater proliferation reduction in endometriosis EEOs following  GEN1  knockdown (49.7% vs. 39.5%,  p  = 0.05) is consistent with synthetic lethality, where pre-existing DNA repair defects sensitise cells to further pathway inhibition [ 30 , 31 ]. The already reduced basal  GEN1  expression in endometriosis EEOs may fall below a critical threshold upon RNAi, severely compromising homologous recombination capacity.\nIron released during retrograde menstruation leads to excessive production of reactive oxygen species through the Fenton reaction, causing DNA damage [ 7 , 8 ]. The landmark study by Anglesio et al. detected somatic mutations in 79% of deep infiltrating endometriosis lesions and identified mutations in known cancer driver genes such as  ARID1A ,  PIK3CA ,  KRAS , and  PPP2R1A  in 26% of patients [ 32 ]. These findings suggest that reduced DNA repair capacity may predispose to genomic instability and progressive accumulation of somatic mutations [ 9 , 10 ]. The reduction in  GEN1  expression observed in our study indicates that a specific defect in the homologous recombination pathway may play a role in endometriosis pathophysiology.\nOur findings have potential clinical and translational implications. The gold standard for endometriosis diagnosis remains invasive laparoscopic surgery, with an average diagnostic delay of 7–10 years [ 1 , 2 ]. Alterations in DNA repair gene expression present potential candidates for non-invasive diagnostic biomarker development. The Cochrane review by Nisenblat et al. emphasised that biomarker panels may enhance diagnostic performance, and  GEN1  expression could be evaluated as part of such a panel in future studies [ 33 ]. Furthermore, targeting DNA repair pathways offers new therapeutic opportunities. The successful use of PARP inhibitors in gynaecological cancers [ 31 , 34 ] suggests that similar approaches could be explored in endometriosis patients with DNA repair defects, although this remains speculative and would require extensive further investigation. A notable strength of this study is that endometriosis diagnosis was confirmed both laparoscopically and histopathologically, and controls were verified as disease-free during laparoscopic evaluation, ensuring robust group classification.\nThis study has several limitations. First, the sample size is relatively small ( n  = 3 per group), which limits statistical power; however, similar sample sizes are commonly employed in organoid studies [ 20 , 23 , 24 ]. This sample size is consistent with the exploratory nature of the study and comparable to other patient-derived endometriosis organoid studies that have employed limited numbers of independently derived organoid lines for functional analyses [ 24 ]. Additionally, detailed menstrual history parameters, including age of menarche and menstrual duration, were not systematically recorded; age of menarche was subject to recall bias and was not consistently available across participants, while menstrual duration demonstrated intra-individual variability that precluded reliable retrospective assessment. Furthermore, one control participant had hypertension and one had uterine myoma; although these conditions are not directly implicated in  GEN1  regulation, their potential influence on endometrial gene expression cannot be entirely excluded. Second, only advanced-stage (stage III–IV) endometriosis patients were included, and  GEN1  expression changes in early-stage patients could not be evaluated. Third, samples were collected only in the proliferative phase, and the effects of hormonal changes on  GEN1  expression could not be investigated. Fourth, endometrial organoids contain only epithelial cells and do not include stromal cells and immune cells [ 20 ]; future studies employing assembloid co-culture systems integrating stromal and immune components, or organ-on-chip platforms recapitulating the endometriotic microenvironment, would provide a more comprehensive understanding of  GEN1  function in disease pathophysiology [ 26 ]. Fifth, although γH2AX analysis was extended to include post-knockdown comparisons demonstrating increased DNA damage following  GEN1  silencing, the sample size remains small and further validation in larger cohorts is warranted. Finally, our study focused on a single DNA repair gene, and simultaneous evaluation of other homologous recombination pathway genes was not performed. Future studies should validate these findings in larger patient cohorts and across different menstrual cycle phases.\n\nThis study was approved by the Institutional Review Board of Biruni University Ethics Committee (Approval No: 2024-BİAEK/10-49; Date: 26 May 2025). Written informed consent was obtained from all participants prior to tissue sampling. The study was conducted in accordance with the principles of the Declaration of Helsinki (2013 revision). A total of six women were enrolled in this study between July and November 2025. The study population consisted of two groups: patients with laparoscopically confirmed endometriosis staged according to the revised American Society for Reproductive Medicine (rASRM) classification ( n  = 3; stage III,  n  = 1; stage IV,  n  = 2) and a control group without evidence of endometriosis confirmed by laparoscopic visualisation ( n  = 3). Inclusion criteria for both groups were as follows: women of reproductive age (18–45 years) with regular menstrual cycles (21–35 days). For the endometriosis group, a prior diagnosis confirmed by laparoscopic surgery and histopathological examination was required. For the control group, the absence of endometriosis was confirmed during laparoscopic surgery performed for benign indications. Exclusion criteria included hormonal therapy within the preceding three months (oral contraceptives, progestins, or GnRH analogues), suspected or confirmed endometrial hyperplasia or malignancy, active pelvic inflammatory disease or genital infection, pregnancy or lactation, history of autoimmune disease, diabetes mellitus or uncontrolled systemic disease, and use of medications that may affect the endometrium. Eutopic endometrial tissue was selected as the primary cell source for endometrial epithelial organoid (EEO) derivation.\nEndometrial tissue samples were obtained by pipelle biopsy (Pipelle de Cornier, Laboratoire CCD, Paris, France) from the uterine cavity. Menstrual cycle phase was determined based on the calculated cycle day from the last menstrual period and confirmed by histological evaluation of endometrial dating by an experienced gynaecological pathologist. All samples were collected during the proliferative phase of the menstrual cycle (cycle days 6–14). Fresh tissue specimens were immediately placed in DMEM/F12 transport medium (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 1% penicillin–streptomycin and transported to the laboratory at 4 °C for processing within 2–4 h of collection.\nEndometrial tissue samples were processed using a combination of controlled mechanical dissociation and sequential enzymatic digestion. All reagents were thawed on ice prior to use, and Matrigel handling was performed at 4 °C to prevent premature polymerisation. Tissue samples were first minced into approximately 1–2 mm 3  fragments using sterile surgical scissors in DMEM/F12 (Gibco) supplemented with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco). Tissue fragments were washed three times in warm Dulbecco’s phosphate-buffered saline (DPBS; Gibco) to remove blood and debris (300×  g , 5 min, 4 °C).\nTissue fragments were subsequently transferred to a sterile enzymatic digestion solution containing 1 mg/mL collagenase type IV (Cat. No: C5138; Sigma-Aldrich, St. Louis, MO, USA) and 50 U/mL DNase I (Cat. No: 10104159001; Roche, Basel, Switzerland) in DMEM/F12. Digestion was performed at 37 °C in a shaking water bath for 60–90 min until partial dissociation was achieved. Following centrifugation (300×  g , 5 min, 4 °C), residual extracellular matrix was further dissociated with 0.05% trypsin-EDTA (Gibco) for 10 min at 37 °C. Enzymatic activity was immediately neutralised using a solution containing 0.5 mg/mL soybean trypsin inhibitor (Sigma-Aldrich).\nThe resulting cell suspension was washed twice with complete DMEM/F12 medium and sequentially passed through 100 µm and 40 µm cell strainers (Corning, Corning, NY, USA). Epithelial glandular fragments retained on the 40 µm strainer were collected by inverting the strainer and backwashing with phenol red-free Advanced DMEM/F12 (Gibco). Cell number and viability were assessed using trypan blue exclusion with a haemocytometer. Isolated cells and glandular fragments were resuspended in ice-cold Matrigel Matrix Basement Membrane (Cat. No: 356234; Corning; lot number: 15525015; protein concentration: 10.6 mg/mL). Twenty-microlitre domes of the cell–Matrigel mixture were plated onto pre-warmed 48-well plates and allowed to polymerise at 37 °C for 15 min.\nFollowing polymerisation, 250 µL of organoid expansion medium was added to each well. The expansion medium consisted of phenol red-free Advanced DMEM/F12 (Gibco) supplemented with 1% GlutaMAX (Gibco), 1% HEPES (Gibco), 1% penicillin–streptomycin, 1× B27 supplement (Gibco), 1× N2 supplement (Gibco), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 50 ng/mL recombinant human EGF (PeproTech, Cranbury, NJ, USA), 100 ng/mL recombinant human FGF-10 (PeproTech), 100 ng/mL recombinant human Noggin (PeproTech), 500 ng/mL recombinant human R-spondin 1 (PeproTech), 500 nM A83-01 (Tocris, Bristol, UK), and 10 mM nicotinamide (Sigma-Aldrich). Y-27632 ROCK inhibitor (10 µM; Sigma-Aldrich) was included during the initial 48 h following plating and after each passage, then withdrawn from the culture medium for the remainder of the expansion period. Cultures were maintained under standard conditions (37 °C, 5% CO 2 , humidified atmosphere), and medium was changed every 48 h. Organoids were monitored daily for morphological development using an inverted phase-contrast microscope and passaged every 7–10 days by mechanical dissociation or using TrypLE Express (Gibco). The organoid isolation, culture, and expansion procedures described above were performed in accordance with the WERF EPHect-EM-Organoids standardisation guidelines for endometrial organoid research [ 26 ]. Organoid cultures were confirmed to be free of mycoplasma contamination by PCR-based testing prior to experimental use. All experiments were performed using EEOs between passages 2 and 5.\nFor  GEN1  gene silencing, EEOs were grown in culture for 7 days to allow the formation of compact, functional spheroids. Prior to transfection, EEOs were released from Matrigel by incubation with Cell Recovery Solution (Corning) for 30 min at 4 °C and dissociated into a single-cell suspension by gentle pipetting. Cells were then transfected with  GEN1  -specific small interfering RNA (siRNA) duplexes or non-targeting control siRNA at a final concentration of 20 nM using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions.\nThe  GEN1 -targeting siRNA sequences were as follows: s132626, sense 5′-GGACUUAACAUUUAUGAGAtt-3′ and antisense 5′-UCUCAUAAAUGUUAAGUCCaa-3′; s132627, sense 5′-GGACAGUGCUAUGCUCGAAtt-3′ and antisense 5′-UUCGAGCAUAGCACUGUCCtt-3′ (Ambion, Thermo Fisher Scientific, Austin, TX, USA). A non-targeting siRNA duplex (Silencer Select Negative Control 1 siRNA, Cat. No: 4390843; Ambion) served as the negative control. These duplexes were selected based on pilot experiments demonstrating higher knockdown efficacy compared with other siRNA sequences.\nFor transfection, siRNA–Lipofectamine complexes were prepared in Opti-MEM (Gibco) and incubated at room temperature for 20 min. Cells were incubated with transfection complexes in suspension for 4 h at 37 °C, then re-embedded in fresh Matrigel and cultured in organoid expansion medium. In selected experiments, cells were co-transfected with 1 nM siGLO Red transfection indicator (Cat. No: D-001630-02; Dharmacon, Lafayette, CO, USA) to monitor transfection efficiency. Cells were harvested 48 h post-transfection, and knockdown efficiency was verified by RT-qPCR and Western blot analysis.\nTo assess cell proliferation, a BrdU incorporation assay was performed. siRNA-treated EEOs were labelled with 10 µM BrdU (Sigma-Aldrich) for 2 h at 48 h post-transfection. Following labelling, EEOs were released from Matrigel, dissociated into single-cell suspensions, and fixed with ice-cold 70% ethanol. DNA was denatured with 2 M HCl containing 0.5% Triton X-100 and neutralised with 0.1 M Na 2 B 4 O 7  (pH 8.5). Cells were stained with FITC-conjugated anti-BrdU antibody (Cat. No: 11-5071-42; eBioscience, Thermo Fisher Scientific, San Diego, CA, USA) and propidium iodide (PI). BrdU incorporation was analysed by flow cytometry (BD FACSCanto II), and data were evaluated using FlowJo software (version 11; BD Biosciences). Each experiment was performed with at least three biological replicates.\nFor immunofluorescence analysis, EEOs were first released from Matrigel by incubation with Cell Recovery Solution (Corning) at 4 °C for 30 min. Released EEOs were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. For whole-mount staining, fixed EEOs were washed three times with PBS and permeabilised overnight at 4 °C with PBS containing 0.5% Triton X-100 and 1% BSA. For primary cell cultures, cells were grown on glass coverslips and fixed with methanol for 10 min on ice. Fixed cells were permeabilised with 0.1% Triton X-100 and 0.5% NP-40 for 10 min on ice. To block non-specific binding, samples were incubated with PBS containing 5% normal goat serum and 1% BSA for 1 h at room temperature.\nSamples were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: rabbit anti- GEN1  (1:400; Abcam, ab198989, Cambridge, UK) and mouse anti-E-cadherin (1:200; BD Biosciences, 610182, Franklin Lakes, NJ, USA). Following three washes with PBS, samples were incubated with secondary antibodies for 1 h at room temperature in the dark. The secondary antibodies were as follows: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500; Invitrogen). Following a final wash with PBS, samples were mounted with Vectashield antifade mounting medium containing DAPI (Vector Laboratories, Newark, CA, USA).\nConfocal images were acquired using a Leica TCS SP5 microscope equipped with an HCX PL APO 63×/1.4 oil immersion objective. Dual-colour images were obtained using laser excitation at 488 nm and 561 nm for Alexa Fluor 488 and Alexa Fluor 594 dyes, respectively. Emission signals were collected through 500–550 nm and 590–650 nm band-pass filters. For DAPI, 405 nm laser excitation and 420–480 nm emission filter were used. Image acquisition and processing were performed using Leica LAS AF software (version 3.3; Leica Microsystems, Wetzlar, Germany). For fluorescence intensity quantification, ImageJ software (version 1.54q; NIH, Bethesda, MD, USA) was used, and at least 10 organoids or 50 cells per condition were analysed. For DNA damage assessment, γH2AX immunofluorescence was performed to evaluate baseline DNA double-strand break levels in control and endometriosis EEOs. EEOs were stained with rabbit anti-γH2AX (1:150; Thermo Scientific, MA5-33062, Waltham, MA, USA) following the same protocol described above. γH2AX immunofluorescence intensity was quantified in at least 50 nuclei per condition from three independent experiments using ImageJ software. In addition to baseline comparisons, γH2AX immunofluorescence was also assessed in EEOs at 48 h following transfection with  GEN1  -specific siRNA or non-targeting control siRNA to evaluate the effect of  GEN1  knockdown on DNA damage accumulation.\nTotal RNA was isolated from EEOs and primary cell cultures using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Prior to RNA extraction, EEOs were released from Matrigel using Cell Recovery Solution (Corning) and washed with PBS. RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific), and samples with a 260/280 ratio between 1.8 and 2.0 were accepted for subsequent analyses. RNA integrity was verified by 1% agarose gel electrophoresis.\nFor complementary DNA (cDNA) synthesis, 1 µg of total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a 20 µL reaction volume. The reaction mixture contained 50 U MultiScribe Reverse Transcriptase, 1× RT Buffer, 4 mM dNTP mix, 2.5 µM random hexamers, and RNase inhibitor. Reverse transcription was performed in a 2720 Thermal Cycler (Applied Biosystems) under the following conditions: 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C. The resulting cDNA samples were diluted 1:5 with nuclease-free water and stored at −20 °C.\nQuantitative real-time PCR (RT-qPCR) was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems).  GEN1  gene expression was analysed using a TaqMan Gene Expression Assay ( GEN1 , Hs00260513_m1; Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). S16 (Hs04332240_s1; Applied Biosystems) served as the internal control. Each 20 µL reaction mixture contained 10 µL TaqMan Universal PCR Master Mix II (No UNG), 1 µL TaqMan Gene Expression Assay (20×), 2 µL cDNA template, and 7 µL nuclease-free water. Thermal cycling conditions were as follows: 2 min at 50 °C, initial denaturation at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Each sample was run in technical duplicate, and nuclease-free water was included on each plate as a no-template control. Relative gene expression was calculated using the 2 −ΔΔCt  method and normalised to the control group. Samples with cycle threshold (Ct) values above 35 were excluded from the analysis. Data were obtained from at least three independent biological replicates. To confirm hormonal receptor status of EEOs, oestrogen receptor alpha ( ESR1 , Hs00174860_m1; Applied Biosystems) and progesterone receptor (PGR, Hs01556702_m1; Applied Biosystems) expression was also assessed.\nEEOs were released from Matrigel using Cell Recovery Solution (Corning) and lysed in RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail (Roche). Protein concentrations were determined using the BCA Protein Assay Kit (Pierce). Equal amounts of protein (20–30 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk in TBS-T for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies: rabbit anti- GEN1  (1:1000; Abcam, ab198989, London, UK) and mouse anti-β-actin (1:5000; Sigma-Aldrich, A5441). After washing with TBS-T, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature: goat anti-rabbit IgG-HRP (1:5000; Cell Signaling) and goat anti-mouse IgG-HRP (1:5000; Cell Signaling). Protein bands were visualised using ECL substrate (Bio-Rad, Hercules, CA, USA) and quantified by densitometry using ImageJ software (NIH). β-Actin served as the loading control.\nAll statistical analyses were performed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 9.0 (GraphPad Software). Due to the small sample size ( n  = 3 per group), the non-parametric Mann–Whitney U test was used for between-group comparisons. Continuous variables were expressed as mean ± standard deviation (SD). With  n  = 3 per group, the exact Mann–Whitney U test yields a minimum two-tailed  p -value of 0.05 only when complete separation between groups is observed (U = 0). Therefore, results were interpreted considering both statistical significance and the magnitude of observed effects. For RNAi-mediated knockdown experiments, silencing efficiency was reported descriptively as percentage reduction ± SD, as paired within-subject comparisons with  n  = 3 do not provide sufficient statistical power for formal hypothesis testing.\nFor gene expression analyses, control group values were normalised to 1.00, and endometriosis group values were calculated relative to this reference. RT-qPCR data were analysed using th 2 −ΔΔCt  method with S16 as the reference gene. Western blot band intensities were measured densitometrically using ImageJ software (National Institutes of Health) and normalised to β-actin. EEO formation efficiency was calculated as the ratio of formed EEOs to total seeded cells and expressed as a percentage. EEO diameters were determined by measuring at least 50 EEOs per group using ImageJ. For proliferation analyses, BrdU incorporation was quantified by flow cytometry and expressed as a percentage of BrdU-positive cells.  GEN1  knockdown efficiency was determined as the percentage reduction in mRNA and protein levels compared with the control siRNA group.\nAll experiments were performed with at least three independent biological replicates, and technical duplicates were included for each experiment. Statistical significance was defined as  p  ≤ 0.05. Given the small sample size, exact  p -values are reported throughout. With  n  = 3 per group, the minimum achievable  p -value for the Mann–Whitney U test is 0.05, corresponding to complete rank separation between groups.\nDuring the preparation of this manuscript, the authors used ChatGPT Plus, a large language model–based artificial intelligence tool, solely for language editing and refinement, and manuscript for-matting. All AI-assisted outputs were reviewed, verified, and, where necessary, revised by the authors. Full responsibility for the scientific content, interpretations, and conclusions of this manuscript rests with the authors.\n\nTo our knowledge, this study provides the first organoid-based evidence that  GEN1 , a critical Holliday junction resolvase, is downregulated in endometriosis and functionally associated with reduced cell proliferation in vitro. Endometriosis-derived EEOs exhibited elevated baseline DNA damage and heightened sensitivity to  GEN1  knockdown compared with controls, suggesting an underlying genomic fragility that may contribute to disease pathogenesis. The convergence of reduced  GEN1  expression, elevated baseline DNA damage, and heightened sensitivity to further  GEN1  depletion supports internal consistency of the observed phenotype. These findings suggest a potential contribution of homologous recombination dysregulation to endometriosis pathogenesis and support further investigation of DNA repair mechanisms in endometriosis. Patient-derived EEOs represent a powerful translational platform for elucidating disease mechanisms and developing personalised treatment strategies.","source_license":"CC-BY-4.0","license_restricted":false}