Standardized method to develop an endometriosis model in Sprague-Dawley rats: A step-by-step description

To establish a standardized and reproducible rat model of endometriosis by refining the classic Vivian technique, with the aim of minimizing methodological variability and replicating key features of human disease.

Sixty female Sprague–Dawley rats (6 weeks old) were evaluated for estrous-cycle phase using vaginal cytology, and only animals in proestrus or estrus were selected. Under ketamine–xylazine anesthesia, a 1-cm segment of the uterine horn was excised, opened longitu- dinally, and sutured to the peritoneal wall with the endometrial surface facing the peritoneum. Protocol refinements included strict graft ori- entation, fixation with four interrupted sutures, and perioperative ceftriaxone prophylaxis. A second laparotomy was performed one month later to assess lesion development, followed by histological confirmation.

All animals (100%) developed macroscopically visible lesions. These lesions presented as cystic, fluid-filled masses with hyperemic surfaces and neovascularization; adhesions to the bowel or omentum were also observed. Lesion size was quantified using digital imaging software. Histological analysis confirmed the presence of endometrial-like tissue composed of cuboidal epithelium, glandular structures, and stromal elements. No perioperative mortality occurred.

This standardized rat model reliably reproduces hallmark features of human endometriosis, including angiogenesis and adhe- sion formation. The protocol refinements address common sources of variability in rodent models and provide a stepwise, reproducible framework for mechanistic investigation and preclinical evaluation of novel therapies.

Adhesion; Angiogenesis; Animal model; Endometriosis; Experimental surgery; Histology; Homologous transplantation; Preclini- cal research; Reproducibility; Sprague–Dawley rat

Endometriosis is a chronic, estrogen-dependent disorder charac- terized by the presence of functional endometrial glands and stroma outside the uterine cavity. Ectopic lesions most commonly occur on the ovaries, pelvic peritoneum, and uterosacral ligaments, although extrapelvic manifestations—such as involvement of the diaphragm or abdominal wall—have also been reported [1,2]. The condition af- fects an estimated 10% of women of reproductive age and is partic- ularly prevalent among those with infertility, in whom rates may reach 30% to 50%, and among those with chronic pelvic pain, where prevalence may be as high as 70% to 80% [1,3]. Beyond its clinical manifestations, endometriosis imposes substantial psychological and socioeconomic burdens, underscoring its importance as a major public health concern. Despite decades of investigation, the pathogenesis of endometri- osis remains incompletely understood. Several theories have been proposed to explain the ectopic implantation and persistence of en- dometrial tissue. Sampson’s theory of retrograde menstruation re- adapted the Jones procedure in Wistar rats using a full-thickness uterine flap sutured to the abdominal wall, improving graft handling and lesion reproducibility. Collectively, these foundational models established the key methodological principles—autologous trans- plantation, stable tissue fixation, and hormonal responsiveness— that continue to underpin contemporary induction techniques. Nev- ertheless, variability persists in implantation success, tissue orienta- tion, suture configuration, infection control, estrous-cycle timing, and postoperative outcomes, with reported success rates ranging from 60% to 80%. Inconsistent graft adherence remains a significant limitation, often leading to epithelial loss or incomplete lesion estab- lishment. In the absence of a rigorously standardized rodent model with consistently reproducible results, a methodological gap persists that limits comparability across studies. The present study addresses these shortcomings by refining the Vivian technique through strict estrous-phase synchronization, verified endometrial orientation, four-point graft fixation, and prophylactic antibiotic administration. These modifications are intended to reduce interoperator variability, enhance reproducibility, and provide a clearly defined standardized protocol suitable for mechanistic and translational research.

1. Laboratory animals and ethical approval statement The experiment was conducted between December 2019 and May 2020 at the Animal Research Facility (ARF), Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, Uni- versitas Indonesia. The study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Indonesia (approval no. KET- 227/UN2.F1/ETIK/PPM.00.02/2020, issued February 24, 2020). All procedures were performed in compliance with national regulations and international standards for the care and use of laboratory ani- mals. Sixty 6-week-old female Sprague–Dawley rats (Rattus norvegi- cus) were used for model induction. Animals were housed at the ARF , IMERI, Faculty of Medicine, Universitas Indonesia, in groups of five per cage under controlled temperature and humidity and a 12-hour light/dark cycle, with free access to standard chow and water. 2. Estrous-cycle determination Estrous cyclicity was assessed to ensure hormonal synchronization before surgery. Vaginal smears were obtained by gently inserting ei- ther a plastic pipette containing 10 μL of sterile 0.9% sodium chlo- ride or a saline-moistened cotton swab into the vaginal canal. Secre- tions were transferred to glass slides, fixed in 90% ethanol, and stained with Giemsa. Slides were examined using a standard light microscope. Estrous phases were classified as proestrus, estrus, mains the most widely cited; however, alternative hypotheses—in- cluding coelomic metaplasia and Müllerian remnant theories—also retain relevance [4,5]. These mechanisms are increasingly viewed not as mutually exclusive but as interconnected processes involving im- mune dysfunction, hormonal dysregulation, angiogenesis, and ge- netic susceptibility that collectively drive lesion establishment and persistence [4,5]. This multifactorial complexity has hindered the de- velopment of definitive diagnostic strategies and targeted therapies. Clinically, endometriosis presents heterogeneously. Although some women remain asymptomatic, many experience cyclic pelvic pain, dysmenorrhea, dyspareunia, dyschezia, or urinary pain. Nota- bly, symptom severity does not consistently correlate with disease stage or depth of invasion [1]. Infertility, often resulting from pelvic adhesions and distorted pelvic anatomy, further highlights the pro- found reproductive consequences of the disease [2]. Laparoscopy with histopathological confirmation remains the diagnostic gold standard, and current treatment strategies include hormonal sup- pression, surgical excision, or a combination of both. However, none of these approaches guarantees long-term remission, and recurrence remains common. The limitations of current diagnostic and therapeutic options un- derscore the need for reliable experimental models of endometriosis to elucidate disease mechanisms and facilitate therapeutic develop- ment. Because spontaneous endometriosis occurs almost exclusively in nonhuman primates—models limited by ethical and financial constraints—rodents have become indispensable in preclinical re- search. Among these species, the Sprague–Dawley rat is particularly advantageous because of its predictable estrous cycles, ease of han- dling, and ability to generate reproducible lesions through surgical induction [6]. However, unlike menstruating species, rats do not un- dergo cyclic endometrial shedding, limiting replication of Sampson’s retrograde menstruation theory. Consequently, surgically induced homologous models remain the primary experimental approach in rats [7]. The earliest systematic rat model of endometriosis was described by Jones [8], who introduced a homologous autotransplantation technique in Sprague–Dawley rats. Their method involved excising a uterine horn segment, separating the myometrium from the endo- metrium, and transplanting a standardized 5×5 mm endometrial fragment onto the peritoneal wall with the epithelial surface facing the cavity. The graft was secured at four corners, and viability was as- sessed 3 weeks later [8]. Building on this approach, Vernon and Wil- son [ 9] compared three induction strategies—sutured uterine squares, intraperitoneal luminal lavage, and dispersed endometrial scrapings—and demonstrated that only sutured autotransplants yielded viable, cystic lesions, thereby establishing surgical implanta- tion as the most reliable method. do Amaral et al. [10] subsequently https://doi.org/10.5653/cerm.2025.086902 Clin Exp Reprod Med [Epub ahead of print] metestrus, or diestrus based on the relative proportions of epithelial cells and leukocytes (Figure 1) [11]. Cycles were considered normal when they repeated every 4–7 days. Only animals in proestrus or es- trus underwent surgical induction to maximize implantation success. 3. Induction of endometriosis Anesthesia was induced with intramuscular ketamine hydrochlo- ride (90 mg/kg) and xylazine (10 mg/kg). Prior to surgery, animals were weighed to calculate anesthetic dosage. After induction, the abdominal wall hair was removed using clippers, and rats were posi- tioned supine on a surgical board with limbs abducted. Routine asepsis and antisepsis were performed. A 4-cm midline incision was made below the umbilicus to expose the peritoneal cavity. The bicornuate uterus was identified (Figure 2), and a 1-cm segment of uterine horn was excised after securing he- mostasis by proximal and distal ligation with 3–0 silk sutures. The ex- cised segment was opened longitudinally to expose the endometrial surface (Figure 3). The flap of uterine tissue was prepared and su- tured to the anterior peritoneal wall of the right lower quadrant, ap- proximately 2 cm from the abdominal incision, with the endometrial side facing the peritoneum. Fixation was achieved using four inter- rupted 6–0 nylon sutures (Figure 4). After confirming hemostasis, the abdominal wall was closed with 4–0 chromic catgut in one layer, and the skin was closed with 4–0 silk sutures. Rats were returned to their cages after recovery from an- esthesia. Prophylactic antibiotic treatment was administered as in- Figure 1 Figure 1. Estrous cycle in rats. (A) Diestrus: Predominance of leukocytes with few or no epithelial cells. (B) Proestrus: Decreased leukocytes with increasing epithelial cells. (C) Estrus: Field composed entirely of epithelial cells, with no leukocytes observed. (D) Metestrus: Postestrus phase characterized by declining epithelial cells with a concomitant increase in leukocytes. Original micrographs reproduced from Zenclussen et al. [11]. Figure 2 Figure 2. Identification of the uterus. Figure 3 Figure 3. Excision of a uterine horn segment. Figure 4. Suturing of the uterine tissue to the peritoneal wall.Figure 4 www.eCERM.org 3 Situmorang H Standardized endometriosis model in Sprague–Dawley rats AA CC BB DD traperitoneal ceftriaxone at a dose of 10 mg per rat on postoperative days 0, 1, and 2. 4. Lesion assessment One month later, a second laparotomy was performed under the same anesthesia and asepsis protocol. A 4-cm midline incision was made, and the peritoneal cavity was inspected. Macroscopic features of endometriotic lesions were documented and photographed using an iPhone 7 Plus camera (Apple Inc.) with a millimeter scale for cali- bration (Figure 5). 5. Histological analysis Lesions were excised and immediately fixed in 10% buffered for- malin. Tissues were processed through graded alcohols, cleared in xylene, and embedded in paraffin. Serial sections were cut using a microtome, mounted on glass slides, and stained with hematoxylin and eosin (H&E). Microscopic evaluation was performed using a standard light microscope at 40× to 400× magnification. 6. Postoperative care Animals were monitored daily for signs of distress, wound healing, and general health status. No intraoperative or postoperative mor- tality occurred. At the conclusion of the study, all rats were eutha- nized by decapitation under deep anesthesia.

All 60 rats (100%) developed macroscopically visible endometriot- ic lesions, indicating that each implanted endometrial fragment suc- cessfully formed a lesion using the modified Vivian technique. At the second laparotomy, performed 1 month after implantation, cystic le- sions were consistently identified on the peritoneal surface. Lesions appeared as fluid-filled, cystic masses with smooth surfaces and a hyperemic coloration compared with the surrounding peritoneal tis- sue. Prominent blood vessels converged toward the lesions, and ad- hesions to adjacent bowel or omentum were observed in several an- imals (Figure 6). The surface area of each lesion was documented by digital photography with a millimeter scale and subsequently mea- sured using SketchAndCalc software (iCalc Inc.). Quantitative measurements demonstrated expected biological variability, with lesion areas ranging from 0.83 to 131.50 mm² (mean, 45.27 mm²; median, 37.34 mm²) across the cohort (Table 1). This dis- tribution aligns with prior reports of surgically induced endometrio- sis, in which lesion heterogeneity is typical despite standardized im- plantation techniques (Figure 7). The uniformly successful lesion for- mation observed in the present study compares favorably with earli- er models, which often report 60% to 80% viability because of incon- sistent graft orientation, limited suture fixation, or suboptimal es- trous timing at implantation. Histological analysis of excised lesions further confirmed the es- tablishment of endometriotic tissue. H&E staining demonstrated cystic structures lined by a single layer of cuboidal epithelial cells, with surrounding endometrial-like glands and stromal components. Stromal cellularity was most pronounced adjacent to the cyst wall and gradually decreased toward the periphery, consistent with orga- nized tissue architecture within the implants (Figures 8 and 9).

This study established a standardized rat model of endometriosis with a 100% lesion induction rate, exceeding the success typically re- ported in classical models such as Vernon and Wilson [9] and the original Vivian technique. The high reproducibility observed here likely reflects several purposeful refinements. First, implantation was Figure 5. Macroscopic appearance of the lesion and area measurement. (A) Representative macroscopic image of the lesion. (B) Representative macroscopic image after area measurement using SketchAndCalc software (iCalc Inc.).Figure 5A Figure 5B Figure 5A Figure 5B https://doi.org/10.5653/cerm.2025.086904 Clin Exp Reprod Med [Epub ahead of print] AA BB Figure 6. (A) Endometriotic lesion and (B) endometriotic lesion with intestinal adhesion. Figure 6A Figure 6B Figure 6A Figure 6B AA BB Table 1. Lesion area distribution after surgical induction of endo- metriosis in rats Parameter Value Number a) 60 Minimum area (mm 2 ) 0.83 Maximum area (mm 2 ) 131.50 Mean area (mm 2 ) 45.27 Median area (mm 2 ) 37.34 Lesion areas were measured from standardized digital photographs con- taining a millimeter reference marker and analyzed using SketchAndCalc planimetry software. a) The number of rats with successfully established lesions at second laparot- omy (100% induction rate). Figure 7. Distribution of endometriotic lesion area in the rat model. Scatter plot showing the distribution of lesion area measurements. performed exclusively during the proestrus or estrus phase, when endogenous estrogen levels peak, thereby providing a hormonally favorable environment that supports endometrial cell survival, at- tachment, and subsequent growth [9,10,12]. Second, the uterine im- plant was fixed using four interrupted 6–0 nylon sutures, ensuring uniform apposition of the endometrial surface to the peritoneum and reducing the risk of graft detachment, a recognized limitation of earlier two-suture techniques. This more comprehensive anchoring likely contributed substantially to consistent lesion establishment across animals. Third, perioperative prophylactic antibiotic adminis- tration may have reduced postoperative infection, thereby limiting inflammatory complications that can compromise graft viability. Col- Size of endometriosis lesion (mm 2 ) 120 100 80 60 40 20 0 10 20 30 40 50 60 Number of rats www.eCERM.org 5 Situmorang H Standardized endometriosis model in Sprague–Dawley rats lectively, these modifications improved procedural stability, ad - dressed key limitations of prior rodent endometriosis models, and enabled reliable lesion formation. Rodent models of endometriosis are broadly categorized as ho- mologous or heterologous. Homologous models, such as ours, use autologous or syngeneic uterine tissue transplanted into the perito- neal cavity, whereas heterologous models use human tissue in im- munocompromised hosts [13]. Despite phylogenetic limitations, rat models remain widely used because they balance affordability, sur- gical feasibility, and preservation of immunological integrity. Our findings support the use of standardized homologous models as re- liable preclinical platforms and complement menstrual-mimicry ap- proaches, such as those proposed by Persoons et al. [14]. A defining feature of this model was the consistent development of neovascularization and adhesions, both of which are hallmarks of clinically significant endometriotic disease. Angiogenesis is essential for the survival and expansion of ectopic endometrial implants and is largely mediated by vascular endothelial growth factor (VEGF), which has been repeatedly implicated as a central driver of lesion vascularization in rodent and human studies [15,16]. Although VEGF expression was not quantified, the pronounced macroscopic neovas- cularization observed aligns with established angiogenic mecha- nisms and supports the biological validity of the induced lesions. Similarly, the frequent formation of adhesions—often underreport- ed in rodent models despite their contribution to infertility, dyspa- reunia, and chronic pelvic pain—enhances the translational rele - vance of this approach [13]. The inflammatory microenvironment also underpins these patho- logical processes. Activated macrophages and pro-inflammatory cy- tokines, particularly tumor necrosis factor alpha (TNF-α), promote Figure 7A Figure 7b Figure 8. Histologic appearance of rat endometriotic lesions (hematoxylin and eosin stain). (A) Tissue section from the endometriosis model (40×). Scale bar: 0.1 mm. (B) Cystic lesion containing endometrial cells and leukocytes; the cyst wall is lined by a single layer of cuboidal epithelium (yellow arrow) (40×). Cystic lesion surrounded by endometrial-like stroma and gland-like structures (pink arrow) (40×). Scale bar: 0.1 mm. Figure 8A Figure 8b Figure 8A Figure 8b Figure 9. (A) High-power view of the cyst wall and surrounding endometrial-like tissue (hematoxylin and eosin stain). Cystic lesion containing endometrial cells and leukocytes; the cyst wall is lined by a single layer of cuboidal epithelium (yellow arrow) (400×). (B) Cystic lesion surrounded by endometrial-like stroma and gland-like structures (arrowhead) (400×). Scale bar: 0.1 mm. https://doi.org/10.5653/cerm.2025.086906 Clin Exp Reprod Med [Epub ahead of print] AA AA BB BB implantation, persistence, and progression of ectopic endometrium, while emerging mediators such as interleukin 37 correlate with dis- ease severity and may serve as biomarkers of lesion activity [17,18]. Although cytokine profiles were not measured in this study, the macroscopic and histological findings—cystic morphology, promi- nent vascularization, and well-preserved glandular and stromal ar- chitecture—are consistent with inflammatory cascades described in contemporary rodent endometriosis models. H&E staining provided morphological confirmation; however, current evidence suggests that optimal validation of experimentally induced lesions includes immunohistochemical (IHC) markers to substantiate tissue identity, distinguish endometriotic implants from nonspecific peritoneal reac- tions, and assess angiogenic or inflammatory activity [19]. The ab- sence of molecular and IHC analyses therefore represents a method- ological limitation, particularly because current standards emphasize evaluation of mediators such as TNF-α, VEGF , and oxidative stress markers (e.g., malondialdehyde) to strengthen mechanistic interpre- tation and translational relevance. Future applications of this stan- dardized model should incorporate targeted molecular and IHC as- says to enhance lesion characterization, support reproducibility, and improve comparability with human disease. Oxidative stress also plays a central role in endometriosis patho- physiology. Reactive oxygen species and byproducts such as malondialdehyde contribute to peritoneal inflammation and cellular stress, which may promote lesion persistence and impair reproduc- tive function [6]. The interaction between oxidative stress and in- flammation has been linked to infertility in both clinical and experi- mental studies, underscoring the value of incorporating oxidative stress biomarkers in future applications of this model. These findings are also consistent with evidence showing repro- ductive consequences of endometriosis in rats, including impaired ovarian reserve and reduced fertility [6]. By reliably inducing lesions with adhesion formation, this model provides a robust framework for investigating how endometriosis disrupts reproductive function and for evaluating targeted interventions. Pharmacological studies have shown that agents such as rosiglitazone can reduce lesion pro- gression in surgically induced rat models, further illustrating the utili- ty of standardized systems for preclinical drug testing [20]. Several limitations should be acknowledged. First, rodents do not menstruate, which limits the extent to which retrograde menstrua- tion can be modeled as a mechanism of disease establishment [7,14]. Second, pain assessment—an essential translational end - point—was not included and should be prioritized in future studies [5]. Third, molecular and IHC evaluation of angiogenic, inflammatory, and oxidative stress pathways would complement the morphologi- cal findings reported here. In addition, the present method specifi- cally generates peritoneal wall endometriosis, whereas human dis- ease encompasses multiple subtypes, including superficial peritone- al lesions, deep infiltrating endometriosis, and ovarian endometrio- mas. Developing parallel protocols to model these phenotypes, and clearly subclassifying experimental models accordingly, would im- prove translational applicability and better reflect clinical heteroge- neity [21]. In conclusion, this modified rat model of endometriosis reliably re- capitulates essential disease features, including vascularization, cyst formation, and adhesion development. The reproducibility achieved in the present study supports its use as a platform for mechanistic studies and preclinical evaluation of novel therapies. Future research should extend this framework by incorporating molecular validation and pain phenotyping to better align experimental outcomes with the clinical complexity of endometriosis. Conflict of interest No potential conflict of interest relevant to this article was reported. ORCID Herbert Situmorang https://orcid.org/0000-0001-7370-5819

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