The development of a preclinical swine model for endometriosis†

To generate a preclinical swine model for endometriosis, we surgically induced endometriosis in gilts one day after estrus ( Figure 1A ). After 1 and 6 -7 months of endometriosis induction, we examined the development of endometriosis by searching for endometriotic lesions at anatomically matching sites in humans [ 2 ]. After 1 month of endometriosis induction, we detected a total of 15 endometriotic lesions in the Pig #1 (4 on the outside of the uterus, 1 on the peritoneal wall and 10 on the intestines), the Pig #2 had a total of 8 lesions which were on the surface of the uterus, and Pig #3 had a total of 27 endometriotic lesions (8 on intestines, 2 on the peritoneal wall and 17 outside the uterus) ( Figure 1B ). In order to examine the development and progression of endometriosis, 3 additional pigs were euthanized at 6 -7 months post endometriosis induction. Pig #4 had 11 endometriotic lesions on the outside of the uterus and 6 endometriotic lesions on the intestines, Pig #5 had 3 endometriotic lesions on the intestine, and 9 on the outside of the uterus, and Pig #6 had 2 endometriotic lesions on the outside of the uterus, 5 on the peritoneal wall, and 8 on the intestines ( Figure 1B ). We observed the typical appearance of endometriotic lesions, red, black and white lesions, which is seen in endometriosis patients [ 30 ] ( Figure 1B ). The presence of endometriotic lesions was confirmed by histopathological examination by H&E analysis ( Figure 2. A – D ). All endometriotic lesions contained endometrial-like epithelial and stromal cells similar to those found in patients with endometriosis ( Figure 2E – L ). The endometriotic lesions and histological confirmations demonstrate that we have successfully developed a swine model representing signs of endometriosis. We confirmed endometriotic lesions by histological analysis ( Figure 2 ). To determine whether the endometriotic lesions were endometrial-like tissue, we examined the expression of E-cadherin, an endometrial epithelial cell marker [ 31 , 32 ], and Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a endometrial stroma cell marker [ 33 ]. Our immunohistochemistry (IHC) results revealed that E-cadherin was expressed in the luminal and glandular epithelium of both the eutopic endometrium ( Figure 2E ) and the endometriotic lesions ( Figure 2. F – H ). Similarly, COUP-TFII expression was confirmed in the stromal cells of both the eutopic endometrium ( Figure 2I ) and the endometriotic lesions ( Figure 2 J – L ). These histological and IHC findings demonstrate the presence of endometrial-like epithelial and stromal cells in these endometriotic lesions, resembling those observed in human endometriosis. Ideal preclinical animal models for endometriosis research should allow researchers to distinguish endometriotic lesions from surrounding normal tissues. Therefore, we developed a swine model of endometriosis by using ex vivo labeling with fluorescein isothiocyanate (FITC) dye-dope silica nanoparticles. To optimize ex vivo labeling condition in endometrial tissue fragments with FITC dye-dope silica nanoparticles, we examined the impact of the concentration of FITC dye-doped silica nanoparticles on ex vivo labeling. Endometrial tissue was collected in the pig one day after estrus. An equal amount of uterine fragments (5g/well) was put into each well, and 0, 20, 40, 60, and 80% of FITC dye-doped silica nanoparticles in 2ml of RPMI-1640 complete media (10% FBS) was added. After 2 hours of incubation, the fragments were washed and the intensity of FITC dye-doped silica nanoparticles was then examined using IVIS. FITC signaling (Total Radiant Efficiency [p/s]/ [ μW/cm 2 ]) was significantly higher at 40% (2.25×10 8 ±2.46×10 7 ; p < 0.05 ), 60% (3.97×10 8 ±8.04×10 7 ; p <0.01) and 80% (4.09×10 8 ±1.76×10 7 ; p <0.01) compared to 0% (3.61×10 6 ±3.45×10 6 ) ( Figure 3 A and B ). To determine an optimal incubation time, swine uterine fragments were incubated with 60% of FITC dye-doped silica nanoparticles for 0, 30, 60, 90 and 120 minutes. Radiant efficacy was significantly higher at 30 (3.47×10 8 ± 1.06×10 7 ; p < 0.01), 60 (3.30×10 8 ± 7.22×10 6 ; p < 0.001), 90 (4.89×10 8 ± 5.23×10 7 ; p < 0.001), and at 120 minutes (4.41×10 8 ± 7.94×10 7 ; p < 0.001) compared to 0 minutes (3.92×10 7 ± 1.95×10 6 ) ( Figure 3 C and D ). Therefore, we decided the optimal labeling condition was 60% concentration and 90 minutes of incubation. The optimal labeling condition of 60% at 90 minutes was used to ex-vivo label swine uterine fragments before they were introduced into the swine’s uterine cavity. One month after endometriosis induction, endometriotic lesions were examined and excised ( Figure 4A ). Fluorescence microscopy revealed FITC positive endometriotic lesions, but the adjacent normal tissues did not fluoresce ( Figure 4 B and C ). We confirmed that the endometrial tissue fragments were labeled with FITC dye-doped silica nanoparticles by histologic analysis. FITC signal was detected in induced endometriotic lesions ( Figure 4 D – F ).

Pigs carrying commercial genetic background were maintained in the designated animal care facility according to institutional guidelines of the University of Missouri. All experimental procedures were approved by the Animal Care and Use Committee (ACUC; protocol number 37561) of the University of Missouri. The reproductive anatomy of pigs closely resembles that of humans, featuring comparable structures such as ovaries, uterine horns, and the overall arrangement of the reproductive system ( Supplementary Figure 1 ). The technique for surgical induction of endometriosis in pigs was modified from our previously published method of inducing endometriosis in mice [ 20 ]. Endometriosis was induced in adult female pigs one day after standing estrous. Cycling gilts (heat + 1 day) were placed under general anesthesia via intravenous injection of Ketamine Hydrochloride (100 mg/ml, Dechra) and Xylazine (Rompun ® 100 mg/mL, Dechra) at a dosage rate of 1.4 / 0.4 mg/kg prior to endotracheal intubation and pigs were maintained on isoflurane for the duration of the surgery. For analgesia and systemic antibiotics, intramuscular injections of Flunixin Meglumine (Prevail 50 mg/mL, VetOne) at a dosage rate of 2.2 mg/kg and Ceftiofur crystalline free acid (Excede ® 100 mg/mL, Zoetis Animal Health) at a dosage rate of 100 mg/44 lbs were given at the start of surgery. A midline abdominal incision was made and a piece of uterus about 6 cm was removed, the remaining uterine horn was sutured closed. The endometrium was dissected from the myometrium until approximately 5g of endometrial tissue was obtained. The endometrium was cut into small fragments of about 1 mm 3 in a petri dish using sterile scissors. The small fragments were then mixed with 20ml HBSS (Gibco, Waltham, MA), and were dispensed into the abdominal cavity by a sterile serological pipette and the abdominal incision was closed with sutures. Sham female pigs were included to observe any effects from the surgery. They underwent the same procedure at the same time as the endometriosis group, and the only difference was no endometrium fragments were flushed into their peritoneal cavity. All pigs remained healthy and did not have any surgery complications during recovery. The pigs were euthanized 15 days after estrus to excise the endometriotic lesions at 1 (n=3) and 6 –7 (n=3) months post induction with matched number of sham group. Uterine tissue was then immediately processed in 10% (vol/vol) paraformaldehyde for histology or immunohistochemistry or snap frozen and stored at −80°C for RNA/protein extraction. FITC Dye-Doped Silica Nanoparticles were synthesized as in the previous study [ 19 ]. Briefly, tetraethyl orthosilicate (TEOS, 99%, Cat #86578), (3-aminopropyl)-triethoxysilane (APTES, 99%, Cat # 440140), Triethylamine (TEA, ≥99% Cat #T0886), and Fluorescein (FITC, Cat 46950) were purchased from Sigma-Aldrich Chemicals (Atlanta, GA, USA). For nanoparticle synthesis, FITC dye-doped silica nanoparticles were prepared in two steps for the synthesis of the silica sphere, followed by the covalent incorporation of FITC on the particles. First, we prepared FITC-APTES solution, where FITC (2 mg) was dissolved in 100 μl dimethylsulfoxide (DMSO). Then, 1 μl of APTES was added, followed by the addition of 0.5 μl of TEA. The mixture was stirred at 30°C in the dark. Next, for the silica synthesis, 1 μl of concentrated ammonia solution (28%), 8.5 ml of absolute ethanol, and 350 μl of TEOS were mixed with 15 μL of FITC dye solution and vigorously stirred at room temperature. After overnight reaction in the dark, the final nanoparticle solution was washed three times with ethanol by centrifugation and dispersed in DI water. To optimize the ex-vivo labeling conditions with FITC dye-doped silica nanoparticles in uterine tissue fragments, we excised approximately 6 cm of uterus from a cycling gilt under anesthesia. The myometrium was removed with scissors and from the uterus which was then cut into small fragments of about 1 mm 3 in a petri dish containing RPMI-1640 media (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). Small fragments were placed into a 24 well plate (5g/well) and then incubated with 0, 20%, 40%, 60% and 80% (v/v) FITC dye-doped silica nanoparticles for 3 h of incubation or 60% (v/v) FITC dye-doped silica nanoparticles for 0, 30, 60, 90 and 120 min of incubation at 37°C in 5% CO 2 . Free-floating nanoparticles were removed by washing 3 times with medium. Induction of autologous endometriosis labeled with FITC dye-doped silica nanoparticles was performed one day post standing estrus. The endometrium was dissected from the myometrium, and 5 g of endometrial tissue was cut into approximately 1 mm 3 tissue fragments in a petri dish using sterile scissors. The tissue fragments were incubated in 60% (v/v) FITC dye-doped silica nanoparticles in a 6-well plate containing 2 ml RPMI-1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). After 90 mins of incubation at 37°C, 5% CO 2 the tissue fragments were flushed back into the pig’s abdominal cavity above the uterus. After 1 month, the pig was euthanized and FITC- positive endometriotic lesions were removed and observed under a fluorescence-dissecting microscope. H&E staining was done to confirm morphology, followed by histological analysis to characterize the endometriotic lesions to show endometrial glands, and stroma. FITC fluorescence signaling from the uterine fragments was detected by IVIS ® Spectrum. FITC signaling intensity was quantified using Living Image ® software (PerkinElmer, Waltham, MA, USA) under fluorescence microscope (Leica Microsystems Inc., IL, USA). To assess statistical significance, we used one-way ANOVA followed by Tukey’s post hoc test for more than two group comparisons. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using the GraphPad Prism 10 (San Diego, CA, USA).

In this study, we developed a preclinical swine model of endometriosis by inoculating autologous endometrial tissue into the peritoneal cavity. Despite important advances in our understanding of endometriosis, its pathogenesis remains unclear, no effective diagnostic tools are yet available, and medical treatments are often incompatible with maintaining fertility. Animal models are essential to facilitate the development of novel pharmacotherapeutics and non-invasive diagnostic tools. Rodent models have been popular for this purpose due to their low cost, ease of use, knowledge of their biology, and their amenability to genetic manipulation. However, translating findings in the rodent models to the clinic may be hindered due to their differences in size and physiology to humans [ 34 ]. For example, clinical treatment of endometriosis is heavily focused on surgical or laparoscopic intervention, and rodent models are not suitable to assess the outcomes of these treatments. Our swine model will compensate for these disadvantages of rodent models due to their compatible size and physiology to humans. Notably, the endometriotic lesions observed in the pig model closely resemble the three types of endometriotic lesions found in endometriosis patients: red (beginning of endometriosis), black (advanced endometriosis), and white (quiescent endometriosis), which represent disease progression [ 30 ]. Endometriosis is a chronic inflammatory disease. Chronic pelvic pain is one of the disabling symptoms of endometriosis, affecting approximately 50-60% of endometriosis patients, and the pain leads to poor quality of life [ 34 ]. Inflammation is thought to contribute to pain sensation through activated immune cells, which produce proinflammatory mediators such as cytokines, chemokines, and prostaglandins [ 35 ]. Cytokines such as IL-1β, IL-6, IL-8, IL-17, TNF-α, and COX2 have been found in endometriotic lesions, peritoneal fluid, and serum [ 35 ]. In contrast, there is an altered expression of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β, which further exacerbates the inflammatory environment [ 35 ]. Moreover, NK cells in peritoneal fluid and peripheral blood from patients with diagnosed endometriosis have reduced cytotoxicity against endometrial cells [ 36 ]. Therefore, pain and inflammation makers need to be further studied in our swine endometriosis model. Although our study focused on the development of a new pig endometriosis model and the basic characterization of endometriotic lesions, the molecular analysis of these lesions was limited. Therefore, further molecular characterization of endometriotic lesions at different sites within the same pig will provide valuable insights into the etiology and pathophysiology of endometriosis. Nonhuman primates share many aspects of reproductive physiology with humans, including spontaneous monthly ovulatory cycles, visible menses, and the development of spontaneous peritoneal endometriosis. However, the identification of spontaneous endometriosis in nonhuman primates involves surgical screening of a large population for the disease with low prevalence [ 37 ]. Nonhuman primates are costly to maintain, and specialized housing is needed [ 38 , 39 ]. Social and economic challenges threaten these centers, and the procurement of new primates is highly scrutinized, thus impacting their availability [ 40 ]. Although nonhuman primates offer several advantages, including close physiological reproductive similarities to humans, they also present economic and ethical challenges as an animal model for endometriosis research. Nonhuman primates are expensive to acquire, house, and maintain, requiring specialized facilities, trained personnel, and veterinary support, which makes studies financially burdensome. In contrast, pigs provide a more cost-effective alternative. They are more widely available than nonhuman primates, whose availability is restricted due to breeding regulations and limited supply [ 41 ]. Additionally, housing and feeding pigs are more affordable, while nonhuman primates require specialized diets and enclosures. Pigs also have a shorter reproductive cycle, allowing for faster experimental turnaround compared to nonhuman primates. Furthermore, their larger litter sizes enable researchers to rapidly multiply particular models, whereas nonhuman primates typically produce only one offspring per pregnancy. Due to these economic advantages, pig models offer a practical and scalable alternative to nonhuman primates for endometriosis research and other biomedical studies. The development of this swine model recapitulates endometriosis should assist us in identifying novel treatment options for this disease. As an important food animal species and a suitable biomedical model, the pig is readily available for medical research. First, pigs are physiologically and anatomically similar to humans, making them a valuable model for studying medical conditions such as cardiovascular disease [ 42 ] and endometriosis. Their comparable body size to humans makes them an attractive model for examining endometriotic lesions over time, allowing for a longitudinal assessment of the disease in the same animal [ 43 ]. This is not possible in rodents due to their small size, as examining endometriotic lesions is typically performed at necropsy [ 43 , 44 ]. Second, pigs exhibit high genetic homology with humans, particularly in genes related to disease susceptibility [ 44 ]; incorporation of pathogenic alleles in humans can successfully recapitulate clinical phenotypes in pigs [ 45 ].Third, the pig’s immune system closely resembles that of humans in more than 80% of analyzed parameters, whereas mice share similarities with humans in less than 10% [ 46 ]. Fourth, female pigs reach sexual maturity earlier than the rhesus macaques (3-4 years) or baboons (4-5 years) [ 47 ]. Their estrus cycles are close to humans, making them a valuable model species to study women’s diseases such as endometriosis, polycystic ovary syndrome, and reproductive health. Endometriosis is a hormone-dependent disease, and the endocrine system of pigs should facilitate them to reflect physiological changes during the onset of endometriosis [ 48 ]. Fifth, due to anatomical alikeness, pigs are commonly used for surgical and medical training [ 46 , 49 ], and clinical imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can be applied to them [ 50 ]. Finally, pigs are ethically more acceptable than nonhuman primates. Not only are pigs suitable for biomedical models for human diseases, but they also offer a higher translational relevance for drug safety and efficacy. Due to the closeness of pigs’ anatomy, physiology and metabolism to humans, they are more drug predictive than rodents [ 46 ] .The pig has become a potential model for human drug metabolism as it has cytochrome P450 enzymes (CYP450), which are important in drug metabolism [ 51 ]. Additionally, as pigs are omnivores like humans, they have similar digestive systems, making them a good model for predicting the oral bioavailability of drugs[ 52 ]. The pig endometriosis model can bridge the gap between small rodent models and nonhuman primates and enhance the possibility to translate animal studies to treat endometriosis in the clinic. Some of the key differences between women and pigs’ reproductive cycle is that in the pigs the cycle is shorter as it is 21 days compared to women which is longer at 28 days [ 28 ]. During the reproductive cycle, women release one egg per cycle compared to pigs that can release multiple eggs which enable them to have large litters [ 26 , 53 ]. Moreover, women’s reproductive cycle undergoes sloughing of the endometrium (menstruation) which the pig does not have as it undergoes reabsorption [ 28 ]. These differences in reproductive cycle do not deter the establishment and growth of endometriotic lesions, as they are maintained by the dynamic changes of estrogen and progesterone which are present in the pigs as in humans [ 48 ]. A disadvantage of the porcine animal model has been the limited availability of research centers that house and manage swine. In recognition of this, the US National Institutes of Health has established the National Swine Resource and Research Centers to support translational swine research [ 54 ]. Our unique expertise in pig models should be combined with genetically engineered techniques to accelerate endometriosis studies. Understanding the pathophysiology of endometriosis with preclinical animal models requires easy assays to distinguish endometriotic lesions from surrounding normal tissues. With this in mind, the donor tissues were labeled with fluorescein isothiocyanate dye-doped silica nanoparticles. Over the years, nanotechnology has revolutionized medicine by enhancing the accuracy and sensitivity of diagnostic tests and by facilitating the development of targeted therapeutic interventions. In this study, we have successfully employed nanotechnology to fluorescently label endometriotic lesions as a proof of concept for the use of nanotechnology in endometriosis research. Interest in the use of diagnostic imaging for the detection of endometriosis has increased over time. Nanotechnology holds promise for augmenting the sensitivity of diagnostic imaging by employing lesion-specific labeling in vivo . The availability of sensitive and specific imaging data could provide the means to accurately detect and quantify the presence of endometriosis, guide surgical planning, and evaluate the clinical response to therapeutic interventions. Nanoparticles (NPs), such as silica nanoparticles, have now entered clinical trials for various biomedical applications, including diagnostics and drug delivery. NPs can be formulated in different ways and administered via multiple routes, including intravenous, intertumoral, intraperitoneal, retro-orbital, intranasal, and topical delivery [ 55 ]. With nanoparticle use advancing to clinical trials, these technologies hold promise for enhancing the sensitivity of diagnostic imaging by employing lesion-specific labeling in vivo. In a recent study done by Lin et al, they designed a nanoparticle modified with hyaluronic acid, which can specifically bid to CD44, an overexpressed protein in endometriotic lesions [ 56 ]. Using this nanoparticle they successfully observed endometriotic lesions through photoacoustic imaging [ 56 ]. For our future studies, we aim to apply this emerging biomedical technique in our swine model to further explore their potential applications. In summary, we report our success in inducing endometriosis in swine through peritoneal seeding of prepared endometrial fragments. We have also demonstrated that nanoparticle labeling of prepared endometrial fragments can result in an enhanced ability to identify both large and small fluorescent labeled endometriotic lesions at the time of surgical retrieval. Development of this swine endometriosis model should offer a novel tool to expand our understanding of the pathogenesis of endometriosis and facilitate testing of novel therapeutics and the development of sensitive and specific diagnostic imaging.

Endometriosis is a benign gynecological disease that affects ~10% of reproductive age women; it is estimated 190 million women may be affected worldwide [ 1 ]. It is characterized by the presence of endometrial glands and stroma located outside of uterus and it is associated with varying degrees of debilitating symptoms including pain and infertility [ 2 , 3 ]. Endometriosis not only affects a patient’s quality of life, but it also imposes a financial burden, costing up to $22 billion in medical care and lost wages each year in the United States [ 4 ]. Varying prevalence rates of endometriosis have been reported for women with infertility, with the overall prevalence approximating 50% [ 5 ]. Although laparoscopic excision or ablation of lesions and menstrual cycle suppression may improve symptoms, definitive treatments other than hysterectomy remain to be discovered. Currently available therapeutics focus on treating symptoms of endometriosis to patients without an affirmative diagnosis [ 6 ]. There is a delay in diagnosis which ranges between 4-11 years from the onset of symptoms until laparoscopic confirmation [ 7 ]. Definitive diagnosis of the disease requires direct surgical visualization because effective non-invasive testing is not clinically available [ 8 ]. At the time of surgical diagnosis, excised endometriotic lesions are confirmed with histopathology. Treating endometriosis with surgical intervention is associated with rare complications including bowel injuries, urological injuries, peritonitis, and unplanned hysterectomy [ 9 ]. Furthermore, recurrence of endometriosis is common after surgery or hormonal treatment [ 10 ]. Endometriotic lesions are commonly found in the gravity-dependent portions of the pelvis, and the observation has led to the “Sampson’s Theory” which asserts peritoneal adherence of endometrial particles from retrograde menstruation being the primary cause of the disease [ 11 ]. Today endometriosis is known to be a pleiomorphic condition with a wide range of pathological manifestations (superficial, ovarian, deep infiltrating, scar-associated, extraperitoneal, etc.), and the mechanisms of its pathogenesis are still poorly understood. Dissecting pathogenesis of endometriosis is difficult to be achieved in the clinic due to ethical constraints. Alternatively, disease models are used to understand pathogenesis of endometriosis and to aid in the development of therapeutic tools. Most research models are either in vivo animal models or in vitro cell or tissue models. In vitro studies aid in the understanding of cellular and molecular mechanisms while in vivo animal models allow for the evaluation of functional outcomes like fertility and pain [ 12 , 13 ]. Animal models used to study endometriosis include syngeneic and autologous rodent models, xenotransplantation of human endometrium into immunodeficient mice, and nonhuman primate models, which are capable of developing spontaneous as well as experimentally induced endometriosis [ 14 – 16 ]. Mouse models have proven to be useful because of their accessibility, cost-effectiveness, small size, high breeding rate and amenability to genetic manipulation [ 17 , 18 ]. For instance, a murine model expressing transgenic fluorescent reporters and ex vivo endometrial Cy5.5 dye-dope silica nanoparticle labeling can facilitate the identification of endometriotic lesions in vivo [ 19 , 20 ]. Advantages of murine endometriosis models are offset by the animal’s small size and physiological dissimilarity to humans; mice do not menstruate or develop spontaneous endometriosis. These gaps have been partially filled by nonhuman primate models including baboons ( Papio sp. ) and rhesus macaques ( Macaca mulatta ), which have provided important contributions to our understanding of endometriosis pathophysiology and responses to novel therapeutics [ 21 , 22 ]. However, the use of nonhuman primates for the study of endometriosis is both expensive and restrictive [ 14 , 23 ]. Moreover, there has been a progressive decline in primate populations as human activity has contributed to the disappearance of natural primate habitat [ 24 ]. Swine have been suggested as a suitable preclinical model in understanding different human physiologies because of their anatomical and physiological similarities to humans [ 25 ]. The pig has an estrous cycle which is approximately 21 days in length. During proestrus, developing and growing follicles produce estradiol (E2) stimulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [ 26 , 27 ]. During metestrus, E2 and LH levels decrease but luteinization of the corpus luteum (CL) results in increasing production of progesterone (P4). Without embryonic signals in the uterus, prostaglandin F2 alpha is released causing luteolysis of the CL [ 26 , 28 ]. Loss of the CL and rapid decline of progesterone of the estrous cycle allows the follicular growth to initiate a return to estrus on day 21. The reproductive cycle of pigs is compatible to the menstrual cycle as ovulation occurs between the follicular and luteal phase, spontaneous ovulation happens, and their cyclicity is continuous and under the control of the hypothalamic-pituitary-ovarian-axis [ 28 ]. Pigs do not present sloughing of the endometrium (menstruation) [ 28 ]; therefore, we induced endometriosis one day after standing estrus to mirror retrograde of endometrial tissues during menstruation. A previous report implicates successful induction of endometriosis in pigs is feasible and a proper model to visualize endometriosis lesions using laparoscopic approach and instruments for diagnosing and treating the condition [ 29 ]. As a food animal species, they are easily accessible to perform studies that require a large group of animals. A porcine endometriosis model may contribute to our understanding of disease pathophysiology, and aid in the development of non-invasive clinical diagnostic tools, such as imaging nanoparticle-tagged lesions with high sensitivity and specificity. In this study, we successfully induced endometriosis in adult female pigs by seeding the peritoneum with autologous endometrial fragments. Building on our previous experience with nanoparticle labeling [ 17 ], we incorporated endometriosis tissue labeling and tracking using nanoparticle-fixed fluorescein isothiocyanate (FITC) dye in order to more easily identify lesions. Histological evaluation of these lesions demonstrated close similarity to human endometriosis.