Ovarian endometrioma subtypes defined by the ratio of ciliated cells: implications for endometriosis origin and carcinoma histologic subtype

Ovarian endometrioma subtypes defined by the ratio of ciliated cells: implications for endometriosis origin and carcinoma histologic subtype | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ovarian endometrioma subtypes defined by the ratio of ciliated cells: implications for endometriosis origin and carcinoma histologic subtype Sun Hui, Tetsuya Hirata, Natsuki Nagashima, Marie Nakajima, Mohammed Elsherbini, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8631039/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The implantation (retrograde menstruation) theory hypothesizes that endometriosis arises when refluxed endometrial cells implant at ectopic sites; however, a fallopian tube epithelial origin has also been proposed. Because endometrium and fallopian tube epithelia differ markedly in their balance of ciliated and secretory cells, we asked whether ciliated-cell proportion defines ovarian endometrioma subtypes and associates with the histologic phenotype of endometriosis-associated ovarian carcinoma. Methods: We analyzed 67 specimens: paired eutopic endometrium and fallopian tube (17 pairs), ovarian endometrioma (n=30), deep (n=6) and peritoneal (n=3) endometriosis, and endometriosis-associated cancers (n=11). Ciliated and secretory differentiation were assessed by immunostaining for forkhead box J1 (FOXJ1) and ezrin (EZR), and 3-mercaptopyruvate sulfurtransferase (MPST), respectively; FOXJ1/paired-box gene 8 (PAX8) co-localization was evaluated by double immunofluorescence. Ovarian endometriomas were classified using an 18.71% FOXJ1-positive cut-off. Results: FOXJ1-positive ciliated cells were abundant in fallopian tube epithelium but scarce in eutopic endometrium, deep endometriosis, and peritoneal endometriosis. Ovarian endometriomas showed a bimodal distribution of FOXJ1-positive frequency and were classified as type 1 (<18.71%, endometrium-like, 19/30) or type 2 (≥18.71%, fallopian tube-like; 11/30). FOXJ1-positive frequency was significantly higher in fallopian tube and type 2 endometrioma than in endometrium, type 1 ovarian endometrioma, and deep and peritoneal endometriosis. In type 2 endometrioma, most FOXJ1-positive ciliated cells co-expressed PAX8, whereas FOXJ1-positive cells lacked PAX8 in fallopian tube epithelium. Clear cell carcinoma was EZR-positive and MPST-negative, whereas endometrioid carcinoma was predominantly MPST-positive with a minor FOXJ1/EZR-positive subset; a mixed carcinoma showed the same component-specific profiles. Conclusions: Ovarian endometriomas comprise two subtypes defined by ciliated-cell proportion, whereas deep and peritoneal endometriosis show low ciliated-cell content comparable to eutopic endometrium. The FOXJ1/PAX8 co-expressing ciliated cells in “fallopian tube-like” type 2 lesions indicate a transitional epithelial phenotype distinct from terminally differentiated fallopian tube epithelium and are compatible with an endometrial origin even for ciliated-cell-rich lesions. Along with the EZR/MPST profiles distinguishing clear cell and endometrioid carcinoma, these findings suggest that differentiation states of endometriotic epithelium may be linked to the histologic phenotype of endometriosis-associated ovarian cancer. ovarian endometrioma endometriosis ciliated cell clear cell carcinoma endometrioid carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Endometriosis is an estrogen-dependent chronic inflammatory condition characterized by endometrium-like tissues outside the uterus, primarily in the ovaries and peritoneal surface, and less frequently in the bowel, bladder, and diaphragm [1, 2]. Endometriosis presumably develops when the endometrial tissue refluxing retrogradely into the pelvis through the fallopian tubes engrafts in the pelvis. However, despite the existing theories such as the metaplasia theory and stem cell theory, the mechanism underlying endometriosis development remains unclear [1]. Three main types of endometriosis include peritoneal (PE), ovarian, and deep (DE) endometriosis [3]. The mechanisms of occurrence of each type of endometriosis may vary [4]. PE is thought to arise mainly from superficial implants of refluxed endometrium on the peritoneal surface, ovarian endometriomas from cortical invagination and cystic transformation of ovarian surface implants, whereas DE likely represents adenomyosis externa arising within pelvic fibromuscular tissues [5]. Particularly, the retrograde menstruation theory is believed to play a significant role in the development of PE and ovarian endometriosis. Recent genetic studies have suggested that the cells derived from clonal proliferation of epithelial cells carrying cancer-associated mutations in normal endometrial glands flow into the pelvic cavity with retrograde reflux of menstruation, leading to the development of ovarian endometrioma [6]. However, some studies have reported that endometriosis originates from the fallopian tube epithelium [7, 8]; thus, the origin and developmental mechanisms of endometriosis remain controversial. The endometrial and fallopian tube epithelia are mainly composed of ciliated and secretory cells [9-11]. Ciliated cells are characterized by the presence of cilia on their surface and play a critical role in helping the ovum (egg) or fertilized egg move through the fallopian tube into the uterus. Forkhead box J1 (FOXJ1), which is a transcription factor that plays a crucial role in cilia formation and maintenance, is a useful marker for identifying ciliated cells [12]. Ezrin (EZR) is a member of the ERM (ezrin, radixin, and moesin) protein family that links the actin cytoskeleton to the plasma membrane [13]. EZR is also a marker of ciliated cells in the fallopian tubes and endometrium [14]. Secretory cells produce mucus, contributing to a favorable environment for fertilization and early embryonic development and supporting ovum or fertilized egg movement. 3-mercaptopyruvate sulfurtransferase (MPST), a key enzyme regulating endogenous H 2 S biosynthesis, is a specific marker for secretory cells of the endometrium and fallopian tubes [15]. Ciliated cells of the endometrium constitute 5–20% of the epithelium throughout the menstrual cycle, which is lower than secretory cells [14]. Contrastingly, the ratio of ciliated cells was reportedly higher than that of secretory cells in the fallopian tubes [16]. Because the proportion of ciliated cells differs between the endometrium and fallopian tubes, we hypothesized that analyzing the ratio of ciliated to secretory cells could provide insights into the origin of endometriosis. Although endometriosis is considered a benign condition, histopathological and epidemiological studies have demonstrated ovarian endometriomas (Ov EMS) as the origin of ovarian clear cell (CCC) and endometrioid (ENC) carcinomas, collectively known as endometriosis-related cancers [17]. Although these two diseases show distinct features in their unique morphology, immunohistochemical profiles, and responses to treatment, they both arise from endometriosis and share common mutations in ARID1A, PIK3CA, and CTNNB1. Cochrane et al. proposed models of CCC and ENC originating from ciliated or secretory cells, respectively [14]. Thus, the composition of ciliated and secretory cells in endometriosis should be clarified for gaining a new perspective on the development of endometriosis-related cancers. However, to our knowledge, no studies have addressed the composition of ciliated and secretory cells in endometriotic epithelial cells. Therefore, we aimed to investigate the composition of ciliated and secretory cells in the endometriotic, endometrial, and tubal epithelia. Materials and Methods The experimental procedures were approved by the Institutional Review Board of the University of Tokyo (registration number #324-20). All patients underwent hysterectomy or surgical removal of ovarian, bowel, bladder, and PE and CCC or ENC synchronous with endometriosis at the University of Tokyo Hospital. After reviewing the medical records, we enrolled 67 patients and obtained written informed consent from all individuals. Diagnoses of endometriosis and endometriosis-associated cancer were made by histological examination performed by certified pathologists. Patients who had received hormone therapy within 6 months before surgery were excluded from the study. Ten endometrial samples and corresponding fallopian tube samples from patients with early-stage cervical cancer (carcinoma in situ , stage 1B1 <2 cm) without endometriosis, adenomyosis, uterine fibroma, or other gynecological disorders were collected after surgery (endometrium/fallopian tube without endometriosis, Group A). Of these samples, four and six exhibited the proliferative and secretory phases, respectively. Furthermore, 7 samples of eutopic endometrium and the corresponding fallopian tube were collected from patients with ovarian endometrioma (endometrium/fallopian tube with endometriosis, Group B), 4 and 3 in the proliferative and secretory phases, respectively; 30 samples were collected from patients with ovarian endometrioma (Group C), 17 and 13 in the proliferative and secretory phases, respectively; 6 samples were collected from patients with DE (Group D), 3, 2, and 1 in the proliferative, secretory, and unknown phases, respectively, and 3 samples were collected from patients of peritoneal endometriosis (PE) (Group E); all three were in the proliferative phase. Additionally, we collected 11 endometriosis-related cancer samples, including 5 of ENC (Group F), 5 of CCC (Group G), and 1 of mixed cancer, including ENC and CCC (Group H). Mixed cancer has been described in our earlier case report [18]. Tissue sections from each sample were subjected to immunostaining. Immunohistochemistry Immunostaining was performed as described previously [19-21]. Paraffin-embedded tissues were sliced to 5-μm thickness, deparaffinized, and rehydrated. Antigen retrieval was performed using an antigen retrieval reagent (Immunosaver, Nissin EM, Tokyo, Japan) at 98°C. Immunohistochemistry was performed using the Dako Envision FLEX+ system (Agilent Technologies, Santa Clara, CA, USA). Endogenous peroxidases were blocked by incubation with a peroxidase-blocking reagent (Agilent Technologies) for 5 min at room temperature. Sections were incubated with each primary antibody or rabbit/mouse immunoglobulin G overnight at 4°C and subsequently incubated with peroxidase-conjugated secondary antibody for 30 minutes at room temperature. Primary antibodies used were: EZR (1:2000, HPA021616, Sigma Aldrich, St. Louis, MO, USA), FOXJ1 (1:500, AMAB91255, Sigma Aldrich), and MPST (1:2000, HPA001240, Sigma Aldrich). Staining was detected using a diaminobenzidine chromogen, and all sections were counterstained with hematoxylin and evaluated under a light microscope (BX50; Olympus, Tokyo, Japan). Immunohistochemical results assessment To compare the frequency of FOXJ1 positive epithelial cells among all epithelial cells, five fields of view were randomly selected for each sample and evaluated under a light microscope at ×400 magnification. The frequency of FOXJ1 positive cells was defined as the percentage of FOXJ1-positive epithelial cells relative to the total number of epithelial cells. Immunofluorescence staining Immunofluorescence staining was performed as previously described [21]. Sections were blocked with blocking reagent, incubated with primary antibodies overnight at 4°C, and subsequently incubated with Alexa Fluor 568 goat anti-rabbit IgG (1:400, Invitrogen, Carlsbad, CA) or Alexa Fluor 488 goat anti-mouse IgG (1:400, Invitrogen) antibodies for 2 h at room temperature. Primary antibodies used were: EZR (1:1500, HPA021616, Sigma Aldrich), FOXJ1 (1:400, AMAB91255, Sigma Aldrich), MPST (1:1500, HPA001240, Sigma Aldrich), and paired-box gene 8 (PAX8) (1:500, 10336-1-AP, Proteintech, Rosemont, Illinois, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (1:300, DOJIDO, Tokyo, Japan), and sections were mounted using an anti-fade reagent (Invitrogen). Statistical analysis As described previously [21], continuous data are presented as means and standard deviations. Categorical data are presented as counts and percentages and were analyzed using Chi-square and Fisher’s exact tests. For two-group comparisons, the Student’s t-test or Mann–Whitney U-test were performed. Multiple groups were compared using a one-way analysis of variance (ANOVA). To assess differences in the frequency of FOXJ1 positive cells across all groups, analysis of covariance (ANCOVA) was conducted, followed by Tukey’s honest significant difference (HSD) test for post-hoc comparisons. Statistical significance was set at P < 0.05. Statistical analyses were performed using Prism 7 (version 7.0a; GraphPad, San Diego, CA, USA) and SPSS (v23; IBM Corp, Armonk, New, USA). Results Patient characteristics and clinical background No significant differences were observed between the groups in terms of age, body mass index, gravidity, parity, menstrual phase, or the reASRM stage of endometriosis (Supplemental Table 1). FOXJ1, EZR and MPST expression in the endometrium, fallopian tube, ovarian endometrioma, DE, and PE In all examined tissues, FOXJ1 was localized to the nuclei of ciliated cells, EZR was expressed in the cytoplasm of ciliated cells, and MPST was detected in both the nuclei and cytoplasm of secretory cells (Figure 1). In the endometrium,immunohistochemical staining revealed that FOXJ1- and EZR-positive ciliated cells accounted for only a small proportion of the total epithelial cells, whereas majority of the epithelial cells were MPST-positive secretory cells (Fig. 1A-C). Conversely, in the fallopian tube, FOXJ1- and EZR-positive ciliated cells and MPST-positive secretory cells comprised approximately half of the epithelial cells (Fig. 1D-F). Interestingly, ovarian endometrioma exhibited two distinct expression patterns. In some cases, the expression pattern resembled that of the endometrium (Fig. 1G-I), whereas in others, it closely resembled that of the fallopian tube (Fig. 1J-L). In DE (Fig. 1M-O) and PE (Fig. 1P-R), the distribution of cell types was similar to that observed in the endometrium, with FOXJ1- and EZR-positive ciliated cells representing only a small subset of the epithelial cells. Definition of type 1 and type 2 ovarian endometrioma based on FOXJ1 expression To objectively distinguish between endometrial- and fallopian tube–like expression patterns, we determined the threshold value by calculating the midpoint between the maximum FOXJ1-positive cell frequency in the endometrium (8.50%) and minimum value in the fallopian tubes (28.92%), resulting in a cut-off of 18.71%. This threshold effectively separated the two tissue types and was applied to classify Ov EMS cases: samples with a FOXJ1-positive frequency below 18.71% were defined as type 1 (EM-like; red dots in Fig. 2A) and those above 18.71% were defined as type 2 (FT-like; green dots in Fig. 2A). Based on this classification, 19 of the 30 Ov EMS cases were categorized as type 1 and 11 as type 2 (Fig. 2B). The clinical characteristics of each type are summarized in Supplemental Table 2. No significant differences existed in the age, body mass index, gravidity, parity, or menstrual phase between the groups. Next, we compared the frequency of FOXJ1-positive cells across all the groups. The frequencies in the fallopian tube and type 2 Ov EMS groups were significantly higher than EM, type 1 Ov EMS, DE, and PE groups (P < 0.05) (Figure 2C). Although the median values in the DE and PE were higher than those in the endometrium and type 1 Ov EMS groups, these differences were not statistically significant. ANCOVA confirmed the observed differences in FOXJ1 expression to be independent of potential confounding clinical variables including age, body mass index, gravidity, parity, and menstrual phase. Furthermore, Student’s t-test revealed no significant difference in the FOXJ1-positive frequency between patients with and without endometriosis within the endometrium and fallopian tube groups (Supplementary Fig. 1). Co-localization of PAX8 and FOXJ1 in type 2 Ov EMS, fallopian tube, and endometrium Type 1 Ov EMS exhibited an expression pattern of ciliated cells similar to that observed in the endometrium, whereas type 2 Ov EMS resembled the fallopian tube. This raises the question of whether type 2 Ov EMS originate from the fallopian tube rather than from the endometrium. Thus, we performed double immunofluorescence staining for PAX8 and FOXJ1. In type 2 Ov EMS, PAX8 was strongly expressed not only in FOXJ1-negative but also in the majority of FOXJ1-positive cells (yellow arrows, Fig. 3A-C). A small number of FOXJ1-positive cells exhibited weak PAX8 expression (white arrows). Conversely, FOXJ1-positive cells did not express PAX8 in the fallopian tubes. These two markers were mutually exclusive and localized to entirely different epithelial cell populations in the fallopian tube (Fig. 3D–F). In the endometrium, nuclear PAX8 expression was predominantly observed in FOXJ1-negative cells, while most FOXJ1 positive cells showed weak or absent nuclear PAX8 expression (white arrows), with only a small fraction exhibiting strong nuclear coexpression (yellow arrows, Fig. 3G-I). In type 1 Ov EMS, FOXJ1-positive cells were only rarely observed. In these few FOXJ1-positive cells, weak nuclear PAX8 expression was detected (white arrows, Supplementary Fig. 2A–C). Most epithelial cells were FOXJ1-negative and demonstrated strong nuclear PAX8 expression. DE and PE exhibited expression patterns similar to those observed in the endometrium; PAX8 was strongly expressed in FOXJ1-negative cells and weakly/not expressed in most FOXJ1-positive cells (white arrows), while a few FOXJ1-positive cells demonstrated strong PAX8 expression (yellow arrows, Supplementary Fig. 2D–I). Expression of FOXJ1, EZR, and MPST in ENC, CCC, and mixed endometrioid and CCC CCC may arise from cells exhibiting ciliated cell features, whereas ENC presumably originates from secretory cells or their precursors [14]. We performed immunohistochemical staining for FOXJ1, EZR, and MPST in the endometriosis-associated carcinoma samples for evaluating this model. Our findings demonstrated that FOXJ1 and EZR, markers of the ciliated cell phenotype, and MPST, a marker of the secretory cell phenotype, were differentially expressed in the tumor tissues. In ENC, the expression patterns of these markers closely resembled those of the normal endometrial epithelium. FOXJ1 was localized to the nucleus, EZR to the cytoplasm, and MPST to both the nucleus and cytoplasm. Majority of the endometrioid cancer cells exhibited cell positivity for MPST, whereas only a small subset showed positivity for FOXJ1 and EZR (Fig. 4A-C). In CCC, tumor cells demonstrated strong cytoplasmic expression of EZR, a marker of the ciliated cell phenotype, whereas neither EZR nor MPST expression was detected (Fig. 4D-F). In mixed carcinomas, the endometrioid and clear cell components exhibited distinct marker expression profiles consistent with those observed in pure ENC and CCC, respectively. In the endometrioid component, cells positive for MPST constituted the majority of the tumor cells, whereas FOXJ1- and EZR-positive cells were limited to a minor subset. In the clear cell component, only EZR was expressed in the cytoplasm, and both FOXJ1 and MPST were absent. Discussion In this study, we obtained the following results: First, two distinct types of Ov EMS were detected in terms of the frequency of ciliated cells: the endometrium-like type (type 1) and tubal-like type (type 2). Second, the composition ratio of ciliated cells in the epithelium was low in DE and PE, similar to that in the endometrium. Third, ciliated cells in type 2 ovarian endometrioma and the endometrium were positive for PAX8, whereas ciliated cells in the fallopian tubes were negative for PAX8. Furthermore, in endometriosis-related cancers, CCC was EZR-positive and MPST-negative, and ENC was EZR-negative and MPST-positive, which was also observed in a rare case of mixed cancer arising from a single endometriotic lesion. To our knowledge, this is the first report to demonstrate two types of Ov EMS based on the ratio of ciliated cells. Notably, we found that Ov EMS can be divided into two distinct subtypes based on the composition ratio of ciliated cells: endometrial-like and fallopian tubal-like types, with frequencies of 63% and 37%, respectively. The retrograde menstrual theory is a major hypothesis regarding the pathogenesis of endometriosis, suggesting that ovarian endometriosis originates in the endometrium [6]. However, reports suggesting ovarian endometriosis originates from the fallopian tubes also exist [7]. In a previous report, the expression pattern of FMO3, a molecule strongly expressed in the fallopian tube, revealed that 56% exhibited a fallopian tube epithelial-like pattern and 44% the opposite expression pattern, which aligns with our observation of the two subtypes of ovarian endometriosis. PAX8 is a member of the paired-box family of genes that is expressed during organogenesis of the thyroid gland, kidney, and Müllerian tract and serves as a sensitive and specific marker for Müllerian-derived epithelial cells [22]. PAX8 is a marker of the fallopian tube secretory cell lineage but not of the ciliated cell population [23]. Herein, FOXJ1-positive ciliated cells in type 2 Ov EMS and the endometrium were positive for PAX8, whereas ciliated cells in the fallopian tubes were negative for this molecule. These results indicated that type 2 Ov EMS was similar to the fallopian tube in terms of a high proportion of ciliated cells, but also similar to the endometrium with respect to the expression of PAX8 in ciliated cells. A previous study using genetic cell lineage tracing demonstrated that PAX8+ secretory cells acted as progenitors of the fallopian tube epithelial cells, giving rise to ciliated cells during differentiation [24]. Similarly, Cochrane et al. demonstrated through single-cell RNA sequencing of endometrioid organoids that secretory cells can differentiate into ciliated cells but not vice versa [15], consistent with the view that ciliated cells represent a terminally differentiated cell type. Herein, ciliated cells in both type 2 Ov EMS and endometrium were positive for PAX8, whereas ciliated cells in the fallopian tube lacked PAX8 expression. This contrast suggests that despite the high proportion of ciliated cells resembling the fallopian tube, type 2 Ov EMS is more likely to share an origin with the endometrium, based on its PAX8-positive and FOXJ1-positive ciliated cell profile. This interpretation is further supported by recent single-cell transcriptome data identifying transitional populations that coexpress PAX8 and FOXJ1, suggesting a trajectory from secretory to ciliated differentiation [10]. Therefore, ciliated cells in type 2 Ov EMS may reflect an endometrium-derived transitional phenotype, rather than a true fallopian lineage. Interestingly, estradiol reportedly promotes ciliogenesis in endometrial epithelial cells [25]. In a previous study comparing the estradiol levels in the endometrium, Ov EMS, PE, and DE, the estrogen concentration was markedly higher in Ov EMS than in other tissues [26]. Locally elevated estradiol levels in Ov EMS may facilitate the differentiation of secretory cells into ciliated cells, thereby contributing to the significantly higher proportion of ciliated cells observed in type 2 Ov EMS than in the endometrium, PE, and DE. MPST was identified as a highly specific marker of secretory cells using RNA seq [15]. MPST was strongly expressed in ENC compared to CCC, which is in line with a previous report using methylenetetrahydrofolate dehydrogenase 1 [14]. EZR was strongly expressed in CCC but not in ENC. FOXJ1 was not detected in CCC. These results are consistent with those of a previous report [14]. Ovarian endometriosis presumably originates from ovarian CCC and ENC, which are described as endometriosis-associated cancers [17, 27]. In the ovary, ENC and CCC occur at similar rates, with the incidence of CCC being particularly high in Japan; however, ENC is much more common than CCC in the endometrium. ENC occur frequently in the malignant transformation of extraovarian endometriosis [28-31]. Cochrane et al. proposed that because CCC and ENC that occur in endometriosis share genetic abnormalities and develop in a similar microenvironment, the different histological types could be explained by different cellular origins [14]. Thus, ENC may originate from secretory cells and CCC may originate from ciliated cells. Based on this model, ENC being more common than CCC in DE can be explained by the finding that the proportion of ciliated cells in DE was as low as that in the endometrium. Herein, we considered that epithelial cells in Ov EMS also originated from the endometrium, as we observed a PAX8 expression pattern closer to that of endometrial ciliated cells than that of tubal ciliated cells. Studies tracing somatic mutations from the endometrium to CCC through Ov EMS have suggested that the epithelial cells of Ov EMS and CCC originated from the uterine endometrial epithelium [32]. However, if it is assumed that both subtypes originate from the endometrium, it remains unknown what determines whether these two types of ovarian endometriosis have clearly different ratios of ciliated cells. This study has some limitations. First, the gold standard for identifying a cell origin should be lineage-tracing experiments, which allow tracing the fate of a specific cell type as it develops. However, the existence and locations of stem or precursor cells in the female genital tract remain controversial. Second, stromal cells should be considered when investigating the origin of endometriosis; however, this study focused only on the epithelium and did not find any differences in the stroma. Nevertheless, the epithelium and stroma were reported to develop independently in the same endometriosis because the epithelium is clonal, while the stroma is not [33]. Therefore, tracing the origin of the stroma may be technically challenging. Conclusion We identified two distinct subtypes of Ov EMS based on the proportion of ciliated cells. The proportion of ciliated cells in the DE and PE groups was as low as that of the endometrium. Notably, in type 2 Ov EMS, containing a high proportion of ciliated cells resembling the fallopian tube, expressed PAX8, indicating a transitional phenotype and origin from the endometrial epithelium rather than from the fallopian tube epithelium. These findings provide novel insights into the cellular origin and pathogenesis of endometriosis and endometriosis-associated cancers. Further studies are warranted to elucidate the biological differences between these two types and the mechanisms underlying their divergence. Declarations Acknowledgments: The authors would like to thank Editage (www.editage.jp) for the English language editing. Clinical trial registration Not applicable Funding This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology. Data availability The datasets analyzed during the current study are included in this published article and its supplementary information files. De-identified individual-level data are available from the corresponding author on reasonable request. Author’s contributions S.H. performed experiments, analysis data, writing of the manuscript and contributed to data interpretation. T.H. designed and supervised experiments, raised funding, interpreted the data, and wrote the manuscript. N.N performed experiments, and contributed data interpretation. M.N. and M.E. contributed to collecting samples and data interpretation. Y.H. and Y.O. supervised experiments and data interpretation. K.K. supervised experiments and contributed to data interpretation and critical discussion. 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Saavalainen L, Lassus H, But A, Tiitinen A, Harkki P, Gissler M, Pukkala E, Heikinheimo O: Risk of Gynecologic Cancer According to the Type of Endometriosis. Obstet Gynecol 2018, 131: 1095-1102. Brunson GL, Barclay DL, Sanders M, Araoz CA: Malignant extraovarian endometriosis: two case reports and review of the literature. Gynecol Oncol 1988, 30: 123-130. Modesitt SC, Tortolero-Luna G, Robinson JB, Gershenson DM, Wolf JK: Ovarian and extraovarian endometriosis-associated cancer. Obstet Gynecol 2002, 100: 788-795. Mandai M, Osuga Y, Hirata T, Enomoto T, Nakai H, Honda R, Taniguchi F, Katabuchi H: Cancers associated with extraovarian endometriosis at less common/rare sites: A nationwide survey in Japan. J Obstet Gynaecol Res 2020, 46: 917-923. Poon C, Rome R: Malignant extra-ovarian endometriosis: A case series of ten patients and review of the literature. Aust N Z J Obstet Gynaecol 2020, 60: 585-591. Suda K, Cruz Diaz LA, Yoshihara K, Nakaoka H, Yachida N, Motoyama T, Inoue I, Enomoto T: Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis. Cancer Sci 2020, 111: 3000-3009. Noe M, Ayhan A, Wang TL, Shih IM: Independent development of endometrial epithelium and stroma within the same endometriosis. J Pathol 2018, 245: 265-269. Additional Declarations No competing interests reported. Supplementary Files SupplementalTable1.xlsx SupplementalTable2.xlsx SupplementaryFig12.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8631039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581811749,"identity":"66baaef4-0e16-49eb-9388-f9dc01816eb0","order_by":0,"name":"Sun Hui","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"","lastName":"Hui","suffix":""},{"id":581811750,"identity":"00b3f57e-7d19-4b04-b377-f956ae29b65d","order_by":1,"name":"Tetsuya Hirata","email":"data:image/png;base64,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","orcid":"","institution":"University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Hirata","suffix":""},{"id":581811752,"identity":"f2a01271-c773-4ad5-bcbd-f2fbd12879cc","order_by":2,"name":"Natsuki Nagashima","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Natsuki","middleName":"","lastName":"Nagashima","suffix":""},{"id":581811753,"identity":"8d620337-a8c2-4a83-b1a0-2557c8e05d12","order_by":3,"name":"Marie Nakajima","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Nakajima","suffix":""},{"id":581811755,"identity":"3eb1eb14-64c8-4b8d-9ec4-220e31800d82","order_by":4,"name":"Mohammed Elsherbini","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Elsherbini","suffix":""},{"id":581811756,"identity":"df1b55ad-3001-4ba7-a20b-90942fec0e50","order_by":5,"name":"Yasushi Hirota","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Hirota","suffix":""},{"id":581811757,"identity":"35da2dc0-9001-414f-a2c5-8719ae3809be","order_by":6,"name":"Kaori Koga","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kaori","middleName":"","lastName":"Koga","suffix":""},{"id":581811758,"identity":"7fd8aefa-fda9-4548-ae2c-e3cc5e78dfaa","order_by":7,"name":"Yutaka Osuga","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Osuga","suffix":""}],"badges":[],"createdAt":"2026-01-18 11:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8631039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8631039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101533485,"identity":"c5d1eda0-b36f-4b04-b0ba-465402c7c08a","added_by":"auto","created_at":"2026-01-30 21:18:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1700019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXJ1, EZR, and MPST expression in the endometrium, fallopian tube, ovarian endometrioma, deep endometriosis, and peritoneal endometriosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, D, G, J, M, and P) FOXJ1, (B, E, H, K, N, and Q) EZR, and (C, F, I, L, O, and R) MPST staining in the endometrium, fallopian tube, ovarian endometrioma, deep endometriosis, and peritoneal endometriosis. FOXJ1 expression in the nuclei of ciliated cells, EZR expression in the cytoplasm of ciliated cells, and MPST expression in the nuclei and cytoplasm of secretory cells.\u003cstrong\u003e \u003c/strong\u003eA–C, Endometrium (EM), D–F, fallopian tube (FT), G–L, ovarian endometrioma (Ov EMS), M–O, deep endometriosis (DE), and P–R,\u003cstrong\u003e \u003c/strong\u003eperitoneal endometriosis (PE). Magnification: ×400; ×1000 (inset); scale bars: 50 μm, 20 μm (inset). FOXJ1: Forkhead box J1, EZR: Ezrin, MPST: 3-mercaptopyruvate sulfurtransferase\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/a503ca781cb3cf34f10fb11a.jpg"},{"id":101533490,"identity":"71d2102f-f0d9-4d57-804b-2f352c1044e0","added_by":"auto","created_at":"2026-01-30 21:18:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":639500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassification of ovarian endometrioma into two subtypes based on the proportion of FOXJ1-positive epithelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dot plot showing the FOXJ1-positive cell ratios in endometrial epithelium (EM), fallopian tube epithelium (FT), and ovarian endometriomas (Ov EMS). Ov EMS samples display a bimodal distribution. The dashed line at a value of 18.71 indicates the cutoff threshold, which is defined as the midpoint between the maximum value of EM (8.50) and minimum value of FT (28.92). Using this threshold, Ov EMS samples are classified into type 1 (EM-like, red dots) and type 2 (FT-like, green dots), based on their FOXJ1-positive cell ratios.\u003c/p\u003e\n\u003cp\u003e(B) Representative immunohistochemical staining of FOXJ1 in Type 1 and Type 2 Ov EMS. Type 1 shows few ciliated cells, while type 2 exhibits a high proportion of FOXJ1-positive ciliated cells. Magnification: ×1000; scale bars: 20 μm.\u003c/p\u003e\n\u003cp\u003e(C) Box-and-whisker plots comparing the proportion of FOXJ1-positive cells among type 1 Ov EMS, type 2 Ov EMS, deep endometriosis (DE), and peritoneal endometriosis (PE), along with EM and FT. Type 1 Ov EMS, DE, and PE have low FOXJ1-positive cell ratios comparable to EM, while type 2 Ov EMS exhibit a higher ratio, similar to FT. The data are presented as box-whisker plots with all points showing the median values (center line), interquartile range (lower and upper boxes), and minimum-maximum outliers (lower and upper whiskers). The different letters denote significant differences between groups. (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e\n\u003cp\u003eFOXJ1: Forkhead box J1, EZR: Ezrin, MPST: 3-mercaptopyruvate sulfurtransferase\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/07751d760b6a4b77bb11096f.jpg"},{"id":101533489,"identity":"5d3f661e-0e4b-45e0-9e82-f24cfbfd89e3","added_by":"auto","created_at":"2026-01-30 21:18:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1134962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization of PAX8 and FOXJ1 in type 2 ovarian endometriotic, fallopian tubal, and endometrial tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, D, and G) PAX8 or (B, E, and H) FOXJ1 are labeled with Alexa Fluor 564 (red fluorescence) or 488 (green fluorescence). Both PAX8 and FOXJ1 are expressed in the nucleus of ciliated cells. In type 2 Ov EMS (A-C) and endometrium (G-I), PAX8 is expressed in almost all the epithelial cells. FOXJ1 expressing cells also express PAX8. Yellow and white arrows indicate cells with high or low expression of PAX8, respectively. FOXJ1 colocalizes with both high and low expression of PAX8. In fallopian tube; however, PAX8 and FOXJ1 are localized in different cells (D-F). Scale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003eFOXJ1: Forkhead box J1, PAX8: paired-box gene 8, T2 Ov EMS: type2 ovarian endometrioma, EM: endometrium, FT: fallopian tube\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/1070096f5a61f99d4fba26be.jpg"},{"id":101752169,"identity":"98bead27-4b65-4e72-a703-ef2fe9a19a91","added_by":"auto","created_at":"2026-02-03 10:25:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2411642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXJ1, EZR, and MPST expression in endometrioid carcinoma, clear cell carcinoma, and mixed endometrioid and clear cell carcinoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, D, G and J) FOXJ1, (B, E, H and K) EZR, and (C, F, I and L) MPST staining in endometrioid carcinoma, clear cell carcinoma and mixed endometrioid and clear cell carcinoma. In endometrioid carcinoma, FOXJ1 is expressed in the nuclei of ciliated cells (A), EZR is expressed in the cytoplasm of ciliated cells (B), and MPST is expressed in both nuclei and cytoplasm of secretory cells (C). In clear cell carcinoma, EZR is positively expressed in the cytoplasm of clear cells (E), whereas FOXJ1 (D) and MPST (F) are negatively expressed in the clear cell carcinoma tissues.\u003cstrong\u003e \u003c/strong\u003eIn endometrioid carcinoma part of mixed carcinoma, FOXJ1 is expressed in the nuclei of ciliated cells (G), EZR is expressed in the cytoplasm of ciliated cells (H), and MPST is expressed in both nuclei and cytoplasm of secretory cells (I). In clear cell carcinoma part of mixed carcinoma, EZR is weakly, positively expressed in the cytoplasm of clear cells (K), whereas FOXJ1 (J) and MPST (L) are negatively expressed in this part.\u003c/p\u003e\n\u003cp\u003eA-C, Endometrioid carcinoma; D-F, Clear cell carcinoma; G-I, Endometrioid carcinoma part of the mixed carcinoma; J-L, clear cell carcinoma part of the mixed carcinoma. Magnification: ×400; ×1000 (inset); scale bars: 50 μm, 20 μm (inset).\u003c/p\u003e\n\u003cp\u003eFOXJ1: Forkhead box J1, EZR: Ezrin, MPST: 3-mercaptopyruvate sulfurtransferase\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/40f6e356fd2f1c0ebbc0ed14.jpg"},{"id":103504572,"identity":"906b1ff3-8cb5-45ce-a0dc-a16f6263cf9d","added_by":"auto","created_at":"2026-02-26 13:20:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8038740,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/3bc4daf0-1359-4628-8990-f4646ab29eb9.pdf"},{"id":101533486,"identity":"113fd610-076a-4147-860e-b8cfb3655d66","added_by":"auto","created_at":"2026-01-30 21:18:25","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10434,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/72451a43f2f6ead79f220ecb.xlsx"},{"id":101533488,"identity":"cbe0991c-7234-4d77-98d0-d802222f74af","added_by":"auto","created_at":"2026-01-30 21:18:25","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9729,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/2c7ed9479cb961b2d73cf327.xlsx"},{"id":101533492,"identity":"599ae147-5ce5-4846-a3a4-1e866549cc16","added_by":"auto","created_at":"2026-01-30 21:18:27","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":45846462,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig12.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8631039/v1/3e75db1b830b178347204aa0.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ovarian endometrioma subtypes defined by the ratio of ciliated cells: implications for endometriosis origin and carcinoma histologic subtype","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometriosis is an estrogen-dependent chronic inflammatory condition characterized by endometrium-like tissues outside the uterus, primarily in the ovaries and peritoneal surface, and less frequently in the bowel, bladder, and diaphragm [1, 2]. Endometriosis presumably develops when the endometrial tissue refluxing retrogradely into the pelvis through the fallopian tubes engrafts in the pelvis. However, despite the existing theories such as the metaplasia theory and stem cell theory, the mechanism underlying endometriosis development remains unclear [1]. Three main types of endometriosis include peritoneal (PE), ovarian, and deep (DE) endometriosis [3]. The mechanisms of occurrence of each type of endometriosis may vary [4]. PE is thought to arise mainly from superficial implants of refluxed endometrium on the peritoneal surface, ovarian endometriomas from cortical invagination and cystic transformation of ovarian surface implants, whereas DE likely represents adenomyosis externa arising within pelvic fibromuscular tissues [5]. Particularly, the retrograde menstruation theory is believed to play a significant role in the development of PE and ovarian endometriosis. Recent genetic studies have suggested that the cells derived from clonal proliferation of epithelial cells carrying cancer-associated mutations in normal endometrial glands flow into the pelvic cavity with retrograde reflux of menstruation, leading to the development of ovarian endometrioma [6]. However, some studies have reported that endometriosis originates from the fallopian tube epithelium [7, 8]; thus, the origin and developmental mechanisms of endometriosis remain controversial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe endometrial and fallopian tube epithelia are mainly composed of ciliated and secretory cells [9-11]. Ciliated cells are characterized by the presence of cilia on their surface and play a critical role in helping the ovum (egg) or fertilized egg move through the fallopian tube into the uterus. Forkhead box J1 (FOXJ1), which is a transcription factor that plays a crucial role in cilia formation and maintenance, is a useful marker for identifying ciliated cells [12]. Ezrin (EZR) is a member of the ERM (ezrin, radixin, and moesin) protein family that links the actin cytoskeleton to the plasma membrane [13]. EZR is also a marker of ciliated cells in the fallopian tubes and endometrium [14]. Secretory cells produce mucus, contributing to a favorable environment for fertilization and early embryonic development and supporting ovum or fertilized egg movement. 3-mercaptopyruvate sulfurtransferase (MPST), a key enzyme regulating endogenous H\u003csub\u003e2\u003c/sub\u003eS biosynthesis, is a specific marker for secretory cells of the endometrium and fallopian tubes [15]. Ciliated cells of the endometrium constitute 5–20% of the epithelium throughout the menstrual cycle, which is lower than secretory cells [14]. Contrastingly, the ratio of ciliated cells was reportedly higher than that of secretory cells in the fallopian tubes [16]. Because the proportion of ciliated cells differs between the endometrium and fallopian tubes, we hypothesized that analyzing the ratio of ciliated to secretory cells could provide insights into the origin of endometriosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough endometriosis is considered a benign condition, histopathological and epidemiological studies have demonstrated ovarian endometriomas (Ov EMS) as the origin of ovarian clear cell (CCC) and endometrioid (ENC) carcinomas, collectively known as endometriosis-related cancers [17]. Although these two diseases show distinct features in their unique morphology, immunohistochemical profiles, and responses to treatment, they both arise from endometriosis and share common mutations in ARID1A, PIK3CA, and CTNNB1. Cochrane et al. proposed models of CCC and ENC originating from ciliated or secretory cells, respectively [14]. Thus, the composition of ciliated and secretory cells in endometriosis should be clarified for gaining a new perspective on the development of endometriosis-related cancers. However, to our knowledge, no studies have addressed the composition of ciliated and secretory cells in endometriotic epithelial cells. Therefore, we aimed to investigate the composition of ciliated\u0026nbsp;and secretory cells in the endometriotic, endometrial, and tubal epithelia.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe experimental procedures were approved by the Institutional Review Board of the University of Tokyo (registration number #324-20). All patients underwent hysterectomy or surgical removal of ovarian, bowel, bladder, and PE and CCC or ENC synchronous with endometriosis at the University of Tokyo Hospital. After reviewing the medical records, we enrolled 67 patients and obtained written informed consent from all individuals. Diagnoses of endometriosis and endometriosis-associated cancer were made by histological examination performed by certified pathologists. Patients who had received hormone therapy within 6 months before surgery were excluded from the study. Ten endometrial samples and corresponding fallopian tube samples from patients with early-stage cervical cancer (carcinoma \u003cem\u003ein situ\u003c/em\u003e, stage 1B1 \u0026lt;2 cm) without endometriosis, adenomyosis, uterine fibroma, or other gynecological disorders were collected after surgery (endometrium/fallopian tube without endometriosis, Group A). Of these samples, four and six exhibited the proliferative and secretory phases, respectively. Furthermore, 7 samples of eutopic endometrium and the corresponding fallopian tube were collected from patients with ovarian endometrioma (endometrium/fallopian tube with endometriosis, Group B), 4 and 3 in the proliferative and secretory phases, respectively; 30 samples were collected from patients with ovarian endometrioma (Group C), 17 and 13 in the proliferative and secretory phases, respectively; 6 samples were collected from patients with DE (Group D), 3, 2, and 1 in the proliferative, secretory, and unknown phases, respectively, and 3 samples were collected from patients of peritoneal endometriosis (PE) (Group E); all three were in the proliferative phase. Additionally, we collected 11 endometriosis-related cancer samples, including 5 of ENC (Group F), 5 of CCC (Group G), and 1 of mixed cancer, including ENC and CCC (Group H). Mixed cancer has been described in our earlier case report [18]. Tissue sections from each sample were subjected to immunostaining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunostaining was performed as described previously [19-21]. Paraffin-embedded tissues were sliced to 5-μm thickness, deparaffinized, and rehydrated. Antigen retrieval was performed using an antigen retrieval reagent (Immunosaver, Nissin EM, Tokyo, Japan) at 98°C. Immunohistochemistry was performed using the Dako Envision FLEX+ system (Agilent Technologies, Santa Clara, CA, USA). Endogenous peroxidases were blocked by incubation with a peroxidase-blocking reagent (Agilent Technologies) for 5 min at room temperature. Sections were incubated with each primary antibody or rabbit/mouse immunoglobulin G overnight at 4°C and subsequently incubated with peroxidase-conjugated secondary antibody for 30 minutes at room temperature. Primary antibodies used were: EZR (1:2000, HPA021616, Sigma Aldrich, St. Louis, MO, USA), FOXJ1 (1:500, AMAB91255, Sigma Aldrich), and MPST (1:2000, HPA001240, Sigma Aldrich). Staining was detected using a diaminobenzidine chromogen, and all sections were counterstained with hematoxylin and evaluated under a light microscope (BX50; Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical\u0026nbsp;results assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the frequency of FOXJ1 positive epithelial cells among all epithelial cells, five fields of view were randomly selected for each sample and evaluated\u0026nbsp;under a light microscope at ×400 magnification. The frequency of FOXJ1 positive cells was defined as the percentage of FOXJ1-positive epithelial cells relative to the total number of epithelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining was performed as previously described [21]. Sections were blocked with blocking reagent, incubated with primary antibodies overnight at 4°C, and subsequently incubated with Alexa Fluor 568 goat anti-rabbit IgG (1:400, Invitrogen, Carlsbad, CA) or Alexa Fluor 488 goat anti-mouse IgG (1:400, Invitrogen) antibodies for 2 h at room temperature. Primary antibodies used were: EZR (1:1500, HPA021616, Sigma Aldrich), FOXJ1 (1:400, AMAB91255, Sigma Aldrich), MPST (1:1500, HPA001240, Sigma Aldrich), and paired-box gene 8 (PAX8) (1:500, 10336-1-AP, Proteintech, Rosemont, Illinois, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (1:300, DOJIDO, Tokyo, Japan), and sections were mounted using an anti-fade reagent (Invitrogen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described previously [21], continuous data are presented as means and standard deviations. Categorical data are presented as counts and percentages and were analyzed using Chi-square and Fisher’s exact tests. For two-group comparisons, the Student’s t-test or Mann–Whitney U-test were performed. Multiple groups were compared using a one-way analysis of variance (ANOVA). To assess differences in the frequency of FOXJ1 positive cells across all groups, analysis of covariance (ANCOVA) was conducted, followed by Tukey’s honest significant difference (HSD) test for post-hoc comparisons. Statistical significance was set at P \u0026lt; 0.05. Statistical analyses were performed using Prism 7 (version 7.0a; GraphPad, San Diego, CA, USA) and SPSS (v23; IBM Corp, Armonk, New, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient characteristics and clinical background\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo significant differences were observed between the groups in terms of age, body mass index, gravidity, parity, menstrual phase, or the reASRM stage of endometriosis (Supplemental Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFOXJ1, EZR and MPST expression in the endometrium, fallopian tube, ovarian endometrioma, DE, and PE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all examined tissues, FOXJ1 was localized to the nuclei of ciliated cells, EZR was expressed in the cytoplasm of ciliated cells, and MPST was detected in both the nuclei and cytoplasm of secretory cells (Figure 1). In the endometrium,immunohistochemical staining revealed that FOXJ1- and EZR-positive ciliated cells accounted for only a small proportion of the total epithelial cells, whereas majority of the epithelial cells were MPST-positive secretory cells (Fig. 1A-C). Conversely, in the fallopian tube, FOXJ1- and EZR-positive ciliated cells and MPST-positive secretory cells comprised approximately half of the epithelial cells (Fig. 1D-F). Interestingly, ovarian endometrioma exhibited two distinct expression patterns. In some cases, the expression pattern resembled that of the endometrium (Fig. 1G-I), whereas in others, it closely resembled that of the fallopian tube (Fig. 1J-L). In DE (Fig. 1M-O) and PE (Fig. 1P-R), the distribution of cell types was similar to that observed in the endometrium, with FOXJ1- and EZR-positive ciliated cells representing only a small subset of the epithelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDefinition of type 1 and type 2 ovarian endometrioma based on FOXJ1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo objectively distinguish between endometrial- and fallopian tube–like expression patterns, we determined the threshold value by calculating the midpoint between the maximum FOXJ1-positive cell frequency in the endometrium (8.50%) and minimum value in the fallopian tubes (28.92%), resulting in a cut-off of 18.71%. This threshold effectively separated the two tissue types and was applied to classify Ov EMS cases: samples with a FOXJ1-positive frequency below 18.71% were defined as type 1 (EM-like; red dots in Fig. 2A) and those above 18.71% were defined as type 2 (FT-like; green dots in Fig. 2A). Based on this classification, 19 of the 30 Ov EMS cases were categorized as type 1 and 11 as type 2 (Fig. 2B). The clinical characteristics of each type are summarized in Supplemental Table 2. No significant differences existed in the age, body mass index, gravidity, parity, or menstrual phase between the groups.\u003c/p\u003e\n\u003cp\u003eNext, we compared the frequency of FOXJ1-positive cells across all the groups. The frequencies in the fallopian tube and type 2 Ov EMS groups were significantly higher than EM, type 1 Ov EMS, DE, and PE groups (P \u0026lt; 0.05) (Figure 2C). Although the median values in the DE and PE were higher than those in the endometrium and type 1 Ov EMS groups, these differences were not statistically significant. ANCOVA confirmed the observed differences in FOXJ1 expression to be independent of potential confounding clinical variables including age, body mass index, gravidity, parity, and menstrual phase. Furthermore, Student’s t-test revealed no significant difference in the FOXJ1-positive frequency between patients with and without endometriosis within the endometrium and fallopian tube groups (Supplementary Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-localization of PAX8 and FOXJ1 in type 2 Ov EMS, fallopian tube, and endometrium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eType 1 Ov EMS exhibited an expression pattern of ciliated cells similar to that observed in the endometrium, whereas type 2 Ov EMS resembled the fallopian tube. This raises the question of whether type 2 Ov EMS originate from the fallopian tube rather than from the endometrium. Thus, we performed double immunofluorescence staining for PAX8 and FOXJ1. In type 2 Ov EMS, PAX8 was strongly expressed not only in FOXJ1-negative but also in the majority of FOXJ1-positive cells (yellow arrows, Fig. 3A-C). A small number of FOXJ1-positive cells exhibited weak PAX8 expression (white arrows). Conversely, FOXJ1-positive cells did not express PAX8 in the fallopian tubes. These two markers were mutually exclusive and localized to entirely different epithelial cell populations in the fallopian tube (Fig. 3D–F). In the endometrium, nuclear PAX8 expression was predominantly observed in FOXJ1-negative cells, while most FOXJ1 positive cells showed weak or absent nuclear PAX8 expression (white arrows), with only a small fraction exhibiting strong nuclear coexpression (yellow arrows, Fig. 3G-I). In type 1 Ov EMS, FOXJ1-positive cells were only rarely observed. In these few FOXJ1-positive cells, weak nuclear PAX8 expression was detected (white arrows, Supplementary Fig. 2A–C). Most epithelial cells were FOXJ1-negative and demonstrated strong nuclear PAX8 expression. DE and PE exhibited expression patterns similar to those observed in the endometrium; PAX8 was strongly expressed in FOXJ1-negative cells and weakly/not expressed in most FOXJ1-positive cells (white arrows), while a few FOXJ1-positive cells demonstrated strong PAX8 expression (yellow arrows, Supplementary Fig. 2D–I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of FOXJ1, EZR, and MPST in ENC, CCC, and mixed endometrioid and CCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CCC may arise from cells exhibiting ciliated cell features, whereas ENC presumably originates from secretory cells or their precursors [14]. We performed immunohistochemical staining for FOXJ1, EZR, and MPST in the endometriosis-associated carcinoma samples for evaluating this model.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Our findings demonstrated that FOXJ1 and EZR, markers of the ciliated cell phenotype, and MPST, a marker of the secretory cell phenotype, were differentially expressed in the tumor tissues. In ENC, the expression patterns of these markers closely resembled those of the normal endometrial epithelium. FOXJ1 was localized to the nucleus, EZR to the cytoplasm, and MPST to both the nucleus and cytoplasm. Majority of the endometrioid cancer cells exhibited cell positivity for MPST, whereas only a small subset showed positivity for FOXJ1 and EZR (Fig. 4A-C). In CCC, tumor cells demonstrated strong cytoplasmic expression of EZR, a marker of the ciliated cell phenotype, whereas neither EZR nor MPST expression was detected (Fig. 4D-F). In mixed carcinomas, the endometrioid and clear cell components exhibited distinct marker expression profiles consistent with those observed in pure ENC and CCC, respectively. In the endometrioid component, cells positive for MPST constituted the majority of the tumor cells, whereas FOXJ1- and EZR-positive cells were limited to a minor subset. In the clear cell component, only EZR was expressed in the cytoplasm, and both FOXJ1 and MPST were absent.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we obtained the following results:\u0026nbsp;First, two distinct types of Ov EMS were detected in terms of the frequency of ciliated cells: the endometrium-like type (type 1) and tubal-like type (type 2). Second, the composition ratio of ciliated cells in the epithelium was low in DE and PE, similar to that in the endometrium. Third, ciliated cells in type 2 ovarian endometrioma and the endometrium were positive for PAX8, whereas ciliated cells in the fallopian tubes were negative for PAX8. Furthermore, in endometriosis-related cancers, CCC was EZR-positive and MPST-negative, and ENC was EZR-negative and MPST-positive, which was also observed in a rare case of mixed cancer arising from a single endometriotic lesion. To our knowledge, this is the first report to demonstrate two types of Ov EMS based on the ratio of ciliated cells.\u003c/p\u003e\n\u003cp\u003eNotably, we found that Ov EMS can be divided into two distinct subtypes based on the composition ratio of ciliated cells: endometrial-like and fallopian tubal-like types, with frequencies of 63% and 37%, respectively. The retrograde menstrual theory is a major hypothesis regarding the pathogenesis of endometriosis, suggesting that ovarian endometriosis originates in the endometrium [6]. However, reports suggesting ovarian endometriosis originates from the fallopian tubes also exist [7]. In a previous report, the expression pattern of FMO3, a molecule strongly expressed in the fallopian tube, revealed that 56% exhibited a fallopian tube epithelial-like pattern and 44% the opposite expression pattern, which aligns with our observation of the two subtypes of ovarian endometriosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePAX8 is a member of the paired-box family of genes that is expressed during organogenesis of the thyroid gland, kidney, and Müllerian tract and serves as a sensitive and specific marker for Müllerian-derived epithelial cells [22]. PAX8 is a marker of the fallopian tube secretory cell lineage but not of the ciliated cell population\u0026nbsp;[23]. Herein,\u0026nbsp;FOXJ1-positive ciliated cells in type 2 Ov EMS and the endometrium were positive for PAX8, whereas ciliated cells in the fallopian tubes were negative for this molecule. These results indicated that type 2 Ov EMS was similar to the fallopian tube in terms of a high proportion of ciliated cells, but also similar to the endometrium with respect to the expression of PAX8 in ciliated cells. A previous study using genetic cell lineage tracing demonstrated that PAX8+ secretory cells acted as progenitors of the fallopian tube epithelial cells, giving rise to ciliated cells during differentiation\u0026nbsp;[24]. Similarly, Cochrane et al. demonstrated through single-cell RNA sequencing of endometrioid organoids that secretory cells can differentiate into ciliated cells but not vice versa\u0026nbsp;[15], consistent with the view that ciliated cells represent a terminally differentiated cell type. Herein, ciliated cells in both type 2\u0026nbsp;Ov EMS\u0026nbsp;and endometrium\u0026nbsp;were positive for PAX8, whereas ciliated cells in the fallopian tube lacked PAX8 expression. This contrast suggests that despite the high proportion of ciliated cells resembling the fallopian tube, type 2\u0026nbsp;Ov EMS\u0026nbsp;is more likely to share an origin with the endometrium, based on its PAX8-positive and\u0026nbsp;FOXJ1-positive\u0026nbsp;ciliated cell profile. This interpretation is further supported by recent single-cell transcriptome data identifying transitional populations that coexpress PAX8 and FOXJ1, suggesting a trajectory from secretory to ciliated differentiation\u0026nbsp;[10]. Therefore, ciliated cells in type 2 Ov EMS may reflect an endometrium-derived transitional phenotype, rather than a true fallopian lineage. Interestingly, estradiol reportedly promotes ciliogenesis in endometrial epithelial cells\u0026nbsp;[25]. In a previous study comparing the estradiol levels in the endometrium, Ov EMS, PE, and DE, the estrogen concentration was markedly higher in Ov EMS than in other tissues\u0026nbsp;[26]. Locally elevated estradiol levels in Ov EMS may facilitate the differentiation of secretory cells into ciliated cells, thereby contributing to the significantly higher proportion of ciliated cells observed in type 2 Ov EMS than in the endometrium, PE, and DE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMPST was identified as a highly specific marker of secretory cells using RNA seq [15]. MPST was strongly expressed in ENC compared to CCC, which is in line with a previous report using methylenetetrahydrofolate dehydrogenase 1 [14]. EZR was strongly expressed in CCC but not in ENC. FOXJ1 was not detected in CCC. These results are consistent with those of a previous report [14]. Ovarian endometriosis presumably originates from ovarian CCC and ENC, which are described as endometriosis-associated cancers [17, 27]. In the ovary, ENC and CCC occur at similar rates, with the incidence of CCC being particularly high in Japan; however, ENC is much more common than CCC in the endometrium. ENC occur frequently in the malignant transformation of extraovarian endometriosis [28-31]. Cochrane et al. proposed that because CCC and ENC that occur in endometriosis share genetic abnormalities and develop in a similar microenvironment, the different histological types could be explained by different cellular origins [14]. Thus, ENC may originate from secretory cells and CCC may originate from ciliated cells. Based on this model, ENC being more common than CCC in DE can be explained by the finding that the proportion of ciliated cells in DE was as low as that in the endometrium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHerein, we considered that epithelial cells in Ov EMS also originated from the endometrium, as we observed a PAX8 expression pattern closer to that of endometrial ciliated cells than that of tubal ciliated cells. Studies tracing somatic mutations from the endometrium to CCC through Ov EMS have suggested that the epithelial cells of Ov EMS and CCC originated from the uterine endometrial epithelium [32]. However, if it is assumed that both subtypes originate from the endometrium, it remains unknown what determines whether these two types of ovarian endometriosis have clearly different ratios of ciliated cells.\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. First, the gold standard for identifying a cell origin should be lineage-tracing experiments, which allow tracing the fate of a specific cell type as it develops. However, the existence and locations of stem or precursor cells in the female genital tract remain controversial. Second, stromal cells should be considered when investigating the origin of endometriosis; however, this study focused only on the epithelium and did not find any differences in the stroma. Nevertheless, the epithelium and stroma were reported to develop independently in the same endometriosis because the epithelium is clonal, while the stroma is not [33]. Therefore, tracing the origin of the stroma may be technically challenging.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe identified two distinct subtypes of Ov EMS based on the proportion of ciliated cells. The proportion of ciliated cells in the DE and PE groups was as low as that of the endometrium. Notably, in type 2 Ov EMS, containing a high proportion of ciliated cells resembling the fallopian tube, expressed PAX8, indicating a transitional phenotype and origin from the endometrial epithelium rather than from the fallopian tube epithelium. These findings provide novel insights into the cellular origin and pathogenesis of endometriosis and endometriosis-associated cancers. Further studies are warranted to elucidate the biological differences between these two types and the mechanisms underlying their divergence.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors would like to thank Editage (www.editage.jp) for the English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are included in this published article and its supplementary information files. De-identified individual-level data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;S.H. performed experiments, analysis data, writing of the manuscript and contributed to data interpretation. T.H. designed and supervised experiments, raised funding, interpreted the data, and wrote the manuscript. N.N performed experiments, and contributed data interpretation. M.N. and M.E. contributed to collecting samples and data interpretation. Y.H. and Y.O. supervised experiments and data interpretation. K.K. supervised experiments and contributed to data interpretation and critical discussion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental procedures were approved by the Institutional Review Board of the University of Tokyo (registration number #324-20).\u0026nbsp;We obtained written informed consent from all individuals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publications:\u0026nbsp;\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Vigano P: \u003cstrong\u003eEndometriosis.\u003c/strong\u003e\u003cem\u003eNat Rev Dis Primers \u003c/em\u003e2018, 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\u003cstrong\u003e245:\u003c/strong\u003e265-269.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ovarian endometrioma, endometriosis, ciliated cell, clear cell carcinoma, endometrioid carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-8631039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8631039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe implantation (retrograde menstruation) theory hypothesizes that endometriosis arises when refluxed endometrial cells implant at ectopic sites; however, a fallopian tube epithelial origin has also been proposed. Because endometrium and fallopian tube epithelia differ markedly in their balance of ciliated and secretory cells, we asked whether ciliated-cell proportion defines ovarian endometrioma subtypes and associates with the histologic phenotype of endometriosis-associated ovarian carcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We analyzed 67 specimens: paired eutopic endometrium and fallopian tube (17 pairs), ovarian endometrioma (n=30), deep (n=6) and peritoneal (n=3) endometriosis, and endometriosis-associated cancers (n=11). Ciliated and secretory differentiation were assessed by immunostaining for forkhead box J1 (FOXJ1) and ezrin (EZR), and 3-mercaptopyruvate sulfurtransferase (MPST), respectively; FOXJ1/paired-box gene 8 (PAX8) co-localization was evaluated by double immunofluorescence. Ovarian endometriomas were classified using an 18.71% FOXJ1-positive cut-off.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eFOXJ1-positive ciliated cells were abundant in fallopian tube epithelium but scarce in eutopic endometrium, deep endometriosis, and peritoneal endometriosis. Ovarian endometriomas showed a bimodal distribution of FOXJ1-positive frequency and were classified as type 1 (\u0026lt;18.71%, endometrium-like, 19/30) or type 2 (≥18.71%, fallopian tube-like; 11/30). FOXJ1-positive frequency was significantly higher in fallopian tube and type 2 endometrioma than in endometrium, type 1 ovarian endometrioma, and deep and peritoneal endometriosis. In type 2 endometrioma, most FOXJ1-positive ciliated cells co-expressed PAX8, whereas FOXJ1-positive cells lacked PAX8 in fallopian tube epithelium. Clear cell carcinoma was EZR-positive and MPST-negative, whereas endometrioid carcinoma was predominantly MPST-positive with a minor FOXJ1/EZR-positive subset; a mixed carcinoma showed the same component-specific profiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Ovarian endometriomas comprise two subtypes defined by ciliated-cell proportion, whereas deep and peritoneal endometriosis show low ciliated-cell content comparable to eutopic endometrium. The FOXJ1/PAX8 co-expressing ciliated cells in “fallopian tube-like” type 2 lesions indicate a transitional epithelial phenotype distinct from terminally differentiated fallopian tube epithelium and are compatible with an endometrial origin even for ciliated-cell-rich lesions. Along with the EZR/MPST profiles distinguishing clear cell and endometrioid carcinoma, these findings suggest that differentiation states of endometriotic epithelium may be linked to the histologic phenotype of endometriosis-associated ovarian cancer.\u003c/p\u003e","manuscriptTitle":"Ovarian endometrioma subtypes defined by the ratio of ciliated cells: implications for endometriosis origin and carcinoma histologic subtype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 21:18:21","doi":"10.21203/rs.3.rs-8631039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00fbfece-cfdb-4ade-975b-a2f10f29b2e7","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-22T05:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 21:18:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8631039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8631039","identity":"rs-8631039","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}