Optimizing a Translational Mouse Model of Endometriosis

Optimizing a Translational Mouse Model of Endometriosis | 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 Optimizing a Translational Mouse Model of Endometriosis Christina Ann Howe, John Coté, Catherine Stoos, Marley Bredehoeft, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3243174/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 Improved animal models of endometriosis are needed to accurately represent the pathophysiology of human disease and identify new therapeutic targets that do not compromise fertility. Current mouse models of endometriosis that involve ovariohysterectomy and hormone replacement preclude evaluation of fertility. Menstrual phase endometrium includes potentially important immune cells and inflammatory mediators. Our goal was to develop a novel, translationally relevant mouse model of endometriosis by transplanting donor menstrual endometrium into the peritoneal cavity of menstruating, immunocompetent, intact recipients. We tested various paradigms to determine the most effective method for establishing endometriotic lesions. Initially, 4 paradigms were tested to optimize method of induction. To enhance the model further, a novel paradigm implanted discrete menstrual phase endometrium via laparoscopy into menstruating mice. Vaginal cytology was performed to confirm continued estrus cyclicity. Potential lesions were harvested during proestrus and confirmed to be endometriosis based on histopathology. All mice demonstrated normal estrus cyclicity post induction. Incidence of endometriosis and the difference in average number of lesions across groups was compared. The use of laparoscopy to place discrete menstrual phase endometrium was the most effective method of induction of endometriosis. This method was just as effective when used to induce endometriosis in menstruating recipient mice, representing a novel translationally relevant model that can be used to assess immunologic factors and the impact of therapeutic interventions on fertility. endometriosis murine translational preclinical animal model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Endometriosis is an estrogen-dependent gynecological disease that affects an estimated 10–15% of women of reproductive age [ 1 ]. It is characterized by endometrial tissue (glands and stroma) proliferating in ectopic locations, and is associated with dysmenorrhea, chronic pelvic pain, dyspareunia, infertility, and increased risk of ovarian cancer [ 2 ]. The painful symptoms of endometriosis substantially affect quality of life and productivity. The estimated societal burden (direct and indirect costs) of endometriosis was $ 4572-12,079 (USD) per patient per year in 2022 [ 3 ]. Most medical therapies are variably effective and cannot be used long-term, and surgical treatment has a high recurrence rate or results in infertility in the case of hysterectomy [ 4 ]. Challenges with diagnosis and treatment stem from that fact that a definitive etiology and pathogenesis of endometriosis is unknown. Endometriosis is theorized to develop either from uterine endometrium or from extra-uterine tissue. Extra- uterine origin theories include: coelomic metaplasia of peritoneal cells, embryonic Müllerian rest theory, and extra-uterine stem cells originating from bone marrow [ 2 ]. Retrograde menstruation into the abdominal cavity via the fallopian tubes is the most widely accepted hypothesis for the implantation of uterine endometrial tissues to extrauterine locations [ 5 ]. In fact, retrograde menstruation is a common physiologic finding in menstruating women with patent fallopian tubes [ 6 ]. Benign metastasis via hematogenous or lymphatic spread is another proposed theory of endometriosis arising from uterine endometrial tissue [ 7 , 8 ]. Regardless of origin, ectopic endometrial implants must escape immune clearance, invade, and attach to ectopic locations, proliferate, neovascularize and survive [ 2 , 9 ]. Investigation of these factors requires an appropriate translational animal model that recapitulates the human disease process. Naturally occurring endometriosis in animal species other than humans only occurs in some non-human primates at a very low incidence rate [ 10 ]. While endometrial lesions in non-human primates compared to women are histologically identical and located in similar sites, the use of non-human primates as an animal model comes with the disadvantages of high costs, need for special infrastructure and training, lack of availability and ethical considerations [ 11 – 13 ]. Additionally, there is a paucity of species that menstruate naturally: humans, some higher order non-human primates, the elephant shrew, some species of bats and the spiny mouse [ 14 – 18 ], none of which are as optimized as mice for use as a preclinical model. Mice are well characterized, cost-effective, prolific, and allow for genetic manipulations. Cummings et al, described the first murine model of endometriosis in 1995 [ 19 ], and although many iterations of murine models have followed, they lack translational relevance. Rodent models of endometriosis that involve ovariohysterectomy and constant levels of hormone replacement are impractical for studies of physiological functions that require natural fluctuations in sex hormones, such as fertility. The use of immunocompromised mice with xenotransplantation of human tissue, preclude the assessment of the role of immune cells in pathogenesis. Models that use full thickness uterine implants include myometrium and serosal layers that are not biologically inert and are not present in human endometrial lesions. Finally, models that implant endometrial into extra-abdominal locations eliminate the assessment of the peritoneal environment. A hypothesized “best fit” model of murine endometriosis has been described, one that uses immunocompetent mice with intact ovaries to approximate spontaneous menstrual endometrial attachment in the peritoneal cavity, allows for longitudinal assessment of lesions and ideally iterative endometrial transplantation [ 20 ]. Mice do not menstruate naturally and have a closed tubo-ovarian junction resulting in the need for significant manipulation to model the theory of uterine origin of ectopic endometrial lesions due to retrograde menstruation. An effective model of mouse menstruation mimicking the human menstrual cycle in a pseudo-pregnant mouse is well established. Pseudo-pregnant menstruating mice display similar gene expression, overt vaginal bleeding, and histologic changes when compared to humans, with the uterus also displaying reepithelization of the luminal endometrial surface [ 21 ]. Using this method of induction of menstruation, menstrual endometrial implants must be placed into the peritoneal cavity. Published methods include surgical engraftment of discrete tissue pieces, and intra-peritoneal injection or surgical injection of endometrial slurry. Surgical engraftment using suture or adhesives introduces variability due to their effects on tissues and bypasses natural attachment of tissues, one of the early factors of the disease that needs elucidation. Laparoscopic implantation of biopsies described and developed by Peterse et al. allows for adhesion of implants to peritoneal surfaces without the need for suture or artificial adhesives [ 22 ]. Both injection techniques more closely mimic true retrograde menstruation and allow for study of early disease stages, but the number of lesions generated is variable and tissue slurry composition fluctuates based on technique. In this study, we compared 5 different paradigms of induction of endometriosis in immunocompetent, intact mice. Initially, 4 paradigms were used to optimize the tissue type and method of induction. Menstrual endometrium harvested day 9.5 post vaginal plug from B6(Cg)-Tyrc-2JTg(UBC-mCherry)1Phbs/J mice was implanted intraperitoneally into naïve B6(Cg)- Tyr c−2J mice using discrete implants via (1) laparoscopy or (2) laparotomy or tissue slurry injection via (3) laparoscopy, or (4) laparotomy. To optimize the model further, a novel paradigm (5) used discrete implants via laparoscopy into menstruating B6(Cg)- Tyr c−2J mice. Using syngeneic, immunocompetent, intact, menstruating donor and recipient mice, we have optimized a minimally invasive, laparoscopic surgical model of implantation of discrete endometrial biopsies. This novel, translationally relevant murine model addresses the majority of recommendations in the “best fit” model. Materials and Methods Animals, housing conditions, diet . All procedures were performed at Creighton University, an AAALAC-accredited facility. This study was performed in accordance with the Guide for Care and Use of Laboratory Animals and was approved by Creighton University’s Institutional Animal Care and Use Committee. All mice were maintained in environmentally controlled rooms (20–26°C; humidity 30–55%) with diurnal lighting (12:12-h light:dark cycle; light on, 0700). 8 ± 8-week old female B6(Cg)- Tyr c− 2J Tg(UBC-mCherry)1Phbs/J (stock #017614) were used as donor mice and female B6(Cg)- Tyr c− 2J /J (stock # 000058) were used as recipient mice. Hereafter, these strains will be referred to as mCherry for B6(Cg)- Tyr c− 2J Tg(UBC-mCherry)1Phbs/J and B6 for B6(Cg)- Tyr c− 2J /J Mice were maintained in individually ventilated cages on 7099 TEK-Fresh (paper) bedding and fed soy protein-free extruded autoclavable diet 2020SX (Teklad Global Rodent Diets®) ad libitum. to decrease exposure to any exogenous estrogens (i.e., the mycotoxin zearalenone in corncob bedding, and/or phytoestrogens in soy based rodent diets) [ 23 – 25 ]. Environmental enrichment was provided as 8-grams of nesting material (Enviro-dri® Shepherd Specialty Paper, Watertown, TN, and Rodent Nesting Sheets ™, Bio-Serv, Flemington, NJ), wood gnawing block, mouse trapeze and mouse tunnel (Bio-Serv, Flemington, NJ). Female mice were group housed until day of vaginal plug, and then single housed until sacrifice to avoid Lee-Boot effect [ 26 ]. Vasectomized male mice were obtained from JAX and were single housed on corncob bedding and fed Teklad global 18% protein 2018S ad libitum . Induction of Menstruation/Decidualization Induction of menstruation was performed using the Rudolph et. al. protocol with modifications [ 21 ]. Naïve donor mCherry (Paradigms 1–5) and B6 recipient female mice (Paradigm 5) were placed with vasectomized males overnight (day 0) and checked for the presence of a vaginal plug the next morning (day 0.5). Females with a confirmed vaginal plug were individually housed. On day 4.5 pseudo-pregnant mice were anesthetized using isoflurane, and decidualization was induced by intrauterine injection of 100 µl sterile sesame oil using a blunt 27G x0.86” needle (ALZET®) followed by mechanical stimulation. Sustained release buprenorphine was administered post procedure. Menstruation was confirmed using vaginal cytology and/or the visual presence of overt vaginal bleeding (Fig. 1 ). Induction of Endometriosis On day 7.5–9.5 decidualized/menstruating donor mCherry mice were anesthetized with isoflurane, blood collected via submandibular bleed using a Goldenrod animal bleeding lancet (5mm), and cervically dislocated. The uterus was removed en bloc using sterile technique, placed in sterile cold (4 ◦ C) PBS (0.01 M PBS composed of 13.7 mM NaCl, 0.27mM KCl, 0.15mM KH 2 PO 4 and 0.8mM Na 2 HPO 4 ; pH 7.4). Each uterine horn was opened longitudinally and decidualized endometrium was dissected from the myometrium, then sectioned into approximately 2 mm 3 discrete biopsies. 10 biopsies were implanted into recipient mice in paradigms 1, 2, and 5. For paradigms 3 and 4, endometrial slurry was made using the Greaves et. al. protocol with modifications [ 27 ]. 10 decidualized endometrial discrete biopsies were weighed, minced, and mixed with 0.2 ml of sterile saline (0.9% NaCl). This slurry was then drawn into a syringe through a 19 G hypodermic needle to ensure uniform suspension. The average wet weight of endometrium implanted was 94.16 mg per recipient mouse. Paradigm 1–4 used intact B6 recipient mice (n = 10). Paradigm 5 used decidualized/menstruating B6 recipients (n = 10) that were time-matched for date of vaginal plug and decidualization with donor decidualized/menstruating mCherry mice. On day 9.5 post vaginal plug, donor mCherry mice were sacrificed and endometrial tissue harvested as described and implanted into the decidualized/menstruating B6 recipients (Fig. 2 ). For all 5 paradigms, recipient B6 mice were weighed, and anesthetized with ketamine (87.5mg/kg) and xylazine (12.5mg/kg) given intraperitoneally. Hair was removed from the abdomen using depilatory cream (Nair ®) and heat support was provided using a Homeothermic Monitoring System (Harvard Apparatus, Holliston, MA). Stage of estrus of recipient was determined via cytological evaluation of vaginal smears for paradigms 1–4. For paradigm 5, decidualization/menstruation of recipient B6 mice was confirmed via cytological evaluation of vaginal smears and/or presence of overt vaginal bleeding. If laparoscope was used, mice were intubated using a 22G x 1 inch IV catheter (Pivetal®) and ventilated with Minivent (Model 845, Harvard Apparatus, Holliston, MA) with stroke volume calculated at 10µl/kg and stroke rate of 200 strokes/minute. Sustained release buprenorphine was administered post procedure for analgesia. Laparotomy (Paradigms 2 and 4) A 5 mm cranial ventral midline incision was made caudal to the xiphoid process. The discrete endometrial biopsies or endometrial slurry were placed intra-abdominally through this incision. The incision was closed using 5 − 0 polyglactin 910 suture (Patterson Veterinary, Loveland, CO). Laparoscopy (Paradigms 1, 3 and 5) Laparoscopic guided implantation of decidualized endometrial biopsies was performed using the Peterse et. al. protocol with modifications [ 22 ]. A 5 mm cranial ventral midline incision was made caudal to the xiphoid process. A 2 mm endoscope covered with a 3 mm insufflation shield (Stryker, Kalamazoo, MI) was placed into the peritoneal cavity. The abdomen was insufflated with warmed CO 2 at a flow of 1L/minute to maintain an intra-abdominal pressure of 5mm Hg. For implantation of discrete endometrial biopsies, a 14 G intravenous catheter (SurFlash™. Patterson Veterinary, Loveland, CO) was inserted into the right lower abdominal quadrant to serve as a port. The biopsies were placed into the tip of the catheter and advanced into the abdomen using a blunt stylet. For implantation of endometrial slurry, the suspension was injected intraperitoneally with a 19G needle through the right lower abdominal quadrant. The incision and port opening were closed using 5 − 0 polyglactin 910 suture (Patterson Veterinary, Loveland, CO) (Fig. 3 ). Lesion Harvest Recipient mice were followed with cytologic evaluation of vaginal smears post-operatively to confirm return to estrus cyclicity. Mice were sacrificed in proestrus at least 30 days post return to normal estrus cyclicity. Mice were anesthetized with isoflurane, blood collected via submandibular bleed using a Goldenrod animal bleeding lancet (5mm), and cervically dislocated. The abdominopelvic cavity was closely examined for endometriosis-like lesions; number and location of each lesion was recorded, along with a detailed description of gross appearance. Lesions were photographed, excised from surrounding tissue, and immediately fixed in 10% neutral-buffered formalin. Endometriosis was induced in an additional 10 decidualized/menstruating recipient mice using paradigm 5 methodology. These mice were followed for 60 and 90 days (n = 5 per timepoint) post return to normal estrus cyclicity and sacrificed as described. Histological assessment Lesions were processed in the Creighton University Histology Core Facility using the Excelsior ES Tissue Processor (Thermo Fisher Scientific, Waltham, MA) and embedded in paraffin. Tissue sections (5 microns) were cut on a Leica RM 2135 microtome (Leica Biosystems, Deer Park, IL) and placed on Fisherbrand™ Superfrost™ Plus Microscope Slides ( Thermo Fisher Scientific, Waltham, MA). The Gemini Auto Stainer was used for hematoxylin and eosin (H&E) staining. Lesion sections were evaluated by histopathologist in a blinded fashion. Immunohistochemistry Protocol CD31/Ki67/Cd68 Immunohistochemical staining was performed on a Discovery Ultra advanced staining system (Roche Diagnostics, Ventana Medical Systems, Inc.). Sections were deparaffinized using a mild detergent solution and vortex mixing at 69°C for 24 min (cat no. 950 − 102, Roche Diagnostics). Tris based reaction buffer, pH 7.6, used throughout the protocol to maintain aqueous conditions and rinse slides (cat no. 950 − 300, Roche Diagnostics). Antigen retrieval by heat, cell conditioning CC1, 8.2 pH tris-borate-EDTA buffer, at 95°C for 24 min (cat no. 950 − 124, Roche Diagnostics). Treated with Discovery ChromoMap RUO Inhibitor for 8 min (cat no. 760 − 159, Roche Diagnostics). Primary antibody for CD31, rabbit polyclonal, 1:100, incubation at 37°C for 44 min (cat no. ab28364, Abcam), primary antibody for Ki67, rabbit polyclonal, 1:200, incubation at 37°C for 32 min (cat no. ab16667, Abcam) or primary antibody for CD68, rabbit polyclonal, 1:200, incubation at 37°C for 32 min (cat no. ab125212, ABCAM). Secondary antibody HRP detection, Discovery anti-rabbit HQ RTU, incubation at 37°C for 16 min (cat no. 760–4815, Roche Diagnostics). Enzyme conjugate biotin-free Discovery anti-HQ horse-radish peroxidase RTU, incubation at 37°C for 16 min (cat no. 760–4820, Roche Diagnostics). Chromogen staining with Discovery Purple Kit RUO for 32 min and counterstained with Hematoxylin (cat no. 760 − 229, 790–2208 & 760–2037, Roche Diagnostics). Toluidine Blue Staining for Mast Cells Sections were stained with Toluidine blue resulting in red-purple (metachromatic staining) mast cells on a blue background. Statistical Analysis Endometriosis Incidence In order to test for differences in the incidence of endometriosis among groups, we first conducted a 2(yes/no) x 5(induction method) omnibus Fisher’s exact test, followed by individual 2 x 2 Fisher’s exact tests. Confirmed Endometrial Lesions To test for differences in confirmed lesions among different endometriosis induction methods, we used a generalized linear model with a Poisson distribution and a log link function using the glm function in R (R Core Team, 2022). Significant differences are defined as p < .05, and marginal differences are defined as p < .10. To test for differences in the presence of lesions on the uterus as well as the presence of glands in confirmed lesions, we conducted Fisher’s exact tests comparing Paradigm 1 (Laparoscopy and endometrial biopsies) with Paradigm 5 (Laparoscopy and endometrial biopsies in decidualized, menstruating recipients). Results Induction of Menstruation/Decidualization We achieved successful decidualization in 34/44 (77.27% ±12.38%, 95% CI) of mCherry mice. We considered decidualization successful if enough tissue could be harvested to implant into 1 recipient mouse. In general, one donor mouse provided enough tissue to implant into 2 recipient mice. decidualization occurred unilaterally, bilaterally, and segmentally throughout the uterine horns. In the 10/44 (22% ±12.38%, 95% CI) mice that failed to decidualize, the uterus was either normal in appearance, or the decidualization was incomplete and the endometrium could not be adequately separated from the myometrium. Estrus Cyclicity Post Surgery All recipient mice returned to normal estrus cyclicity post-surgery. B6 menstruating recipient mice in paradigm 5 returned to estrus cyclicity between 3–14 days (mean = 6.8 days, SD = 3.4, SEM = 1.07, 95% CI(4.70, 8.90)) post operatively. Endometriosis Incidence The incidence of endometriosis differed across induction methods (p < .001, Table 1 ). Post hoc Fisher’s exact tests showed that Paradigm 1 produced higher rates of endometriosis than Paradigms 3–4 (p .47). For all five paradigms, the average days of disease from day of induction to lesion harvest was 34.85 days (SD = .05, SEM = 1.07, 95% CI (34.45, 35.25)). We induced endometriosis at a rate of 100% in paradigms 1 and 5. (Table 1 ) Endometriosis was also confirmed in 100% of recipient mice (paradigm 5 methodology) at 60 and 90 days. Table 1 Individual mouse data per paradigm Paradigm Recipient estrus stage Donor sacrifice days post vaginal plug Days of Disease Individual Take Rate a , % Mean counts of confirmed lesions b Endometriosis Incidence c , % 1: Laparoscopy w/discrete endometrial biopsies Estrus Estrus Estrus Estrus Estrus Estrus Estrus Proestrus Diestrus Metestrus 8.5 8.5 9.5 9.5 10.5 9.5 9.5 8.5 9.5 9.5 41 42 36 34 36 34 34 35 33 34 20 (2/10) 40 (4/10) 20 (2/10) 30 (3/10) 25 (2/8) 20 (2/10) 20 (2/10) 10 (1/10) 60 (6/10) 30 (3/10) 2.7 100 2: Laparotomy w/discrete endometrial biopsies Proestrus Estrus Proestrus Proestrus Proestrus Proestrus Estrus Estrus Estrus Estrus 9.5 9.5 9.5 9.5 9.5 9.5 9.5 9.5 9.5 9.5 35 35 36 35 34 35 34 35 37 37 10 (1/10) 10 (1/10) 10 (1/10) 0 (0/10) 20 (2/10) 0 (0/10) 2 (2/10) 10 (1/10) 10 (1/10) 20 (2/10) 1.1 80 5: Laparoscopy w/ discrete endometrial biopsies in menstruating recipients Menstruating 9.5 35 35 35 44 36 20 (2/10) 40 (4/10) 20 (2/10) 30 (3/10) 30 (3/10) 20 (2/10) 40 (4/10) 20 (2/10) 40 (4/10) 10 (1/10) 2.6 100 Individual Lesion Count d 3: Laparoscopy w/ endometrial slurry Estrus Estrus Estrus Estrus Proestrus Diestrus Diestrus Diestrus Diestrus Diestrus 8.5 8.5 8.5 8.5 8.5 8.5 9.5 9.5 9.5 8.5 36 34 33 33 36 32 32 35 34 34 1 1 0 0 0 0 0 0 0 0 0.2 20 4: Laparotomy w/ endometrial slurry Estrus Estrus Estrus Estrus Estrus Estrus Estrus Proestrus Diestrus Diestrus 8.5 8.5 8.5 8.5 9.5 9.5 9.5 8.5 8.5 8.5 34 35 38 5 34 35 33 33 33 33 1 0 0 0 0 0 0 0 0 0 0.1 10 Average days of Disease 34.85 a Individual take rate = number confirmed lesions/number of implanted biopsies x 100, expressed as a percentage b Mean counts of confirmed lesions per mouse c Endometriosis incidence per paradigm, expressed as percentage of animals (n = 10) per paradigm that had confirmed endometriosis lesion(s) histologically. d Individual confirmed endometrial lesion count(s) for slurry implanted animals Histological Confirmation The method of endometriosis induction significantly impacted the presence of confirmed (χ 2 = 55.16, df = 4, p < .001; Fig. 4 ) lesions. Paradigms 1 (mean = 2.7 lesions, SD = 1.4, SE = 0.45, 95% CI) and 5 (mean = 2.6 lesions, SD = 1.17, SE = 0.37, 95% CI) produced significantly more confirmed lesions than Paradigms 3 (mean = 0.2 lesions, SD 0.42, SE = 0.13, 95% CI) and 4 (mean = 0.1 lesion, SD 0.32, SE 0.1, 95% CI). Paradigm 1 also produced marginally more confirmed lesions than Paradigm 2 (mean = 1.1 lesions, SD 0.74, SE 0.23, 95% CI) (p = .088). Endometriosis was confirmed by the presence of stroma, and hemosiderin histologically(Figs. 5 and 6 ). In some lesions, glands, epithelium, and calcifications were also present. Most endometrial lesions were found on skeletal muscle (body wall), adipose tissue, mesentery, on/near the bladder and on broad ligaments of uterine horns. Lesions on the uterine serosal surface and lesions with glands were found in paradigms 1 and 5, however, we found no evidence for differences in the number of animals developing lesions in the uterus between paradigm 1 and 5 (1/9 vs 5/5; p = .141), nor did we find evidence for differences in the number of animals developing lesions with glands between Paradigm 1 and 5 (2/8 vs 5/5, p = .350). Immunohistochemistry Immunohistochemical staining performed on confirmed lesions indicated the presence of angiogenesis (CD31), proliferation (Ki67), mast cells (toluidine blue) and macrophages (Cd68) (Fig. 7 ). Discussion In this study, we developed and optimized a translationally relevant preclinical murine model of endometriosis by analyzing, adapting, and modifying previously published methods [ 21 , 22 , 27 ]. Currently, there is no standardized preclinical animal model for endometriosis. A murine model is ideal due to size, fecundity, and amenability to genetic manipulation. We used immunocompetent, intact mice with implantation of menstrual phase endometrium that spontaneously attached. Additionally, we felt implantation into menstruating mice improved model fidelity. We used soy-free diet and paper bedding to limit exposure to exogenous estrogens (i.e., the mycotoxin zearalenone in corncob bedding, and/or phytoestrogens in soy based rodent diets)[ 23 – 25 ] because supplemental estrogen has been shown to promote endometrial lesion growth in rats and mice [ 19 , 28 ]. Future studies examining the effects of exogenous estrogens or endocrine disrupters should consider feed, bedding, and caging choice. We selected mCherry donor mice to allow longitudinal, non-invasive monitoring of developing endometrial lesions in recipient B6 females using fluorescent imaging (IVIS Lumina XR®), but resolution was inadequate with a low signal to background ratio. This differed from the success reported using non-invasive fluorescent monitoring with endometrial tissue labeled with mCherry adenoviral vectors (AAV) [ 29 ]. However, in that study, the strongest, most reliable signal was generated from endometrial tissue implanted subcutaneously on the ventral abdomen compared to intraperitoneal implantation, the more translationally relevant location. Additionally, the endpoint was only 20 days, and the use of the AAV vector would increase signal compared to the transgenic mouse alone. Immunohistochemical staining of harvested endometrial lesions demonstrated angiogenesis (CD31) and proliferation (Ki67). The average number of days of disease was 34.85, with confirmed endometriotic lesions also harvested at 60 and 90 days post implantation indicating prolonged survival with this model. With optimization of imaging and the use of transgenic strains expressing fluorophores, non-invasive longitudinal assessment is possible. All menstruating recipient mice returned to normal estrus cyclicity. This allowed the implanted menstrual phase endometrium to undergo cyclical hormonal exposure to estrogen and progesterone, a more translational model than supplementation with supraphysiologic doses of estrogen and/or progesterone in ovariectomized mice. All mice were sacrificed in proestrus, to decrease variability of lesion size and appearance. Infertility is a common complaint of women suffering from endometriosis and 30–50% of women with endometriosis seek care due to infertility alone [ 30 ]. With the use of intact mice, future studies can examine fertility in recipient mice. Pathologists have classically defined endometriosis in women as the presence of endometroid glands and stroma in biopsied lesions. Paradigm 1 had 20% (2/10) of mice and paradigm 5 had 50% (5/10) of mice with confirmed lesions containing glands. Paradigms 2, 3 and 4 had mice with lesions that only contained stroma and hemosiderin. Although there was no significant difference detected between paradigm 1 and 5 for lesions with glands or uterine lesions, increased group size may allow for detection of differences. Stromal endometriosis in women is characterized as small, microscopic nodules or plaques of endometrioid-type stroma often containing arteriole-like vascular channels, hemosiderin pigment, inflammatory cells, microcalcifications and reactive mesothelial proliferation. It is a common form of endometriosis and can occur with or without the presence of typical endometriosis [ 31 ]. Many of the confirmed lesions in this study demonstrated one or more of these characteristics. Additionally, we evaluated lesions at a much earlier timepoint of disease in the mouse (approximately 3 human equivalent years), compared to histological evaluation of suspected lesions in women, as women are typically diagnosed an average of 8 years after symptoms appear. There is tremendous heterogeneity in endometriosis lesions, and evidence of ordered progression is lacking. It is possible that stromal endometriosis is an earlier phenotype of the disease or is a result of a type of metastatic process. Use of an appropriate mouse model, displaying the potential for prolonged lesion survival (90 days) would allow for further evaluation of lesion progression, the role mesothelial inflammation and proliferation plays, and better characterization of endometriosis phenotypes. In addition to mCherry donor + B6 recipient mice, we have successfully induced endometriosis using decidualized/menstruating B6(Cg)- Tyr c−2J /J or B6.129S6- Gper1 tm1Cwan /J females as both donors and recipients (unpublished). It is critical that this model is evaluated in other widely used strains of mice, as differences in endometrial lesion phenotype have been demonstrated between C57BL/6 and BALB/c mice [ 32 ] and inbred mouse lines can differ in their immunologic and inflammatory pathways. Immune cell dysfunction in endometriosis is associated with impaired phagocytosis/clearance of menstrual debris, increased survival/proliferation of ectopic tissue, neuro-angiogenesis, and pain. Macrophages (Mφ) are the most abundant immune cells present in endometriotic lesions, and, in response to estrogen, adopt a ‘wound-healing’ (anti-inflammatory) M2 phenotype compatible with survival and growth of ectopic tissue [ 33 ]. Mast cells (MC) are also present, and the MC stabilizer ketotifen effectively reduces hyperalgesia in a rat model of endometriosis, indicating MC may play a role in endometriosis pain [ 34 ]. Ovarian cycle, estradiol, neuroimmune/neuroendocrine factors, and a nervous system responsive to hormones all play a role in the pain a woman with endometriosis experiences [ 35 ]. The use of an ovariectomized and/or immunodeficient mouse would preclude the study of these factors in a mouse model. Immunohistochemical staining of confirmed endometriotic lesions revealed the presence of both macrophages (Cd68) and mast cells (toluidine blue) within or in close proximity to ectopic endometrial tissue, validating this model for use in future studies of these immune cells and for evaluation of pain and pain modulation. We found confirmed lesions in translationally relevant locations such as the bladder, uterine ligaments, and serosal surface of the uterus (5/10 recipients in paradigm 5). Lesions in women are generally found in gravity dependent areas such as the posterior cul de sac [ 2 ] and consistent with the quadruped nature of the mouse, most lesions were located on the ventral aspect of serosal surfaces. Although the ovaries are a common site of endometriosis in women, no lesions were found on the ovaries in recipient mice. This is not surprising as mice have an ovarian bursa and closed tubo-ovarial junction and there is no bursa surrounding the ovaries in women [ 36 ]. The closed tubo-ovarial junction also precludes the study of spontaneous endometriosis development due to retrograde menstruation in a decidualized mouse, thus requiring the use of both a donor and recipient mouse and a surgical or injectable method of implantation. Many lesions were attached to adipose tissue. The significance of this needs further exploration, as there is a strong relationship between estrogen and adipose tissue in women [ 37 ] and endometriosis is associated with lean body mass index and low waist-to-hip ratio [ 1 ]. Additionally, higher levels of gene expression characteristic of M2 phenotype macrophages are associated with adipose tissue in lean mice [ 38 ], and endometriosis alters adipose stem cell population and metabolic gene expression [ 39 ]. Using laparoscopy, we implanted discrete biopsies of menstrual endometrium into immunocompetent, intact menstruating mice which resulted in a 100% incidence of endometriosis and mean confirmed lesion counts of more than 2 per mouse. Implantation of discrete biopsies via laparoscopy was successful in a previous study with the same incidence of endometriosis (100%) and higher reported take rate (60%); however, uterine biopsies included non-menstrual endometrium and myometrium versus menstrual phase endometrium in our study [ 22 ]. Additionally, in paradigm 5 we utilized menstruating recipient mice that were time matched to menstruating donor mice ensuring that both mice transitioned from the proliferative to the secretory phase simultaneously and were in the menstrual phase of the cycle at the time of transplant surgery. Consequently, both mice were subject to the same hormonal milieu and the recipient mice were in the same phase physiologically as women experiencing retrograde menstruation. (Fig. 8 ). In paradigm 5, we optimized date of sacrifice of donor and implantation into recipient mice to day 9.5 post induction of pseudopregnancy and 4.5 days post decidualization. In menstruating mice, day 9.5, correlates with highest vaginal bleeding scores, largest decrease in systemic progesterone levels, and maximum expression of mRNA profiles. Notably, prostaglandin-endoperoxide synthase 2 (Ptgs2) is 10-fold higher on day 9.5 and vascular endothelial growth factor (Vegfa) and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression are also upregulated. [ 21 ] Ptgs2 and Vegfa expression are elevated in ectopic lesions in women suffering from endometriosis and PECAM-1 is highly expressed in pelvic endometriotic lesions [ 40 – 42 ]. The use of decidualized menstrual endometrium in animal models is critical to attempt to replicate expression of potentially important factors that could be therapeutic targets or participate in the etiology and pathogenesis of endometriosis. Injection of decidualized endometrial slurry was largely unsuccessful when compared to placement of discrete biopsies and compared to reported results injecting decidualized endometrium [ 27 ]. However, to have similar conditions between groups, we made a 5 mm incision in mice injected with slurry, comparable to the incision made for placement of laparoscope. This differs from other reported models that perform an intraperitoneal injection without laparotomy. The physiologic effects of anesthesia and laparotomy cannot be ignored, and although the laparotomy incision was small and should decrease stress responses compared to larger incisions [ 43 ] this could have negatively affected the development of endometriotic lesions with slurry injection model. All three paradigms utilizing discrete endometrial biopsies had higher group incidences of endometriosis and higher mean confirmed lesions counts compared to the slurry injection paradigms. It has been previously reported in a chicken chorioallantoic membrane (CAM) model of implantation that larger biopsies (> 1mm 3 ) resulted in lesion formation compared to no lesion development when endometrial cells were transplanted [ 44 ]. This suggests that intact tissue architecture is crucial to implantation, survival, and proliferation, and could explain the lack of lesions when endometrial slurry was used. However, further exploration of injected menstrual phase endometrial slurry into menstruating mice is warranted, as this could be a non-invasive model allowing for iterative transplantation. Paradigms 1 and 5, (discrete biopsies with laparoscopy) resulted in 100% incidence of endometriosis, while paradigm 2 (discrete biopsies with laparotomy) had an 80% incidence. Additionally, paradigms 1 and 5 had mean lesion counts of 2.7 and 2.6 respectively, with paradigm 2 having 1.1. The discrete endometrial biopsies adhered to abdominal tissues without the use of suture or glue. The spontaneous attachment of menstrual phase endometrium allows for early study of disease progression including angiogenesis, apoptosis, proliferation, inflammation, and chemotactic homing response. There are reported differences in the morphology of mouse peritoneum and post-operative adhesion formation post laparoscopy or laparotomy [ 45 , 46 ]. Bulging of mesothelial cells with the presence of intercellular clefts occurs 1–2 hours after CO 2 insufflation [ 47 ]. This phenomenon has been hypothesized to play a role in peritoneal metastasis and infiltration of tumor cells into the sub mesothelial connective tissue matrix [ 48 ]. The biological response of the peritoneum to laparoscopy aids in endometrial biopsy attachment and proliferation, as a compromised mesothelial barrier may play a role in the pathogenesis of the disease [ 49 ]. Surgical treatment has also been reported as a potential risk factor for recurrence of ovarian endometriomas [ 50 ]. Further investigation into the role diagnostic and therapeutic laparoscopy potentially plays in recurrence and progression of endometriosis is warranted and would be possible using this model. Continued validation and use of this close approximation of “best fit” animal model of endometriosis will allow for translational exploration of the etiology and pathogenesis of endometriosis and future studies in fertility by using a menstruating, sexually intact and immunocompetent mouse. Declarations Acknowledgments We would like to thank Alex Hall, Research Statistician II and Jack Taylor, PhD Biostatistician for their consultation on statistics, the Dept. of Pharmacology & Neuroscience, Dept. of Obstetrics & Gynecology, CUSOM for their support, the Stryker Corporation for their donation of equipment, Ms. Melissa Holzapfel, UNMC Tissue Sciences Facility, Ms. Toni Howard, Creighton University Histology Core Facility, and the Creighton Animal Resource Facility and animal husbandry staff. Author Statements and Declarations Christina Howe, John Coté, and Janee Gelineau- van Waes contributed to the study conception and design. Material preparation, data collection and analysis were performed by Christina Howe, Jodi Hallgren, Marley Bredehoeft, and Janee Gelineau-van Waes. Catherine Stoos performed histopathology The first draft of the manuscript was written by Christina Howe and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conflicts of Interest/Competing Interests Financial interests: Christina Howe, Janee Gelineau-van Waes and Catherine Stoos declare they have no financial interests. John Coté was a Principal Investigator on endometriosis industry sponsored trials for AbbVie and Myovant. He has received compensation as a member of the endometriosis speaker’s bureau for AbbVie and Pfizer. Non-financial interests : none. Ethics Approval/Compliance with Ethical Standards : This study was performed in accordance with the Guide for Care and Use of Laboratory Animals and was approved by Creighton University’s Institutional Animal Care and Use Committee. IRB approval is not applicable . Consent to Participate : Not Applicable Consent for Publication: Not Applicable Data Availability : The original contributions presented in the study and data supporting the conclusions are included in the article; further inquiries can be directed to the corresponding author. Code Availability : Not Applicable Funding: This work was supported by: George F. Haddix President’s Faculty Research Fund 04/01/19 – 03/31/20 Partial financial support was received from Robin Farias-Eisner, MD, PhD, MBA, FACOG and the OB/GYN Department, and Peter Abel, PhD, and Dept of Pharmacology & Neuroscience, CUSOM Health Sciences Strategic Investment Fund (HSSIF) 07/01/21 – 06/30/23 References Zondervan KT, Becker CM, Missmer SA. Endometriosis. 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Gynecol. 2007;109(6):p 1411-1420. https://doi.org/ 10.1097/01.AOG.0000265215.87717.8b 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3243174","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":228959963,"identity":"4dcdd1fe-b7d8-461f-bc5a-66c4ec2dec1d","order_by":0,"name":"Christina Ann Howe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie3OsUrDQBjA8a8cZPog65W25BWuHLg01NcouCQcnIuo0KWTFFyrXa+Tr9AHcLhwoK9QqYOh0FkXSad6SSy6XLoK3h+SC8n3yx2Az/cHCxWAtmtir9abvSHQ+ktr6iB0VZKkIoTVhDQTNoJqj5IE9f+Pks5dnu0KcxlS8TTZPcbd6Gyeba4h7i21g3SfmcHEjNtKypfZVmL/1RCuQHInoRIMJCZdri9O1qAN9pUIOgj2TQPJiopcfY5B7w9k7yYjCRrrXQICWmNEK6KdhK7swVCep4uHLW/PtEBGBefIBF84SKgk+SjiQTpHk78XengaqTTf4GTYu3eQ725/HbWaZI3jZTc/j9H06LTP5/P9s74AGjZiCrSXfvkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0009-1571-9097","institution":"Creighton University","correspondingAuthor":true,"prefix":"","firstName":"Christina","middleName":"Ann","lastName":"Howe","suffix":""},{"id":228959964,"identity":"e8234fcd-46ee-4545-8752-70a0b2ce6118","order_by":1,"name":"John Coté","email":"","orcid":"","institution":"Creighton University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Coté","suffix":""},{"id":228959965,"identity":"74536bcd-7bde-4149-b777-8000ce3942bc","order_by":2,"name":"Catherine Stoos","email":"","orcid":"","institution":"CHI Health Creighton University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Stoos","suffix":""},{"id":228959966,"identity":"c89b6d19-8977-4761-baab-42333e005cf5","order_by":3,"name":"Marley Bredehoeft","email":"","orcid":"","institution":"Creighton University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Marley","middleName":"","lastName":"Bredehoeft","suffix":""},{"id":228959967,"identity":"3ed71204-1f0d-4dff-81d3-f7df0af0f305","order_by":4,"name":"Jodi Hallgren","email":"","orcid":"","institution":"Creighton University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jodi","middleName":"","lastName":"Hallgren","suffix":""},{"id":228959968,"identity":"5d80d898-5753-4313-9575-ad4028015ba7","order_by":5,"name":"Janee Gelineau-van Waes","email":"","orcid":"","institution":"Creighton University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Janee","middleName":"Gelineau-van","lastName":"Waes","suffix":""}],"badges":[],"createdAt":"2023-08-07 19:26:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3243174/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3243174/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42393402,"identity":"b9b888c7-a291-4832-8bfb-db10e0c7f474","added_by":"auto","created_at":"2023-08-30 23:44:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218665,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Menstruating mouse displaying overt vaginal bleeding; (b) Vaginal cytology of menstruating mouse depicting frank blood ;(c) Decidualized uterus d9.5 post vaginal plug, indicated by enlarged, swollen, dark pink uterine horns, day 5.5 post decidualization procedure\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/90908583f144b1ed11a1ee5b.png"},{"id":42394035,"identity":"428d1d9e-d50b-40a1-91ba-86f426a04435","added_by":"auto","created_at":"2023-08-31 00:00:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":210498,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline of induction of menstruation, endometriosis, and lesion harvest for (A) paradigms 1-4 and (B) paradigm 5\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/85179e6007ef1996f42997a1.png"},{"id":42393397,"identity":"1c362a7a-27fd-4669-bcc6-ba901666229f","added_by":"auto","created_at":"2023-08-30 23:44:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":186928,"visible":true,"origin":"","legend":"\u003cp\u003eLaparoscopic Surgery (a) and (b) optimizing laparoscopic procedure; (c) discrete endometrial tissue biopsy implanted onto body wall (arrow) via laparoscopy\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/77063ca722e90709146bad8c.png"},{"id":42393404,"identity":"ce1e8ed7-abc3-4334-a242-aa826ba0ef1f","added_by":"auto","created_at":"2023-08-30 23:44:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":251321,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±95% CI) counts of confirmed endometriotic lesions across the 5 induction paradigms. Bars with no letters in common are significantly (p\u0026lt;.05) different based on \u003cem\u003epost hoc\u003c/em\u003etests from the Poisson GLM model of the log difference between groups. Figures were created in ggplot2 (H. Wickham. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.) using R version 4.2.2\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/eafcc7644d4bdef2c3ecff5f.png"},{"id":42393879,"identity":"195b6391-9617-494b-a64f-1f4b5d64d77b","added_by":"auto","created_at":"2023-08-30 23:52:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184892,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Bladder, (b) Uterine body, and (c) Adipose: Gross endometriotic lesions in situ with hemosiderin (arrows)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/d5d0799c813d5c937ed186e9.png"},{"id":42393877,"identity":"196b5af2-40a4-46c8-ab3c-56d07d685c80","added_by":"auto","created_at":"2023-08-30 23:52:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":255398,"visible":true,"origin":"","legend":"\u003cp\u003eConfirmed endometriotic lesions (arrows) showing hemosiderin and stroma on (a) adipose tissue; (b) skeletal muscle; (c) bladder, magnification 20X\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/1943808f7c7e36b7107c492d.png"},{"id":42393398,"identity":"03dd2c48-3f1a-417b-bbf1-28a9c729394d","added_by":"auto","created_at":"2023-08-30 23:44:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1098045,"visible":true,"origin":"","legend":"\u003cp\u003eEndometriotic lesions with immunohistochemical staining performed on a Discovery Ultra advanced staining system (Roche Diagnostics, Ventana Medical Systems, Inc.). Sections were stained using primary antibodies for (a) the endothelial cell marker CD31 (rabbit polyclonal, 1:100, Abcam ab28364) , (b) the cell proliferation marker Ki67 (rabbit polyclonal, 1:200, Abcam ab16667) or (c) the macrophage marker Cd68 followed by secondary Ab HRP detection (discovery anti-rabbit HQ RTU) enzyme conjugate biotin-free Discovery anti-HQ HRP RTU, chromogen staining with Discovery purple kit RUO, and a hematoxylin counterstain. Additionally, lesions were stained with Toluidine blue (d) for mast cells. Arrows indicate antigen positive cells, magnification 20X\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/7144348b8e357d101fd1d652.png"},{"id":42393401,"identity":"5cc1ce34-0178-4310-a7d8-dec1e40a0504","added_by":"auto","created_at":"2023-08-30 23:44:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142143,"visible":true,"origin":"","legend":"\u003cp\u003eTiming of menstrual cycle phases in women versus pseudo-pregnant decidualized mice\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/141a013794c9fe7d9b5a55a3.png"},{"id":54531480,"identity":"2bdf89d0-c820-443d-b64e-c9c9e5da193e","added_by":"auto","created_at":"2024-04-12 00:42:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3569801,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3243174/v1/3b66d9d4-8348-48e7-bcd1-96418854403d.pdf"}],"financialInterests":"","formattedTitle":"Optimizing a Translational Mouse Model of Endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is an estrogen-dependent gynecological disease that affects an estimated 10\u0026ndash;15% of women of reproductive age [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is characterized by endometrial tissue (glands and stroma) proliferating in ectopic locations, and is associated with dysmenorrhea, chronic pelvic pain, dyspareunia, infertility, and increased risk of ovarian cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The painful symptoms of endometriosis substantially affect quality of life and productivity. The estimated societal burden (direct and indirect costs) of endometriosis was \u003cspan\u003e$\u003c/span\u003e4572-12,079 (USD) per patient per year in 2022 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Most medical therapies are variably effective and cannot be used long-term, and surgical treatment has a high recurrence rate or results in infertility in the case of hysterectomy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Challenges with diagnosis and treatment stem from that fact that a definitive etiology and pathogenesis of endometriosis is unknown.\u003c/p\u003e \u003cp\u003eEndometriosis is theorized to develop either from uterine endometrium or from extra-uterine tissue. Extra- uterine origin theories include: coelomic metaplasia of peritoneal cells, embryonic M\u0026uuml;llerian rest theory, and extra-uterine stem cells originating from bone marrow [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Retrograde menstruation into the abdominal cavity via the fallopian tubes is the most widely accepted hypothesis for the implantation of uterine endometrial tissues to extrauterine locations [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In fact, retrograde menstruation is a common physiologic finding in menstruating women with patent fallopian tubes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Benign metastasis via hematogenous or lymphatic spread is another proposed theory of endometriosis arising from uterine endometrial tissue [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Regardless of origin, ectopic endometrial implants must escape immune clearance, invade, and attach to ectopic locations, proliferate, neovascularize and survive [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Investigation of these factors requires an appropriate translational animal model that recapitulates the human disease process.\u003c/p\u003e \u003cp\u003eNaturally occurring endometriosis in animal species other than humans only occurs in some non-human primates at a very low incidence rate [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While endometrial lesions in non-human primates compared to women are histologically identical and located in similar sites, the use of non-human primates as an animal model comes with the disadvantages of high costs, need for special infrastructure and training, lack of availability and ethical considerations [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, there is a paucity of species that menstruate naturally: humans, some higher order non-human primates, the elephant shrew, some species of bats and the spiny mouse [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], none of which are as optimized as mice for use as a preclinical model. Mice are well characterized, cost-effective, prolific, and allow for genetic manipulations.\u003c/p\u003e \u003cp\u003eCummings et al, described the first murine model of endometriosis in 1995 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and although many iterations of murine models have followed, they lack translational relevance. Rodent models of endometriosis that involve ovariohysterectomy and constant levels of hormone replacement are impractical for studies of physiological functions that require natural fluctuations in sex hormones, such as fertility. The use of immunocompromised mice with xenotransplantation of human tissue, preclude the assessment of the role of immune cells in pathogenesis. Models that use full thickness uterine implants include myometrium and serosal layers that are not biologically inert and are not present in human endometrial lesions. Finally, models that implant endometrial into extra-abdominal locations eliminate the assessment of the peritoneal environment. A hypothesized \u0026ldquo;best fit\u0026rdquo; model of murine endometriosis has been described, one that uses immunocompetent mice with intact ovaries to approximate spontaneous menstrual endometrial attachment in the peritoneal cavity, allows for longitudinal assessment of lesions and ideally iterative endometrial transplantation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMice do not menstruate naturally and have a closed tubo-ovarian junction resulting in the need for significant manipulation to model the theory of uterine origin of ectopic endometrial lesions due to retrograde menstruation. An effective model of mouse menstruation mimicking the human menstrual cycle in a pseudo-pregnant mouse is well established. Pseudo-pregnant menstruating mice display similar gene expression, overt vaginal bleeding, and histologic changes when compared to humans, with the uterus also displaying reepithelization of the luminal endometrial surface [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Using this method of induction of menstruation, menstrual endometrial implants must be placed into the peritoneal cavity. Published methods include surgical engraftment of discrete tissue pieces, and intra-peritoneal injection or surgical injection of endometrial slurry. Surgical engraftment using suture or adhesives introduces variability due to their effects on tissues and bypasses natural attachment of tissues, one of the early factors of the disease that needs elucidation. Laparoscopic implantation of biopsies described and developed by Peterse et al. allows for adhesion of implants to peritoneal surfaces without the need for suture or artificial adhesives [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Both injection techniques more closely mimic true retrograde menstruation and allow for study of early disease stages, but the number of lesions generated is variable and tissue slurry composition fluctuates based on technique.\u003c/p\u003e \u003cp\u003eIn this study, we compared 5 different paradigms of induction of endometriosis in immunocompetent, intact mice. Initially, 4 paradigms were used to optimize the tissue type and method of induction. Menstrual endometrium harvested day 9.5 post vaginal plug from B6(Cg)-Tyrc-2JTg(UBC-mCherry)1Phbs/J mice was implanted intraperitoneally into na\u0026iuml;ve B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;2J\u003c/em\u003e\u003c/sup\u003e mice using discrete implants via (1) laparoscopy or (2) laparotomy or tissue slurry injection via (3) laparoscopy, or (4) laparotomy. To optimize the model further, a novel paradigm (5) used discrete implants via laparoscopy into menstruating B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;2J\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eUsing syngeneic, immunocompetent, intact, menstruating donor and recipient mice, we have optimized a minimally invasive, laparoscopic surgical model of implantation of discrete endometrial biopsies. This novel, translationally relevant murine model addresses the majority of recommendations in the \u0026ldquo;best fit\u0026rdquo; model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eAnimals, housing conditions, diet\u003c/b\u003e. All procedures were performed at Creighton University, an AAALAC-accredited facility. This study was performed in accordance with the \u003cem\u003eGuide for Care and Use of Laboratory Animals\u003c/em\u003e and was approved by Creighton University\u0026rsquo;s Institutional Animal Care and Use Committee. All mice were maintained in environmentally controlled rooms (20\u0026ndash;26\u0026deg;C; humidity 30\u0026ndash;55%) with diurnal lighting (12:12-h light:dark cycle; light on, 0700). 8\u0026thinsp;\u0026plusmn;\u0026thinsp;8-week old female B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;\u0026thinsp;2J\u003c/em\u003e\u003c/sup\u003eTg(UBC-mCherry)1Phbs/J (stock #017614) were used as donor mice and female B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;\u0026thinsp;2J\u003c/em\u003e\u003c/sup\u003e/J (stock # 000058) were used as recipient mice. Hereafter, these strains will be referred to as mCherry for B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;\u0026thinsp;2J\u003c/em\u003e\u003c/sup\u003eTg(UBC-mCherry)1Phbs/J and B6 for B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;\u0026thinsp;2J\u003c/em\u003e\u003c/sup\u003e/J Mice were maintained in individually ventilated cages on 7099 TEK-Fresh (paper) bedding and fed soy protein-free extruded autoclavable diet 2020SX (Teklad Global Rodent Diets\u0026reg;) \u003cem\u003ead libitum.\u003c/em\u003e to decrease exposure to any exogenous estrogens (i.e., the mycotoxin zearalenone in corncob bedding, and/or phytoestrogens in soy based rodent diets) [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Environmental enrichment was provided as 8-grams of nesting material (Enviro-dri\u0026reg; Shepherd Specialty Paper, Watertown, TN, and Rodent Nesting Sheets \u0026trade;, Bio-Serv, Flemington, NJ), wood gnawing block, mouse trapeze and mouse tunnel (Bio-Serv, Flemington, NJ). Female mice were group housed until day of vaginal plug, and then single housed until sacrifice to avoid Lee-Boot effect [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Vasectomized male mice were obtained from JAX and were single housed on corncob bedding and fed Teklad global 18% protein 2018S \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInduction of Menstruation/Decidualization\u003c/h2\u003e \u003cp\u003eInduction of menstruation was performed using the Rudolph et. al. protocol with modifications [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Na\u0026iuml;ve donor mCherry (Paradigms 1\u0026ndash;5) and B6 recipient female mice (Paradigm 5) were placed with vasectomized males overnight (day 0) and checked for the presence of a vaginal plug the next morning (day 0.5). Females with a confirmed vaginal plug were individually housed. On day 4.5 pseudo-pregnant mice were anesthetized using isoflurane, and decidualization was induced by intrauterine injection of 100 \u0026micro;l sterile sesame oil using a blunt 27G x0.86\u0026rdquo; needle (ALZET\u0026reg;) followed by mechanical stimulation. Sustained release buprenorphine was administered post procedure. Menstruation was confirmed using vaginal cytology and/or the visual presence of overt vaginal bleeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInduction of Endometriosis\u003c/h2\u003e \u003cp\u003eOn day 7.5\u0026ndash;9.5 decidualized/menstruating donor mCherry mice were anesthetized with isoflurane, blood collected via submandibular bleed using a Goldenrod animal bleeding lancet (5mm), and cervically dislocated. The uterus was removed en bloc using sterile technique, placed in sterile cold (4\u003csup\u003e◦\u003c/sup\u003eC) PBS (0.01 M PBS composed of 13.7 mM NaCl, 0.27mM KCl, 0.15mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 0.8mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e; pH 7.4). Each uterine horn was opened longitudinally and decidualized endometrium was dissected from the myometrium, then sectioned into approximately 2 mm\u003csup\u003e3\u003c/sup\u003e discrete biopsies. 10 biopsies were implanted into recipient mice in paradigms 1, 2, and 5. For paradigms 3 and 4, endometrial slurry was made using the Greaves et. al. protocol with modifications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 10 decidualized endometrial discrete biopsies were weighed, minced, and mixed with 0.2 ml of sterile saline (0.9% NaCl). This slurry was then drawn into a syringe through a 19 G hypodermic needle to ensure uniform suspension. The average wet weight of endometrium implanted was 94.16 mg per recipient mouse. Paradigm 1\u0026ndash;4 used intact B6 recipient mice (n\u0026thinsp;=\u0026thinsp;10). Paradigm 5 used \u003cem\u003edecidualized/menstruating\u003c/em\u003e B6 recipients (n\u0026thinsp;=\u0026thinsp;10) that were time-matched for date of vaginal plug and decidualization with donor decidualized/menstruating mCherry mice. On day 9.5 post vaginal plug, donor mCherry mice were sacrificed and endometrial tissue harvested as described and implanted into the decidualized/menstruating B6 recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor all 5 paradigms, recipient B6 mice were weighed, and anesthetized with ketamine (87.5mg/kg) and xylazine (12.5mg/kg) given intraperitoneally. Hair was removed from the abdomen using depilatory cream (Nair \u0026reg;) and heat support was provided using a Homeothermic Monitoring System (Harvard Apparatus, Holliston, MA). Stage of estrus of recipient was determined via cytological evaluation of vaginal smears for paradigms 1\u0026ndash;4. For paradigm 5, decidualization/menstruation of recipient B6 mice was confirmed via cytological evaluation of vaginal smears and/or presence of overt vaginal bleeding. If laparoscope was used, mice were intubated using a 22G x 1 inch IV catheter (Pivetal\u0026reg;) and ventilated with Minivent (Model 845, Harvard Apparatus, Holliston, MA) with stroke volume calculated at 10\u0026micro;l/kg and stroke rate of 200 strokes/minute. Sustained release buprenorphine was administered post procedure for analgesia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLaparotomy (Paradigms 2 and 4)\u003c/h2\u003e \u003cp\u003eA 5 mm cranial ventral midline incision was made caudal to the xiphoid process. The discrete endometrial biopsies or endometrial slurry were placed intra-abdominally through this incision. The incision was closed using 5\u0026thinsp;\u0026minus;\u0026thinsp;0 polyglactin 910 suture (Patterson Veterinary, Loveland, CO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLaparoscopy (Paradigms 1, 3 and 5)\u003c/h2\u003e \u003cp\u003eLaparoscopic guided implantation of decidualized endometrial biopsies was performed using the Peterse et. al. protocol with modifications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A 5 mm cranial ventral midline incision was made caudal to the xiphoid process. A 2 mm endoscope covered with a 3 mm insufflation shield (Stryker, Kalamazoo, MI) was placed into the peritoneal cavity. The abdomen was insufflated with warmed CO\u003csub\u003e2\u003c/sub\u003e at a flow of 1L/minute to maintain an intra-abdominal pressure of 5mm Hg. For implantation of discrete endometrial biopsies, a 14 G intravenous catheter (SurFlash\u0026trade;. Patterson Veterinary, Loveland, CO) was inserted into the right lower abdominal quadrant to serve as a port. The biopsies were placed into the tip of the catheter and advanced into the abdomen using a blunt stylet. For implantation of endometrial slurry, the suspension was injected intraperitoneally with a 19G needle through the right lower abdominal quadrant. The incision and port opening were closed using 5\u0026thinsp;\u0026minus;\u0026thinsp;0 polyglactin 910 suture (Patterson Veterinary, Loveland, CO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLesion Harvest\u003c/h3\u003e\n\u003cp\u003eRecipient mice were followed with cytologic evaluation of vaginal smears post-operatively to confirm return to estrus cyclicity. Mice were sacrificed in proestrus at least 30 days post return to normal estrus cyclicity. Mice were anesthetized with isoflurane, blood collected via submandibular bleed using a Goldenrod animal bleeding lancet (5mm), and cervically dislocated. The abdominopelvic cavity was closely examined for endometriosis-like lesions; number and location of each lesion was recorded, along with a detailed description of gross appearance. Lesions were photographed, excised from surrounding tissue, and immediately fixed in 10% neutral-buffered formalin.\u003c/p\u003e \u003cp\u003eEndometriosis was induced in an additional 10 decidualized/menstruating recipient mice using paradigm 5 methodology. These mice were followed for 60 and 90 days (n\u0026thinsp;=\u0026thinsp;5 per timepoint) post return to normal estrus cyclicity and sacrificed as described.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistological assessment\u003c/h2\u003e \u003cp\u003eLesions were processed in the Creighton University Histology Core Facility using the Excelsior ES Tissue Processor (Thermo Fisher Scientific, Waltham, MA) and embedded in paraffin. Tissue sections (5 microns) were cut on a Leica RM 2135 microtome (Leica Biosystems, Deer Park, IL) and placed on Fisherbrand\u0026trade; Superfrost\u0026trade; Plus Microscope Slides ( Thermo Fisher Scientific, Waltham, MA). The Gemini Auto Stainer was used for hematoxylin and eosin (H\u0026amp;E) staining. Lesion sections were evaluated by histopathologist in a blinded fashion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry Protocol CD31/Ki67/Cd68\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed on a Discovery Ultra advanced staining system (Roche Diagnostics, Ventana Medical Systems, Inc.). Sections were deparaffinized using a mild detergent solution and vortex mixing at 69\u0026deg;C for 24 min (cat no. 950\u0026thinsp;\u0026minus;\u0026thinsp;102, Roche Diagnostics). Tris based reaction buffer, pH 7.6, used throughout the protocol to maintain aqueous conditions and rinse slides (cat no. 950\u0026thinsp;\u0026minus;\u0026thinsp;300, Roche Diagnostics). Antigen retrieval by heat, cell conditioning CC1, 8.2 pH tris-borate-EDTA buffer, at 95\u0026deg;C for 24 min (cat no. 950\u0026thinsp;\u0026minus;\u0026thinsp;124, Roche Diagnostics). Treated with Discovery ChromoMap RUO Inhibitor for 8 min (cat no. 760\u0026thinsp;\u0026minus;\u0026thinsp;159, Roche Diagnostics). Primary antibody for CD31, rabbit polyclonal, 1:100, incubation at 37\u0026deg;C for 44 min (cat no. ab28364, Abcam), primary antibody for Ki67, rabbit polyclonal, 1:200, incubation at 37\u0026deg;C for 32 min (cat no. ab16667, Abcam) or primary antibody for CD68, rabbit polyclonal, 1:200, incubation at 37\u0026deg;C for 32 min (cat no. ab125212, ABCAM). Secondary antibody HRP detection, Discovery anti-rabbit HQ RTU, incubation at 37\u0026deg;C for 16 min (cat no. 760\u0026ndash;4815, Roche Diagnostics). Enzyme conjugate biotin-free Discovery anti-HQ horse-radish peroxidase RTU, incubation at 37\u0026deg;C for 16 min (cat no. 760\u0026ndash;4820, Roche Diagnostics). Chromogen staining with Discovery Purple Kit RUO for 32 min and counterstained with Hematoxylin (cat no. 760\u0026thinsp;\u0026minus;\u0026thinsp;229, 790\u0026ndash;2208 \u0026amp; 760\u0026ndash;2037, Roche Diagnostics).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eToluidine Blue Staining for Mast Cells\u003c/h2\u003e \u003cp\u003eSections were stained with Toluidine blue resulting in red-purple (metachromatic staining) mast cells on a blue background.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eEndometriosis Incidence\u003c/h2\u003e \u003cp\u003eIn order to test for differences in the incidence of endometriosis among groups, we first conducted a 2(yes/no) x 5(induction method) omnibus Fisher\u0026rsquo;s exact test, followed by individual 2 x 2 Fisher\u0026rsquo;s exact tests.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConfirmed Endometrial Lesions\u003c/h2\u003e \u003cp\u003eTo test for differences in confirmed lesions among different endometriosis induction methods, we used a generalized linear model with a Poisson distribution and a log link function using the glm function in R (R Core Team, 2022). Significant differences are defined as p\u0026thinsp;\u0026lt;\u0026thinsp;.05, and marginal differences are defined as p\u0026thinsp;\u0026lt;\u0026thinsp;.10.\u003c/p\u003e \u003cp\u003eTo test for differences in the presence of lesions on the uterus as well as the presence of glands in confirmed lesions, we conducted Fisher\u0026rsquo;s exact tests comparing Paradigm 1 (Laparoscopy and endometrial biopsies) with Paradigm 5 (Laparoscopy and endometrial biopsies in decidualized, menstruating recipients).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInduction of Menstruation/Decidualization\u003c/h2\u003e \u003cp\u003eWe achieved successful decidualization in 34/44 (77.27% \u0026plusmn;12.38%, 95% CI) of mCherry mice. We considered decidualization successful if enough tissue could be harvested to implant into 1 recipient mouse. In general, one donor mouse provided enough tissue to implant into 2 recipient mice. decidualization occurred unilaterally, bilaterally, and segmentally throughout the uterine horns. In the 10/44 (22% \u0026plusmn;12.38%, 95% CI) mice that failed to decidualize, the uterus was either normal in appearance, or the decidualization was incomplete and the endometrium could not be adequately separated from the myometrium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEstrus Cyclicity Post Surgery\u003c/h2\u003e \u003cp\u003eAll recipient mice returned to normal estrus cyclicity post-surgery. B6 menstruating recipient mice in paradigm 5 returned to estrus cyclicity between 3\u0026ndash;14 days (mean\u0026thinsp;=\u0026thinsp;6.8 days, SD\u0026thinsp;=\u0026thinsp;3.4, SEM\u0026thinsp;=\u0026thinsp;1.07, 95% CI(4.70, 8.90)) post operatively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEndometriosis Incidence\u003c/h2\u003e \u003cp\u003eThe incidence of endometriosis differed across induction methods (p\u0026thinsp;\u0026lt;\u0026thinsp;.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Post hoc Fisher\u0026rsquo;s exact tests showed that Paradigm 1 produced higher rates of endometriosis than Paradigms 3\u0026ndash;4 (p\u0026thinsp;\u0026lt;\u0026thinsp;.001) but did not differ from Paradigms 2 or 5 (p\u0026thinsp;\u0026gt;\u0026thinsp;.47). For all five paradigms, the average days of disease from day of induction to lesion harvest was 34.85 days (SD\u0026thinsp;=\u0026thinsp;.05, SEM\u0026thinsp;=\u0026thinsp;1.07, 95% CI (34.45, 35.25)). We induced endometriosis at a rate of 100% in paradigms 1 and 5. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) Endometriosis was also confirmed in 100% of recipient mice (paradigm 5 methodology) at 60 and 90 days.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIndividual mouse data per paradigm\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParadigm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecipient estrus stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDonor sacrifice days post vaginal plug\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDays of Disease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIndividual Take Rate\u003csup\u003ea\u003c/sup\u003e, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean counts of confirmed lesions\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEndometriosis Incidence\u003csup\u003ec\u003c/sup\u003e, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1: Laparoscopy w/discrete endometrial biopsies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eDiestrus\u003c/p\u003e \u003cp\u003eMetestrus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003cp\u003e8.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e10.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e8.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003cp\u003e42\u003c/p\u003e \u003cp\u003e36\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e36\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e33\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003cp\u003e40 (4/10)\u003c/p\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003cp\u003e30 (3/10)\u003c/p\u003e \u003cp\u003e25 (2/8)\u003c/p\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e60 (6/10)\u003c/p\u003e \u003cp\u003e30 (3/10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2: Laparotomy w/discrete endometrial biopsies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003cp\u003eEstrus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e36\u003c/p\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e34\u003c/p\u003e \u003cp\u003e35\u003c/p\u003e \u003cp\u003e37\u003c/p\u003e \u003cp\u003e37\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e0 (0/10)\u003c/p\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003cp\u003e0 (0/10)\u003c/p\u003e \u003cp\u003e2 (2/10)\u003c/p\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e10 (1/10)\u003c/p\u003e \u003cp\u003e20 (2/10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5: Laparoscopy w/ discrete endometrial biopsies in menstruating recipients\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMenstruating\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e44\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e20 (2/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e40 (4/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e20 (2/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e30 (3/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e30 (3/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e20 (2/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e40 (4/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e20 (2/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e40 (4/10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e10 (1/10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2.6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIndividual Lesion Count\u003c/b\u003e\u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3: Laparoscopy w/ endometrial slurry\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eProestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4: Laparotomy w/ endometrial slurry\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eEstrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eProestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eDiestrus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e9.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e38\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAverage days of Disease\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e34.85\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003e Individual take rate\u0026thinsp;=\u0026thinsp;number confirmed lesions/number of implanted biopsies x 100, expressed as a percentage\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003e Mean counts of confirmed lesions per mouse\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ec\u003c/sup\u003e Endometriosis incidence per paradigm, expressed as percentage of animals (n\u0026thinsp;=\u0026thinsp;10) per paradigm that had confirmed endometriosis lesion(s) histologically.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ed\u003c/sup\u003e Individual confirmed endometrial lesion count(s) for slurry implanted animals\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHistological Confirmation\u003c/h2\u003e \u003cp\u003eThe method of endometriosis induction significantly impacted the presence of confirmed (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;55.16, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) lesions. Paradigms 1 (mean\u0026thinsp;=\u0026thinsp;2.7 lesions, SD\u0026thinsp;=\u0026thinsp;1.4, SE\u0026thinsp;=\u0026thinsp;0.45, 95% CI) and 5 (mean\u0026thinsp;=\u0026thinsp;2.6 lesions, SD\u0026thinsp;=\u0026thinsp;1.17, SE\u0026thinsp;=\u0026thinsp;0.37, 95% CI) produced significantly more confirmed lesions than Paradigms 3 (mean\u0026thinsp;=\u0026thinsp;0.2 lesions, SD 0.42, SE\u0026thinsp;=\u0026thinsp;0.13, 95% CI) and 4 (mean\u0026thinsp;=\u0026thinsp;0.1 lesion, SD 0.32, SE 0.1, 95% CI). Paradigm 1 also produced marginally more confirmed lesions than Paradigm 2 (mean\u0026thinsp;=\u0026thinsp;1.1 lesions, SD 0.74, SE 0.23, 95% CI) (p\u0026thinsp;=\u0026thinsp;.088).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEndometriosis was confirmed by the presence of stroma, and hemosiderin histologically(Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In some lesions, glands, epithelium, and calcifications were also present. Most endometrial lesions were found on skeletal muscle (body wall), adipose tissue, mesentery, on/near the bladder and on broad ligaments of uterine horns. Lesions on the uterine serosal surface and lesions with glands were found in paradigms 1 and 5, however, we found no evidence for differences in the number of animals developing lesions in the uterus between paradigm 1 and 5 (1/9 vs 5/5; p\u0026thinsp;=\u0026thinsp;.141), nor did we find evidence for differences in the number of animals developing lesions with glands between Paradigm 1 and 5 (2/8 vs 5/5, p\u0026thinsp;=\u0026thinsp;.350).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining performed on confirmed lesions indicated the presence of angiogenesis (CD31), proliferation (Ki67), mast cells (toluidine blue) and macrophages (Cd68) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we developed and optimized a translationally relevant preclinical murine model of endometriosis by analyzing, adapting, and modifying previously published methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Currently, there is no standardized preclinical animal model for endometriosis. A murine model is ideal due to size, fecundity, and amenability to genetic manipulation. We used immunocompetent, intact mice with implantation of menstrual phase endometrium that spontaneously attached. Additionally, we felt implantation into menstruating mice improved model fidelity.\u003c/p\u003e \u003cp\u003eWe used soy-free diet and paper bedding to limit exposure to exogenous estrogens (i.e., the mycotoxin zearalenone in corncob bedding, and/or phytoestrogens in soy based rodent diets)[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] because supplemental estrogen has been shown to promote endometrial lesion growth in rats and mice [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Future studies examining the effects of exogenous estrogens or endocrine disrupters should consider feed, bedding, and caging choice.\u003c/p\u003e \u003cp\u003eWe selected mCherry donor mice to allow longitudinal, non-invasive monitoring of developing endometrial lesions in recipient B6 females using fluorescent imaging (IVIS Lumina XR\u0026reg;), but resolution was inadequate with a low signal to background ratio. This differed from the success reported using non-invasive fluorescent monitoring with endometrial tissue labeled with mCherry adenoviral vectors (AAV) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, in that study, the strongest, most reliable signal was generated from endometrial tissue implanted subcutaneously on the ventral abdomen compared to intraperitoneal implantation, the more translationally relevant location. Additionally, the endpoint was only 20 days, and the use of the AAV vector would increase signal compared to the transgenic mouse alone. Immunohistochemical staining of harvested endometrial lesions demonstrated angiogenesis (CD31) and proliferation (Ki67). The average number of days of disease was 34.85, with confirmed endometriotic lesions also harvested at 60 and 90 days post implantation indicating prolonged survival with this model. With optimization of imaging and the use of transgenic strains expressing fluorophores, non-invasive longitudinal assessment is possible.\u003c/p\u003e \u003cp\u003eAll menstruating recipient mice returned to normal estrus cyclicity. This allowed the implanted menstrual phase endometrium to undergo cyclical hormonal exposure to estrogen and progesterone, a more translational model than supplementation with supraphysiologic doses of estrogen and/or progesterone in ovariectomized mice. All mice were sacrificed in proestrus, to decrease variability of lesion size and appearance. Infertility is a common complaint of women suffering from endometriosis and 30\u0026ndash;50% of women with endometriosis seek care due to infertility alone [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. With the use of intact mice, future studies can examine fertility in recipient mice.\u003c/p\u003e \u003cp\u003ePathologists have classically defined endometriosis in women as the presence of endometroid glands and stroma in biopsied lesions. Paradigm 1 had 20% (2/10) of mice and paradigm 5 had 50% (5/10) of mice with confirmed lesions containing glands. Paradigms 2, 3 and 4 had mice with lesions that only contained stroma and hemosiderin. Although there was no significant difference detected between paradigm 1 and 5 for lesions with glands or uterine lesions, increased group size may allow for detection of differences. Stromal endometriosis in women is characterized as small, microscopic nodules or plaques of endometrioid-type stroma often containing arteriole-like vascular channels, hemosiderin pigment, inflammatory cells, microcalcifications and reactive mesothelial proliferation. It is a common form of endometriosis and can occur with or without the presence of typical endometriosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Many of the confirmed lesions in this study demonstrated one or more of these characteristics. Additionally, we evaluated lesions at a much earlier timepoint of disease in the mouse (approximately 3 human equivalent years), compared to histological evaluation of suspected lesions in women, as women are typically diagnosed an average of 8 years after symptoms appear. There is tremendous heterogeneity in endometriosis lesions, and evidence of ordered progression is lacking. It is possible that stromal endometriosis is an earlier phenotype of the disease or is a result of a type of metastatic process. Use of an appropriate mouse model, displaying the potential for prolonged lesion survival (90 days) would allow for further evaluation of lesion progression, the role mesothelial inflammation and proliferation plays, and better characterization of endometriosis phenotypes.\u003c/p\u003e \u003cp\u003eIn addition to mCherry donor\u0026thinsp;+\u0026thinsp;B6 recipient mice, we have successfully induced endometriosis using decidualized/menstruating B6(Cg)-\u003cem\u003eTyr\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u0026minus;2J\u003c/em\u003e\u003c/sup\u003e/J or B6.129S6-\u003cem\u003eGper1\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Cwan\u003c/em\u003e\u003c/sup\u003e/J females as both donors and recipients (unpublished). It is critical that this model is evaluated in other widely used strains of mice, as differences in endometrial lesion phenotype have been demonstrated between C57BL/6 and BALB/c mice [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and inbred mouse lines can differ in their immunologic and inflammatory pathways. Immune cell dysfunction in endometriosis is associated with impaired phagocytosis/clearance of menstrual debris, increased survival/proliferation of ectopic tissue, neuro-angiogenesis, and pain. Macrophages (Mφ) are the most abundant immune cells present in endometriotic lesions, and, in response to estrogen, adopt a \u0026lsquo;wound-healing\u0026rsquo; (anti-inflammatory) M2 phenotype compatible with survival and growth of ectopic tissue [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Mast cells (MC) are also present, and the MC stabilizer ketotifen effectively reduces hyperalgesia in a rat model of endometriosis, indicating MC may play a role in endometriosis pain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Ovarian cycle, estradiol, neuroimmune/neuroendocrine factors, and a nervous system responsive to hormones all play a role in the pain a woman with endometriosis experiences [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The use of an ovariectomized and/or immunodeficient mouse would preclude the study of these factors in a mouse model. Immunohistochemical staining of confirmed endometriotic lesions revealed the presence of both macrophages (Cd68) and mast cells (toluidine blue) within or in close proximity to ectopic endometrial tissue, validating this model for use in future studies of these immune cells and for evaluation of pain and pain modulation.\u003c/p\u003e \u003cp\u003eWe found confirmed lesions in translationally relevant locations such as the bladder, uterine ligaments, and serosal surface of the uterus (5/10 recipients in paradigm 5). Lesions in women are generally found in gravity dependent areas such as the posterior cul de sac [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and consistent with the quadruped nature of the mouse, most lesions were located on the ventral aspect of serosal surfaces. Although the ovaries are a common site of endometriosis in women, no lesions were found on the ovaries in recipient mice. This is not surprising as mice have an ovarian bursa and closed tubo-ovarial junction and there is no bursa surrounding the ovaries in women [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The closed tubo-ovarial junction also precludes the study of spontaneous endometriosis development due to retrograde menstruation in a decidualized mouse, thus requiring the use of both a donor and recipient mouse and a surgical or injectable method of implantation. Many lesions were attached to adipose tissue. The significance of this needs further exploration, as there is a strong relationship between estrogen and adipose tissue in women [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and endometriosis is associated with lean body mass index and low waist-to-hip ratio [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Additionally, higher levels of gene expression characteristic of M2 phenotype macrophages are associated with adipose tissue in lean mice [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and endometriosis alters adipose stem cell population and metabolic gene expression [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUsing laparoscopy, we implanted discrete biopsies of menstrual endometrium into immunocompetent, intact menstruating mice which resulted in a 100% incidence of endometriosis and mean confirmed lesion counts of more than 2 per mouse. Implantation of discrete biopsies via laparoscopy was successful in a previous study with the same incidence of endometriosis (100%) and higher reported take rate (60%); however, uterine biopsies included non-menstrual endometrium and myometrium versus menstrual phase endometrium in our study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, in paradigm 5 we utilized menstruating recipient mice that were time matched to menstruating donor mice ensuring that both mice transitioned from the proliferative to the secretory phase simultaneously and were in the menstrual phase of the cycle at the time of transplant surgery. Consequently, both mice were subject to the same hormonal milieu and the recipient mice were in the same phase physiologically as women experiencing retrograde menstruation. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In paradigm 5, we optimized date of sacrifice of donor and implantation into recipient mice to day 9.5 post induction of pseudopregnancy and 4.5 days post decidualization. In menstruating mice, day 9.5, correlates with highest vaginal bleeding scores, largest decrease in systemic progesterone levels, and maximum expression of mRNA profiles. Notably, prostaglandin-endoperoxide synthase 2 (Ptgs2) is 10-fold higher on day 9.5 and vascular endothelial growth factor (Vegfa) and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression are also upregulated. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Ptgs2 and Vegfa expression are elevated in ectopic lesions in women suffering from endometriosis and PECAM-1 is highly expressed in pelvic endometriotic lesions [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The use of decidualized menstrual endometrium in animal models is critical to attempt to replicate expression of potentially important factors that could be therapeutic targets or participate in the etiology and pathogenesis of endometriosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInjection of decidualized endometrial slurry was largely unsuccessful when compared to placement of discrete biopsies and compared to reported results injecting decidualized endometrium [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, to have similar conditions between groups, we made a 5 mm incision in mice injected with slurry, comparable to the incision made for placement of laparoscope. This differs from other reported models that perform an intraperitoneal injection without laparotomy. The physiologic effects of anesthesia and laparotomy cannot be ignored, and although the laparotomy incision was small and should decrease stress responses compared to larger incisions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] this could have negatively affected the development of endometriotic lesions with slurry injection model. All three paradigms utilizing discrete endometrial biopsies had higher group incidences of endometriosis and higher mean confirmed lesions counts compared to the slurry injection paradigms. It has been previously reported in a chicken chorioallantoic membrane (CAM) model of implantation that larger biopsies (\u0026gt;\u0026thinsp;1mm\u003csup\u003e3\u003c/sup\u003e) resulted in lesion formation compared to no lesion development when endometrial cells were transplanted [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This suggests that intact tissue architecture is crucial to implantation, survival, and proliferation, and could explain the lack of lesions when endometrial slurry was used. However, further exploration of injected menstrual phase endometrial slurry into menstruating mice is warranted, as this could be a non-invasive model allowing for iterative transplantation.\u003c/p\u003e \u003cp\u003eParadigms 1 and 5, (discrete biopsies with laparoscopy) resulted in 100% incidence of endometriosis, while paradigm 2 (discrete biopsies with laparotomy) had an 80% incidence. Additionally, paradigms 1 and 5 had mean lesion counts of 2.7 and 2.6 respectively, with paradigm 2 having 1.1. The discrete endometrial biopsies adhered to abdominal tissues without the use of suture or glue. The spontaneous attachment of menstrual phase endometrium allows for early study of disease progression including angiogenesis, apoptosis, proliferation, inflammation, and chemotactic homing response. There are reported differences in the morphology of mouse peritoneum and post-operative adhesion formation post laparoscopy or laparotomy [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Bulging of mesothelial cells with the presence of intercellular clefts occurs 1\u0026ndash;2 hours after CO\u003csub\u003e2\u003c/sub\u003e insufflation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This phenomenon has been hypothesized to play a role in peritoneal metastasis and infiltration of tumor cells into the sub mesothelial connective tissue matrix [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The biological response of the peritoneum to laparoscopy aids in endometrial biopsy attachment and proliferation, as a compromised mesothelial barrier may play a role in the pathogenesis of the disease [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Surgical treatment has also been reported as a potential risk factor for recurrence of ovarian endometriomas [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Further investigation into the role diagnostic and therapeutic laparoscopy potentially plays in recurrence and progression of endometriosis is warranted and would be possible using this model.\u003c/p\u003e \u003cp\u003eContinued validation and use of this close approximation of \u0026ldquo;best fit\u0026rdquo; animal model of endometriosis will allow for translational exploration of the etiology and pathogenesis of endometriosis and future studies in fertility by using a menstruating, sexually intact and immunocompetent mouse.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Alex Hall, Research Statistician II and \u0026nbsp;Jack Taylor, PhD Biostatistician for their consultation on statistics, the Dept. of Pharmacology \u0026amp; Neuroscience, Dept. of Obstetrics \u0026amp; Gynecology, CUSOM for their support, the Stryker Corporation for their donation of equipment, Ms. Melissa Holzapfel, UNMC Tissue Sciences Facility, Ms. Toni Howard, Creighton University Histology Core Facility, and the Creighton Animal Resource Facility and animal husbandry staff.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Statements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChristina Howe, \u0026nbsp; John Cot\u0026eacute;, and Janee Gelineau- van Waes contributed to the study conception and design. Material preparation, data collection and analysis were performed by Christina Howe, \u0026nbsp;Jodi Hallgren, Marley Bredehoeft, and Janee Gelineau-van Waes. Catherine Stoos performed histopathology \u0026nbsp;The first draft of the manuscript was written by Christina Howe and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflicts of Interest/Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests:\u003c/strong\u003e Christina Howe, Janee Gelineau-van Waes and Catherine Stoos declare they have no financial interests. John Cot\u0026eacute; was a Principal Investigator on endometriosis industry sponsored trials for AbbVie and Myovant. He has received compensation as a member of the endometriosis speaker\u0026rsquo;s bureau for AbbVie and Pfizer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Non-financial interests\u003c/strong\u003e: none.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval/Compliance with Ethical Standards\u003c/strong\u003e: This study was performed in accordance with the \u003cem\u003eGuide for Care and Use of Laboratory Animals\u003c/em\u003e and was approved by Creighton University\u0026rsquo;s Institutional Animal Care and Use Committee. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRB approval is not applicable\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e: Not Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: The original contributions presented in the study and data supporting the conclusions are included in the article; further inquiries can be directed to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e: Not Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by:\u003c/p\u003e\n\u003cp\u003eGeorge F. Haddix President\u0026rsquo;s Faculty Research Fund 04/01/19 \u0026ndash; 03/31/20\u003c/p\u003e\n\u003cp\u003ePartial financial support was received from Robin Farias-Eisner, MD, PhD, MBA, FACOG and the OB/GYN Department, and Peter Abel, PhD, and Dept of Pharmacology \u0026amp; Neuroscience, CUSOM\u003c/p\u003e\n\u003cp\u003eHealth Sciences Strategic Investment Fund (HSSIF) 07/01/21 \u0026ndash; 06/30/23\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZondervan KT, Becker CM, Missmer SA. Endometriosis. \u003cem\u003eN. Engl. J. Med\u003c/em\u003e. 2020;382(13):1244-1256. https://doi.org/10.1056/NEJMra1810764\u003c/li\u003e\n\u003cli\u003e\u003cu\u003e2. \u003c/u\u003eSaunders, PT, \u0026amp; Horne, AW. (2021). 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Genes Linked to Endometriosis by GWAS Are Integral to Cytoskeleton Regulation and Suggests That Mesothelial Barrier Homeostasis Is a Factor in the Pathogenesis of Endometriosis. \u003cem\u003eReprod. Sci.\u003c/em\u003e 2017;24, 803\u0026ndash;811. https://doi.org/10.1177/1933719116660847\u003c/li\u003e\n\u003cli\u003e\u003cu\u003e \u003c/u\u003eLiu X, Yuan L, Shen F, Zhu Z, Jiang H, Guo S. Patterns of and Risk Factors for Recurrence in Women with Ovarian Endometriomas. \u003cem\u003eObstet. Gynecol.\u003c/em\u003e 2007;109(6):p 1411-1420. https://doi.org/ 10.1097/01.AOG.0000265215.87717.8b\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":true,"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":"endometriosis, murine, translational, preclinical animal model","lastPublishedDoi":"10.21203/rs.3.rs-3243174/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3243174/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImproved animal models of endometriosis are needed to accurately represent the pathophysiology of human disease and identify new therapeutic targets that do not compromise fertility. Current mouse models of endometriosis that involve ovariohysterectomy and hormone replacement preclude evaluation of fertility. Menstrual phase endometrium includes potentially important immune cells and inflammatory mediators. Our goal was to develop a novel, translationally relevant mouse model of endometriosis by transplanting donor menstrual endometrium into the peritoneal cavity of menstruating, immunocompetent, intact recipients. We tested various paradigms to determine the most effective method for establishing endometriotic lesions. Initially, 4 paradigms were tested to optimize method of induction. To enhance the model further, a novel paradigm implanted discrete menstrual phase endometrium via laparoscopy into menstruating mice. Vaginal cytology was performed to confirm continued estrus cyclicity. Potential lesions were harvested during proestrus and confirmed to be endometriosis based on histopathology. All mice demonstrated normal estrus cyclicity post induction. Incidence of endometriosis and the difference in average number of lesions across groups was compared. The use of laparoscopy to place discrete menstrual phase endometrium was the most effective method of induction of endometriosis. This method was just as effective when used to induce endometriosis in menstruating recipient mice, representing a novel translationally relevant model that can be used to assess immunologic factors and the impact of therapeutic interventions on fertility.\u003c/p\u003e","manuscriptTitle":"Optimizing a Translational Mouse Model of Endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-30 23:43:57","doi":"10.21203/rs.3.rs-3243174/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":"3fbfeb8e-3954-4415-b81b-3da4ef545bf6","owner":[],"postedDate":"August 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-12T00:34:00+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-30 23:43:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3243174","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3243174","identity":"rs-3243174","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}