A novel mouse model of endometriosis: Simulating the recurrent hemorrhagic microenvironment of clinical lesions

A novel mouse model of endometriosis: Simulating the recurrent hemorrhagic microenvironment of clinical lesions Abstract Current models of endometriosis (EMs) still have limitations in replicating the key pathological features of human EMs, particularly the cyclic bleeding associated with ectopic lesions. To address this gap, this study aimed to develop a proof-of-concept mouse model that incorporates repeated retrograde hemorrhagic exposure through the intraperitoneal injection of endometrial fragments, followed by repeated intraperitoneal injections of fresh whole blood. An EMs model was established in female C57BL/6J mice via intraperitoneal injection of endometrial fragments combined with saline or whole blood, respectively. The model was systematically evaluated using ectopic lesion burden, peritoneal adhesion scores, histopathological staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Both models successfully recapitulated the fundamental pathological features of EMs. However, the intraperitoneal injection of whole blood (IPBI) protocol induced a markedly greater lesion burden, together with more pronounced histopathological and molecular alterations, and recapitulated repeated exposure to retrograde menstruation. In addition, the IPBI group exhibited more robust systemic biomarker responses, indicating enhanced clinical relevance. The present study successfully established a proof-of-concept EMs mouse model that introduces repeated retrograde hemorrhagic exposure to create a clinically relevant, blood-stimulated microenvironment. This model provides a reliable tool for investigating the pathogenesis of EMs and developing therapeutic strategies. 1 INTRODUCTION Endometriosis (EMs) is a chronic inflammatory disease characterized by the ectopic growth of endometrial-like tissue outside the uterine cavity, predominantly in pelvic sites such as the ovaries and peritoneum, or in extrapelvic locations. Approximately 10% of women of reproductive age and individuals assigned female at birth (AFAB) worldwide are affected, with a cumulative patient population exceeding 190 million.1, 2 The core clinical manifestations of EMs include persistent pelvic pain (such as dysmenorrhea and dyspareunia) and infertility, often accompanied by fatigue, gastrointestinal disturbances, and urinary symptoms. Notably, approximately 20%–25% of affected individuals remain asymptomatic, whereas the prevalence of EMs rises to as high as 50% among women presenting with pelvic pain.3 The current gold standard for diagnosis relies on laparoscopic surgery to directly visualize the lesions. However, due to the technical challenges of invasive procedures, disparities in medical resource accessibility, and the absence of noninvasive diagnostic methods, diagnosis is frequently delayed by several years.1, 4 Although EMs has been clinically recognized for over a century, its exact pathogenesis remains incompletely elucidated. Current theories mainly include the retrograde menstruation theory, the coelomic metaplasia theory, the vascular and lymphatic dissemination theory, and the induction theory.5-8 Among these, the retrograde menstruation theory, proposed by Sampson in 1921, remains the predominant theoretical framework. This hypothesis posits that during menstruation shed endometrial tissues (containing glandular epithelial-stromal cell complexes) can undergo retrograde transit through the fallopian tubes into the peritoneal cavity. Subsequently, via a cascade of adhesion, invasion, and angiogenesis, these fragments ultimately establish ectopic lesions at sites such as the ovaries and pelvic peritoneum. Animal models of EMs are essential tools for studying the disease. Although nonhuman primates serve as ideal spontaneous models, their application is constrained by high costs and ethical considerations.9 Consequently, rodents have become the predominant choice due to their rapid reproduction and ease of manipulation.10 However, a recent systematic review and meta-analysis demonstrated that due to the lack of a unified gold standard protocol, currently widely utilized immunocompetent mouse models of EMs exhibit significant heterogeneity in terms of animal strains, tissue types, and transplantation methods, which severely hinders the comparability and reproducibility of research findings.11 Current modeling approaches mainly include syngeneic and heterologous implantation. Syngeneic implantation can be further categorized into autologous and allogeneic methods. Autologous implantation is highly invasive and is therefore unsuitable for studies focusing on EMs-associated infertility.12 Traditional allogeneic implantation typically involves the intraperitoneal injection of uterine tissue fragments; however, this method has a relatively low success rate.13 Although heterologous implantation preserves the characteristics of human-derived tissues, it precludes the investigation of normal immune responses.14 Most critically, due to the absence of a menstrual cycle in rodents, existing models fail to simulate the key pathological feature of cyclic bleeding inherent to the retrograde menstruation theory. Therefore, driven by the pressing demand for model standardization and optimization, future research is urgently required to establish a novel animal model that not only preserves the structural integrity of the reproductive tract but also better approximates the recurrent hemorrhagic microenvironment characteristic of clinical EMs.11 This study aimed to establish a proof-of-concept mouse model of EMs through the intraperitoneal injection of endometrial fragments, followed by repeated intraperitoneal injections of fresh whole blood, to simulate repeated retrograde hemorrhagic exposure. The efficacy of the model was comprehensively validated via macroscopic observation, histopathological analysis, molecular biological detection, and systemic response evaluation. Specifically, the in vivo growth and invasive capacity were evaluated by measuring the ectopic lesion burden and adhesions. The histomorphology and degree of fibrosis of the ectopic lesions were analyzed using hematoxylin and eosin (HE) and Masson's trichrome staining. Immunohistochemistry staining was employed to detect the expression of adhesion factors. Enzyme-linked immunosorbent assay (ELISA) was used to assess the inflammatory status of the local microenvironment and systemic biomarkers. The expression of angiogenesis-related genes was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). 2 METHODS 2.1 Animals A total of 15 female C57BL/6J mice, weighing 18–20 g, were procured from Jiangsu Huachuang sino Pharma Tech Co., Ltd. All mice had free access to standard chow and water and were maintained in a controlled environment at a temperature of 20–24°C, humidity of 40%–60%, and a 12-h light/dark cycle. After a 2-week acclimatization period, the mice were randomly divided into the donor group (n = 5) and the recipient group (n = 10), using a random number sequence generated by Excel software. To confirm the adequacy of this sample size, a post-hoc power analysis for primary lesion parameters was conducted (Table S5). All animal experiments were performed in accordance with the ARRIVE 2.0 guidelines and were approved by the Ethics Committee of the Experimental Animal Department of Nanjing University of Chinese Medicine (no.: 202410A019). Except for the researchers responsible for postoperative intervention, all personnel involved in subsequent data collection and analysis were blinded to the animal group allocation. 2.2 Drug preparation β-Estradiol 3-benzoate (EB) (1.5 mg) (Macklin Biochemical, Shanghai, China) was weighed and dissolved in 5 mL of dimethyl sulfoxide (Beyotime, Biotechnology, Shanghai, China) and mixed with 45 mL of corn oil to prepare an EB solution with a concentration of 30 μg/mL. The solution was then stored at 4°C, protected from light, for later use. 2.3 Model preparation 2.3.1 Preimplantation preparation All mice were subcutaneously injected with EB solution at a dose of 150 μg/kg body weight at the nape of the neck (one injection every 4 days, for a total of 2 injections).15 From the first injection until the day of intraperitoneal implantation, vaginal cytology smears were performed daily to monitor the estrous cycle of the mice, ensuring that all mice were synchronized on the day of implantation. 2.3.2 Implantation procedure Donor mice were anesthetized with isoflurane and subsequently euthanized by cervical dislocation. The abdominal cavity was promptly opened, and the uterus was excised and placed in prechilled sterile salinebetween two recipient mice (0.5 mL per recipient). To eliminate subjective bias during the injection process, the specific donor-recipient pairings were preassigned based on the initial random allocation sequence (e.g., the suspension from the first donor was injected into the first two recipients on the randomized list). Injections were performed without anesthesia using a restrained technique, per institutional approval. After disinfecting the abdomen of the recipient mice with povidone-iodine, the suspension was injected into the peritoneal cavity at a site approximately 0.5 cm superior to the urethral orifice. After the injection, gentle pressure was briefly applied to the puncture site, and erythromycin ointment was administered to prevent infection. The entire experimental procedure was performed under sterile conditions on ice and completed within 5 min to rinse off blood and mucus. Subsequently, the uteri were longitudinally incised, and the bilateral uterine horns were placed in separate Petri dishes, each containing 0.5 mL of ice-cold phosphate-buffered saline (PBS). The tissue was rapidly minced into fragments of ≤1 mm3. To control for interdonor variation, the tissue fragments and PBS from both horns were pooled and mixed, resulting in a total suspension volume of approximately 1.0 mL. This suspension was subsequently distributed randomly and equally. 2.3.3 Postoperative management After the implantation in all recipient mice was completed, they were subcutaneously injected twice with EB solution at the nape of the neck on the same day according to the original protocol. Starting one day postimplantation, mice in the intraperitoneal injection of whole blood (IPBI) group were administered 0.2 mL of syngeneic whole blood via intraperitoneal injection every 3 days for a total of five doses, aiming to approximate the frequency of hemorrhagic exposure during multiple menstrual cycles within the 14-day establishment period. The control group received intraperitoneal injections of 0.2 mL physiological saline under identical conditions (n = 5 per group). All mice were maintained until day 14 postmodeling to facilitate endometrial growth. Whole blood was collected from 14 female C57BL/6J mice (24–26 g) via the retro-orbital venous plexus. To minimize animal usage and ensure animal welfare, an alternating blood sampling scheme among groups was implemented. All procedures were performed under anesthesia and aseptic conditions. 2.4 Sample collection and macroscopic observation of ectopic lesions On day 14 after model establishment, all mice were euthanized by cervical dislocation under anesthesia. Immediately, 1 mL of saline was injected into the peritoneal cavity, and the abdomen was gently massaged to collect peritoneal fluid. Subsequently, a laparotomy was performed to observe the implantation of ectopic lesions, which were then isolated. The thoracic cavity was then opened, blood was aspirated from the right ventricle, and serum was separated by centrifugation (3000 g, 15 min). 2.5 Assessment of ectopic lesion burden The collected ectopic lesions were evaluated, including parameters such as number, weight, and volume. The volume of ectopic lesions was calculated using a modified ellipsoid formula: V = 1/2 × (length × width2),16 where length and width values were obtained by caliper measurements. 2.6 Peritoneal adhesion scoring The severity of peritoneal adhesions was assessed using the Blauer scoring system, with scores ranging from 0 to 4 (0 = no adhesions; 1 = thin adhesive; 2 = thick adhesive bands limited to one area; 3 = extensive and thick adhesive bands; 4 = adhesions including internal organs).17 2.7 Histological analysis Ectopic lesions were fixed in 4% paraformaldehyde, and samples were subsequently embedded in paraffin. Sections were stained with HE and Masson's trichrome staining and observed under a microscope. HE-stained sections underwent histopathological scoring using the following formula: score = P (explant epithelial cell viability) × I (glandular density). Specific criteria for scoring were referenced from a previously described method.18 Masson's trichrome staining was utilized to assess the degree of fibrosis, where collagen fibers appeared blue, smooth muscle red, and cell nuclei dark brown. Semiquantitative analysis of the blue collagen area was conducted using ImageJ software. 2.8 Immunohistochemistry staining The procedure for preparing tissue sections for immunohistochemistry staining was consistent with that used for HE staining. After antigen retrieval and blocking endogenous peroxidase activity, the sections were incubated overnight at 4°C with diluted intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), integrin alpha5 (ITGA5), and integrin alphaV (ITGAV) (details of the antibodies are provided in Table S1) antibodies. Subsequently, the sections were incubated with secondary antibodies at 37°C for 30 min. 2.9 ELISA analysis Inflammatory indicators included interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and serum biomarkers such as cancer antigen 125 (CA-125). These were measured using ELISA kits (details of the antibodies are provided in Table S2). All procedures were strictly conducted according to the manufacturer's instructions, and the intra-assay and inter-assay precision for the measurements are summarized in Table S4. 2.10 Quantitative reverse transcription PCR Total RNA was extracted from ectopic lesions using TRIzol reagent (Beyotime Biotechnology Shanghai, China) and reverse transcribed using 4 × Hifair III SuperMix plus (Yeasen Biotechnology, Shanghai, China) to generate complementary DNA (cDNA). RT-qPCR analysis was performed on an instrument (Applied Biosystems, USA) using Hieff qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen Biotechnology, Shanghai, China). With beta-actin serving as the housekeeping gene, the expression level of each gene was calculated using the 2−ΔΔCt method. Detailed information on the primers used for RT-qPCR is provided in Table S3. 2.11 Statistical analysis Data were analyzed using SPSS 26.0 and GraphPad Prism 9.0 software. Measurement data were expressed as mean ± standard error of the mean (SEM). Data normality was assessed using the Shapiro–Wilk test. For comparisons between two groups, a t-test was performed if the data were normally distributed; otherwise, the nonparametric Mann–Whitney U test was applied. A p-value of <0.05 was considered statistically significant. 3 RESULTS 3.1 IPBI-induced more severe macroscopic lesions of EMs To investigate the differences in macroscopic lesions between the two groups, we evaluated the morphology, lesion burden, and extent of adhesion in ectopic lesions. On day 14 after model establishment, both the control and IPBI groups developed ectopic lesions (Figure 1A). These ectopic lesions were primarily concentrated on the mesentery, with a few also surrounding the liver, peritoneum, uterus, and ovaries, and adhering to adjacent tissues. Macroscopically, the lesions appeared as irregular cystic nodules with a semitranslucent surface, containing pale yellow purulent fluid. The cyst wall exhibited neovascularization and was encapsulated by connective tissue. Quantitative assessment revealed that, compared to the control group, the IPBI group showed a significant increase in the number, weight, and volume of ectopic lesions (Figure 1B–D). In addition, the peritoneal adhesion score was markedly elevated in the IPBI group (Figure 1E). (Exact numerical values for these and subsequent parameters are provided in Table S6.) Collectively, these findings indicate that IPBI triggers a more severe lesion phenotype in EMs. 3.2 IPBI-induced ectopic lesions exhibit more typical histopathological characteristics and more severe fibrosis To assess the histological architecture and the extent of fibrosis in the ectopic lesions, HE and Masson's trichrome staining were performed. Compared to the control group, the endometrial glands in the lesions of the IPBI group were more abundant and densely arranged (Figure 2A). These glands were composed of numerous glandular epithelial cells and were closely surrounded by stromal cells, with blood vessels observed in some areas. Furthermore, the histopathological score of the IPBI group was significantly higher than that of the control group (Figure 2B). In terms of fibrosis, only minimal collagen deposition was observed in the ectopic lesions of the control group. In contrast, collagen deposition in the IPBI group was more widespread, predominantly distributed in the stromal and epithelial regions (Figure 2C). Semiquantitative analysis of the collagen deposition area revealed a significantly higher degree of collagen fiber deposition in the IPBI group (Figure 2D). The results indicate that IPBI-induced ectopic lesions exhibit more typical histopathological features and more severe fibrotic pathology. 3.3 IPBI-induced severe local lesions accompanied by elevated systemic biomarkers To evaluate critical pathological processes in EMs, including adhesion, inflammation, and angiogenesis, we conducted a comprehensive analysis of the ectopic lesions and their infiltrated peritoneal microenvironment. The expression of adhesion-related factors, including ICAM-1, VCAM-1, ITGA5, and ITGAV, was examined using immunohistochemistry. The results showed that the expression levels of ICAM-1, VCAM-1, ITGA5, and ITGAV were significantly higher in the IPBI group than in the control group (Figure 3A–E). This indicates that the ectopic lesions induced by IPBI possess a strong adhesive capacity, which facilitates their proliferation. Examination of inflammatory cytokines in the peritoneal fluid revealed significantly elevated levels of IL-1β, IL-6, and TNF-α in the IPBI group (Figure 3F–H). These findings suggest that IPBI is a critical factor in inducing a marked inflammatory microenvironment within the peritoneal cavity. Furthermore, the mRNA expression levels of angiogenesis-related genes in the ectopic lesion tissues showed that the expression of vascular endothelial growth factor (VEGF-A) and VEGF-C was significantly upregulated in the IPBI group compared to the control group (Figure 3I,J). Taken together, these results indicate that IPBI enhances the adhesive and angiogenic capabilities of ectopic lesions and triggers a more intense inflammatory response in the peritoneal microenvironment, collectively promoting the key pathological progression of EMs. To investigate whether the severity of local lesions is reflected in the systemic circulation, we measured the levels of CA-125, a key biomarker for EMs. The results demonstrated that the serum concentration of CA-125 in the IPBI group was significantly higher than that in the control group (Figure 3K). This suggests that IPBI exacerbates the local pathological progression of EMs and triggers a corresponding systemic response, further confirming the superiority of this model in holistically simulating the disease. 4 DISCUSSION A core characteristic of EMs is the cyclic bleeding of ectopic endometrium.19 Therefore, establishing an animal model capable of simulating this critical pathological process is pivotal for elucidating its pathogenesis. To overcome the fundamental limitation that rodents lack spontaneous cyclic bleeding, this study innovatively employed a strategy of intraperitoneal whole blood injection. This model simulates repeated retrograde hemorrhagic exposure, thereby establishing a pathological microenvironment enriched with blood components within the peritoneal cavity. Concurrently, endometrial tissue was implanted following estrous cycle synchronization via exogenous hormones and supplemented with continuous estrogen administration to replicate the estrogen-dependent features of EMs. This protocol resulted in more severe and clinically relevant pathology while effectively circumventing the interference of pelvic inflammation often associated with traditional laparotomy. Clinical studies have indicated that patients with EMs exhibit significant progression of pathological features, such as increased volume of ectopic lesions and accelerated expansion of tissue parenchyma.20 Consistent with these findings, we observed in the present study that both models successfully induced the formation of ectopic lesions. Notably, the IPBI group exhibited significant increases in the number, weight, and volume of these lesions. Moreover, pelvic adhesions, a typical clinical manifestation of EMs, were evident in both models. However, evaluation of adhesion severity using the Blauer scoring system revealed that the intraperitoneal adhesion scores in the IPBI group were significantly higher than those in the control group. Collectively, these results indicate that introducing repeated retrograde hemorrhagic exposure significantly exacerbates these key pathological features. Pathologically, EMs is defined by ectopic endometrial glands and stroma, frequently accompanied by fibrosis. Mouse models established to mimic this condition have shown that experimental lesions are histologically comparable to human pathology, containing glands, stroma, and cyst-like structures.9 HE staining confirmed that the IPBI group presented with typical endometrial glandular and stromal architecture, whereas these features were markedly reduced in the control group. Furthermore, fibrosis is widely recognized in all forms of EMs and plays a critical role in EMs-associated pain and infertility.21 Masson's trichrome staining is a classical method for evaluating tissue collagen deposition and fibrosis.22 Although collagen deposition was observed in both groups, it was significantly more pronounced in the IPBI group. Taken together, these results suggest that IPBI successfully recapitulates the characteristic histopathology of human EMs and demonstrates a more advanced fibrotic progression. The progression of EMs is driven by the synergistic action of multiple molecular mechanisms. Among these, chronic inflammation, aberrant adhesion, and angiogenesis are pivotal processes that propel the disease toward deep infiltration and fibrosis.23 Peritoneal inflammation, triggered by retrograde menstruation, serves as the initiating factor for EMs. It recruits immune cells and releases pro-inflammatory cytokines, thereby promoting the growth and invasion of ectopic tissues.24 Aberrant adhesion constitutes a critical step in lesion establishment. Clinical studies have confirmed that various adhesion molecules, including integrins, are potential therapeutic targets for EMs.25, 26 These molecules mediate the interaction between ectopic endometrial tissues and the peritoneal matrix, facilitating migration, adhesion, and invasion.27-29 Furthermore, ectopic endometrial tissues are characterized by extensive vascularization, a feature primarily sustained by the VEGF family. Notably, VEGF-A and VEGF-C exert synergistic effects to promote neovascularization, thereby fueling disease progression.30, 31 The results of the present study showed that the expression levels of the aforementioned inflammatory cytokines, adhesion molecules, and angiogenesis-related genes were significantly higher in the IPBI group compared to the control group. These findings collectively indicate that intraperitoneal injection of whole blood effectively recapitulates the inflammatory-adhesion-angiogenesis pathological axis in EMs. In this model, elevated pro-inflammatory cytokines in the peritoneal fluid create a microenvironment conducive to lesion establishment, and they also directly or indirectly induce the upregulation of adhesion molecules, facilitating abnormal adhesion between the ectopic endometrium and the peritoneum. Subsequently, inflammatory signals and the successfully adhered lesions jointly trigger an angiogenic response, providing nutritional support for sustained growth, ultimately driving the establishment and progression of ectopic lesions. Beyond the critical pathological mechanisms described above, the present model also induced alterations in systemic biomarkers. As a pivotal biomarker for EMs, CA-125 has been clinically demonstrated to possess high diagnostic specificity in symptomatic women, with levels universally and significantly elevated in serum, menstrual blood, and peritoneal fluid.32-34 Consistent with these clinical observations, the serum CA-125 concentration in the IPBI group was significantly higher than that in the control group. Furthermore, Zheng et al.35 reported similar findings in animal models, suggesting its potential utility as a nonsurgical diagnostic tool for EMs. These results further corroborate the robust association between CA-125 and EMs. The synergistic effects among the aforementioned molecular pathways initiate a pathological cascade of inflammation, adhesion, and angiogenesis, thereby accelerating disease progression and its systemic impact and revealing potential therapeutic targets (Figure 4). This study has several limitations. First, as a proof-of-concept model, although significant early pathological changes were observed, the small sample size (n = 5 per group) and short observation period limited the interpretation of chronic features, such as deeply infiltrating endometriosis, long-term fibrosis, or impaired fertility. Future comprehensive studies with expanded sample sizes are imperative to validate these findings and explore long-term disease progression. Second, we utilized full-thickness uterine tissue. Although human retrograde menstruation consists primarily of endometrium, the use of full-thickness tissue optimizes lesion adhesion and achieves a higher lesion maintenance rate and more extensive lesion distribution compared to purified endometrium. Furthermore, given that human menstrual effluent is approximately 50% blood and 50% tissue by volume, future studies should test a refined protocol with purified endometrium suspended in whole blood (1:1 v/v) to better mimic menstrual fluid. Third, the current study lacks a highly specific sham control. Future research should include a sham arm receiving only saline and blood (without endometrial tissue) to clearly delineate the pathological differences between the tissue and blood components. Fourth, evidence regarding “cyclic bleeding” in this model remains indirect. Specifically, as hemosiderin/iron staining or timed bleeding cycle markers are absent in the current study, our findings primarily rest on the pathological effects of repeated blood injections rather than the direct demonstration of cyclic lesion bleeding. Future studies should incorporate these specific markers to further validate the hormonal responsiveness of the lesions. Finally, although inbred mice facilitate experimental standardization, their genetic homogeneity does not fully reflect human diversity. Future validation across different genetic backgrounds is necessary to confirm the generalizability of these findings. Despite these constraints, this exploratory study provides critical insights into the pathological progression of EMs driven by recurrent bleeding exposure. 5 CONCLUSIONS In conclusion, we established a proof-of-concept EMs model via the intraperitoneal injection of endometrial fragments followed by repeated intraperitoneal injections of fresh whole blood. This approach yielded more robust pathological alterations at macroscopic, molecular, and systemic levels compared to the standard injection model alone. By incorporating repeated retrograde hemorrhagic exposure, this model provides a promising and more clinically relevant tool for further investigating pathogenesis and therapeutic strategies. AUTHOR CONTRIBUTIONS Yu Zhuang: Conceptualization; methodology; writing – original draft. Yan Zan: Data curation; methodology. Tiantian Ma: Software. Xiaoquan Huang: Data curation; software. Junwei Li: Software. Liangjun Xia: Conceptualization; funding acquisition; supervision. Yuping Sa: Supervision. Youbing Xia: Formal analysis; funding acquisition; supervision. ACKNOWLEDGMENTS We express our sincere gratitude to our colleagues for providing technical support and valuable advice during the experiments. We are grateful to the laboratory staff for their assistance with experimental equipment and materials. Additionally, we thank Figdraw for providing the drawing tools. FUNDING INFORMATION The study was supported by the National Natural Science Foundation of China (grant numbers: 82205251 and 82274638). CONFLICT OF INTEREST STATEMENT None. ETHICS STATEMENT All animal experiments were performed in accordance with the ARRIVE 2.0 guidelines and were approved by the Ethics Committee of the Experimental Animal Department of Nanjing University of Chinese Medicine (no.: 202410A019).