{"paper_id":"88a7b2f1-ae92-4f38-a4c2-447999e0f211","body_text":"vv\n036\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\nhttps://dx.doi.org/10.17352/jgroDOI: 2581-5288ISSN: \nCLINICAL GROUP\nAbstract\nObjectives: Endometriosis is a common disease that affects about 10% - 15% of women in their reproductive years worldwide with no curative treatment. The most \ncommon symptom of endometriosis is debilitating pelvic/abdominal pain. Current therapeutic options have limited insight into the disease mechanism and include drugs \nand/or surgery, which may be ineffective over the long term with unwanted side effects. We aimed at establishing a translational rodent endometriosis model that can be \nused to identify novel therapies. The validity of the model was conﬁ rmed by investigating the effect of the clinically-used GnRH agonist, leuprolide.\nMethods: Endometriosis was induced by a surgical procedure in adult non-pregnant female Sprague Dawley rats in the diestrus or estrus stage (cycle determination \nby vaginal smear). One group of rats received a subcutaneous injection of leuprolide at 1mg/kg, every 4 weeks. Following the tr eatment period, we performed a direct \nassessment of the endometriosis-induced abdominal pain using the Von-Frey method and spontaneous pain using the abdominal licki ng test. Then, the lesions were \nexcised and measured.\nResults: Abdominal pain threshold was decreased by more than 2 fold in rats with surgically-induced endometriosis compared to sham rats.  Leuprolide treatment \nsigniﬁ cantly increased the threshold force required to elicit a behavioral withdrawal response in rats suffering from endometriosis. The observed pelvic ﬂ oor mechanical \nhyperalgesia has not been correlated to the growth of endometriosis lesions. The hormonal cycle at the surgery induction in ﬂ uenced the endometriosis lesions growth. \nLeuprolide signiﬁ cantly inhibited the growth of endometriosis-like lesions.\nConclusions: we have established, based on previously reported rodent models, a model of endometriosis-associated pain that responds to clinically active drugs and \ncan, therefore, be used to identify novel therapies and investigate some of the pathophysiological mechanisms involved in endometriosis.\nResearch Article\nEstablishment and validation of \na rodent model of endometriosis \nto evaluate the effect of new \ntherapeutic strategies\nRana Assaly1*, Sandrine Compagnie1, Laurine Allimonnier1, \nManon Bracconi1, Francois Giuliano2 and Delphine Behr-\nRoussel1 \n1Pelvipharm, Montigny-le-Bretonneux, France\n2AP-HP, Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation, Raymond Poincaré \nhospital, Garches, France\nReceived: 06 October, 2022\nAccepted: 28 October, 2022\nPublished: 29 October, 2022\n*Corresponding author: Rana Assaly, : PharmD, PhD, \nPelvipharm, Montigny-le-Bretonneux, France, Tel: +33 1 \n70 42 93 47; E-mail: \n \nORCID: https://orcid.org/0000-0001-9521-0956\nKeywords: Endometriosis; Preclinical; Translational \nmodel; In vivo; Pharmacology; Pain; GnRH agonist; \nTreatment\nCopyright License: © 2022 Assaly R, et al. This is an \nopen-access article distributed under the terms of the \nCreative Commons Attribution License, which permits \nunrestricted use, distribution, and reproduction in any \nmedium, provided the original author and source are \ncredited.\nhttps://www.peertechzpublications.com\nIntroduction\nEndometriosis is the presence of ectopic endometrial tissue \noutside the uterine cavity and is a common gynecological \ndisease reported in 10% - 15% of women during their \nreproductive years in the world [1]. The most common \nsymptom of endometriosis is debilitating pelvic/abdominal \npain. In fact, up to 70% of women suffering from pelvic \npain are affected by endometriosis. Other symptoms include \ndyspareunia, severe dysmenorrhea, and dysuria. Women with \nthe condition also suffer from co-occurring painful conditions, \nincluding interstitial cystitis/painful bladder symptoms and \nirritable bowel syndrome and 50% of these women suffer from \ninfertility [2].\nThe disease has a huge negative impact on women’s \nquality of life, work productivity, sexual relationship and self-\nesteem [3]. The most common theory proposed to explain \nthe pathophysiology of the endometriosis is retrograde \n\n037\nhttps://www.peertechzpublications.com/journals/journal-of-gynecological-research-and-obstetrics\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\nmenstruation hypothesis but also other hypotheses have been \ndiscussed like in ﬂ ammatory factors, dysregulated immunity, \nhormones and genetic and epigenetic factors        [4]. None of \nthese mechanisms could explain alone the different types and \nsymptoms of endometriosis. Current therapeutic options have \nlimited insight into the disease mechanisms and include drugs \nand/or surgery to reduce symptoms and manage complications. \nThese available treatments provide short-term solutions but \ntend to be ineffective over the long term with a high incidence \nof unwanted side effects such as premature bone loss and \nvaginal dryness. The most common medical treatment for \nendometriosis is gonadotropin-releasing hormone (GnRH) \nagonists and estrogen/progestin combinations [5,6]. In \nrecent years, the search for more effective and less invasive \ntherapeutic curative strategies to reduce years of suffering \nin women with endometriosis is receiving increased research \nattention. The development and validation of In vivo models \nare necessary to allow the preclinical testing of the ef ﬁ cacy of \npotential new treatment options.\nNon-human primates are considered the most \nphysiologically relevant model of endometriosis since these \nanimals develop spontaneous endometriosis [7]. However, \ntheir use is limited by cost and ethical concerns. Thus, rodent \nmodels of endometriosis [8] have been developed to investigate \nthe effect of the presence of endometriosis lesions on functional \noutcomes and the extent of its reversal by new compounds. \nHowever, it is dif ﬁ cult to develop a unique model that \nreplicates all symptoms and aspects of a complex disease such \nas endometriosis. Indeed, endometriosis is a heterogeneous \ndisease with poorly known etiology and several phenotypes such \nas ovarian endometrioma, super ﬁ cial peritoneal disease and \ndeep inﬁ ltrating endometriosis [4]. In addition, endometriosis \nis multifactorial and has been associated with environmental, \ngenetic, immunological and hormonal factors [9]. Different \nmodels have been developed in mice and rats and recently \nreviewed by Bruner-Tran, et al.  [8], each model emphasizing \none or several features to explore disease-related mechanisms, \nto identify therapeutic targets and/or to provide insight into the \ndevelopment of co-morbidities. Pain is considered a signiﬁ cant \ncontributor to endometriosis morbidity and pelvic pain is one of \nthe most described symptoms [10], even though asymptomatic \ncases are described [11], this study aimed at establishing a \nrodent model of endometriosis to be a useful tool in evaluating \nendometriosis-related pelvic pain. To that end, we developed \na rodent model of endometriosis in immunocompetent rats \nbased on surgically-induced endometriosis originally described \nby Vermon and Wilson [12] to evaluate endometriosis-\ninduced pain by assessing speci ﬁ cally pelvic pain while others \nextensively evaluated generalized pain behavior. In addition, \nendometriosis diagnosis is based on the analysis of the lesions \ncollected during laparoscopic surgery [13] and the American \nSociety of Reproductive Medicine classi ﬁ ed the disease into \n4 stages according to the evaluation of the endometriotic \nlesions [14]. Thus, this study also described the proliferation of \nendometriosis lesions being a major feature of endometriosis. \nEven though the model described in this study is based on \npreviously published studies reviewed by others [8,15], very \nfew authors reported the impact of the estrous stage on \nendometriosis-induced models in rodents. Hence, this study \nwas designed to investigate if any in ﬂ uence of the estrous \nstage at induction of endometriosis on both primary endpoints \ni.e., pelvic pain and endometriosis lesions size. To validate the \ntranslational value of this model, we tested the effect of one of \nthe most clinically used medical options, a GnRH agonist on \npelvic pain and lesions size.\nMaterials and methods\nEndometriosis-induced surgery and treatment adminis-\ntration\nAdult non-pregnant female Sprague Dawley rats (Elevage \nJanvier, Le Genest-St-Isle, France, 6-8 weeks old) were housed \nat least 10 days prior to the beginning of the experiments with \nfree access to standard chow (Chow M20, 841201, SDS, UK) \nand water and maintained on an inversed 12h dark/light cycle \n(10:00/22:00). All procedures were approved by the local ethical \ncommittee (CEE47) and performed in accordance with the \nlegislation on the use of laboratory animals (NIH publication \nN° 85 - 23, revised 1996) and Animal Care Regulations in force \nin France as of 1988 (authorization from competent French \nMinistry of Agriculture - Agreement No. B78-423-1, July 2017).\nAfter the acclimation period, the reproductive status was \ndetermined by vaginal lavage using traditional nomenclature \nfor the 4 estrous stages (proestrus, estrus, metestrus and \ndiestrus). After the con ﬁ rmation of one full cycle by daily \nvaginal lavage, the rats were identi ﬁ ed with a unique \nidentiﬁ cation number; then using a random number table, they \nwere randomly allocated to different experimental groups (n = \n12 rats per group): sham group (SHAM), endometriosis group \n(ENDO) and treated group (ENDO+leuprolide). \nWhen in the diestrus stage, all animals underwent either \nsham the surgical procedure or endometriosis induction by a \nsurgical procedure based on the initial protocol described by \nVernon and Wilson in rats [12] and Cummings and Metcalf [16].\nIn addition, in order to evaluate the inﬂ uence of the estrous \nstage on pelvic pain and lesions size, a group of rats ( n = 12) \nthat underwent endometriosis induction when in the proestrus/\nestrous stage was added to this study.\nWhether surgical induction was conducted in the diestrus \nor proestrus/estrus stage, the rats were anesthetized with \nisoﬂ urane (1% - 1.2%, Centravet, France) under aseptic \nconditions and using a heating pad to maintain a body \ntemperature of the animal at 37 °C. A midline incision was \nmade through the skin and muscle layer to expose the pelvic \nand abdominal organs. A segment of the mid-left uterine \nhorn was excised, six 2x2 pieces of the excised uterus were \nprepared and each piece was sutured onto alternate mesenteric \narteries using 4.0 nylon sutures. The same surgical procedure \nwas applied for the sham group except for the step of suturing \nuterine pieces. Then, the muscle layer and the skin were closed \nusing polyester suture (Vetsure© Bond) and the animal was \nclosely monitored during recovery. Following surgery, animals \nwere carefully monitored for health status and weighed twice \nper week. One week after surgery, the rats allocated to the \n\n038\nhttps://www.peertechzpublications.com/journals/journal-of-gynecological-research-and-obstetrics\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\ntreated group received a single subcutaneous injection of the \nGnRH agonist, leuprolide at 1mg/kg repeated once on day \n28 [17]. All endpoints were reported 6 weeks post-surgical \nprocedure.\nPelvic pain assessments\nA vaginal cytology smear was performed to determine the \nestrous stage at the time of pain assessment.\nA widely accepted method to evaluate mechanical \nhyperalgesia in conscious animals is the Von Frey ﬁ laments. \nThis method is based on behavioral observations of animal \nresponse to mechanical stimuli of increasing forces applied to \nthe plantar area by means of ﬁ laments with grading forces and \nconsists of determining the hind paw withdrawal threshold \nas a referred pain-associated behavior. In this study, we \nadapted the Von Frey method to apply to the pelvic area and \nto determine a pelvic pain nociception threshold. Brieﬂ y, a few \ndays before the Von Frey experiments, the rat abdomen was \nshaved under light iso ﬂ urane anesthesia (Centravet, France). \nOn the day of assessment, the rat bladder was emptied by \nmanual compression and the rat was placed in a metabolic \ncage for a minimum 30-minute acclimatization period before \nevaluation.\nMonoﬁ laments of differing forces ranging from 0.008 to \n4g (Bioseb, Vitrolles, France) were applied in increasing order \nof force and rat behavior was recorded according to a pain \nbehavior rating scale (score 1 to 4). The forces of the von Frey \nﬁ bers used in this study were determined in a preliminary test \nbased on the modiﬁ ed up-and-down method [18]. Five repeated \nstimulations per ﬁ lament with 5 - second intervals were \nperformed, followed by 3-minute intervals before applying \nthe next higher force ﬁ lament. Care was taken to stimulate \ndifferent areas within the lower abdominal region to avoid \ndesensitization. The pain threshold was determined by the \nlower force evoking a pain reaction of the rat characterized by \nabrupt retraction of the abdomen, jumping, and/or immediate \nlicking of the site of application (score 3 and above).\nAlso, spontaneous pain was evaluated by the abdominal \nlicking test which is used as an indicator of abdominal \ndiscomfort in various pelvic pain models [19,20]. This test \nwas performed in an open- ﬁ eld home cage and consists of \nobserving, after allowing the rat to acclimate to the cage, the \nnumber of times the rat licked the abdominal region [21]. The \naverage number of abdominally-directed licking was recorded \nover two 10 min periods by an experimenter blinded to the \ngroups.\nLesions measurement\nFollowing behavioral assessments, the rats were \nanesthetized and then euthanized by an intracardiac injection \nof pentobarbital (Eutasol®, Centravet, France), the abdominal \ncavity opened and the lesions measured by an in-house \ndeveloped method adapted from a described method by Becker, \net al. [22]. Brieﬂ y, the harvested lesions were aligned on graph \npaper, and the image was captured and analyzed using Image \nJ software by measuring for each lesion two perpendicular \ndiameters (D1 and D2) and calculating a cross-sectional area \n(CSA) using the formula for an ellipse: (D1xD2x π/4). Then, \nthe CSA of the lesions harvested from the same animal was \naveraged to calculate a mean CSA for each animal and ﬁ nally \nto compute the average CSA per experimental group for \ncomparison between groups.\nStatistical analysis\nAll results are presented as mean ± SEM: Some rats were \nexcluded if the endometriosis-like lesions were not present at \nharvesting or if the behavior of the rat during pain evaluation \nwas agitated not allowing assessment. A ﬁ nal n = 10 rats/group \nwas included in the ﬁ nal analysis.\nStudent’s t-test or one-way ANOVA followed by Dunnett’s \nmultiple comparisons post-test were used when appropriate \nto compare the means of two groups. Statistical analysis was \nperformed with GraphPad Prism® 6.05 software. p values < \n0.05 were considered signiﬁ cant.\nResults\nInﬂ uence of estrous cycle on pelvic pain and lesions size\nPelvic pain threshold determined by Von Frey ﬁ laments \nin endometriosis rats signi ﬁ cantly decreased by 2 fold \ncompared to the SHAM group 6 weeks post-induction surgery \nindependently of the estrous stage at the time of endometriosis \ninduction (Figure 1).\nHowever, the mean cross-sectional lesion area was \nsigniﬁ cantly larger compared with the mean area of the lesions \nat the time of the surgical induction of endometriosis (3.8 ± 0.8 \nmm\n2 vs. 2.2 ± 0.1 mm2, respectively, Figure 2) when the surgical \ninduction of endometriosis was achieved in diestrus stage but \nnot in proestrus/estrus stage. In fact, when the surgery was \nconducted in the proestrus/estrus stage, the mean cross-\nsectional lesion area was smaller compared with the mean area \nof the lesions at the time of the surgery (1.7 ± 0.2 mm\n2 vs. 3.0 ± \n0.1 mm2, respectively, Figure 2).\nSH\nAM \nEND\nO \ndie\nstrus \nENDO\n pr\no/estr\nus \nPain threshold (g)\nFigure 1: Effect of estrous cycle on pelvic pain threshold determined by Von Frey \nexperiments. Von Frey experiments were performed 6 weeks post sham surgery \n(SHAM, n = 10) or endometriosis induction surgery when rats were at diestrus stage \n(ENDO diestrus, n = 10) or estrus stage (ENDO pro/estrus, n = 10). Data are mean ± \ns.e.m *p < 0.05, Dunnett’s multiple post-test vs. SHAM.\n\n039\nhttps://www.peertechzpublications.com/journals/journal-of-gynecological-research-and-obstetrics\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\nOf note, the lesions prepared from the uterus when the rat \nwas in the estrus/proestrus stage were larger than the lesions \nprepared when the rat was in the diestrus stage.\nConsequently, the establishment and validation of the \nmodel were continued using rats in the diestrus stage at the \ninduction of endometriosis and presented here.\nBody weight evolution\nThe body weight progressively increased, starting from \nthe ﬁ rst-week post-surgery until the end of the experimental \nperiod for all groups of rats. The rats in the ENDO group \nshowed the same body weight evolution compared to sham-\noperated rats. However, the leuprolide-treated endometriosis \nrats (ENDO+leuprolide) showed signi ﬁ cantly higher body \nweight evolution than ENDO rats (Figure 3). Indeed, the rats \nthat received leuprolide showed a sharp increase in body weight \ngain the ﬁ rst 2 weeks after treatment administration compared \nto the day of starting the treatment (12 ± 1% the ﬁ rst week and \nthe second week) compared to the ENDO group (5 ± 1% the ﬁ rst \nweek and the second week)\nOf note, the body weights, before starting the treatment \nperiod, were comparable between all groups of rats (232 ± 9 g, \n226 ± 3 g, and 224 ± 4 g for SHAM, ENDO and ENDO+leuprolide \ngroups, respectively; One-Way ANOVA, ns, p > 0.05).\nPelvic pain assessments\nAs mentioned earlier, pelvic pain threshold in the ENDO \ngroup was signi ﬁ cantly decreased by more than 2 fold \ncompared to the SHAM group 6 weeks post-induction surgery \n(Figure 1). Leuprolide at 1mg/kg administered subcutaneously \nonce every 28 days shifted pain responses towards higher \nforces indicating that rats in the ENDO+leuprolide group are \nless sensitive to pain compared to rats from ENDO group. Thus, \nthe GnRH agonist leuprolide decreased abdominal hyperalgesia \ninduced by endometriosis surgery by signi ﬁ cantly increasing \nthe pain threshold (Figure 4).\nLicking behavior remained almost unchanged in SHAM \nrats before and after surgery (+5%) while a slight increase in \nabdominal-directed licking was observed in ENDO rats after \nsurgery (+15%), however, this increase did not reach statistical \nsigniﬁ cance (Student’s t-test, ns, p > 0.05). Consequently, the \nabdominal-directed licking behavior assessment design should \nbe reconsidered with some modi ﬁ cations to the experimental \nconditions to allow the evaluation of the effect of potential \ntherapies. Indeed, some authors reported abdominal direct \nlicking behavior in mice and rats using different conditions \ncompared to the ones used in this study such as increasing the \nnumber of animals [21], increasing the observation window \n[19] and taking into account abdominal directed licking when \npart of the normal grooming behavior [20].\nLesions measurement\nThe presence of lesions was con ﬁ rmed in 90% of \nendometriosis rats (ENDO and ENDO+leuprolide groups). \nThe mean cross-sectional lesion area was signi ﬁ cantly larger \ncompared with the mean area of the lesions at the time of the \nsurgical induction of endometriosis (3.8 ± 0.8 mm\n2 vs. 2.2 ± \n0.1 mm2, respectively). Treatment with leuprolide suppressed \nby 60% the growth of endometriosis-like lesions ( p < 0.05, \nStudent’s t-test).\nFigure 2: Representative pictures of the different range of lesions at induction of \nendometriosis and day of harvesting when rat was in estrus (A and B) or disterus \n(C, D and E).\nBody weight (g)\n****\nFigure 3: Evolution of body weight post-surgery and during the treatment period. \nRats underwent either sham surgery (SHAM, n = 10), endometriosis induction \nsurgery (ENDO, n = 10) or endometriosis induction surgery and tretad with leuprolide \n(ENDO+leuprolide, n = 10). Data are mean ± s.e.m ****p < 0.01, two-way ANOVA test.\nSHAM \nEN\nDO \nENDO \n+ leuprolide\nPain threshold (g)\nFigure 4:  Effect of subcutaneous leuprolide at 1mg/kg on pelvic pain threshold \ndetermined by Von Frey experiments. Von Frey experiments were performed 6 \nweeks post sham surgery (SHAM, n = 10) or endometriosis induction (ENDO, n = \n10) or endometriosis induction and treatment with leuprolide 1lmg/kg once every \n4 weeks (ENDO +leurpolide, n = 10). Data are mean ± s.e.m * p < 0.05, Dunnett’s \nmultiple post-test vs. SHAM, & p < 0.05, Dunnett’s multiple post-test vs. ENDO.\n\n040\nhttps://www.peertechzpublications.com/journals/journal-of-gynecological-research-and-obstetrics\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\nDiscussion\nThis study described a translational rat model of \nendometriosis-associated pain. The validity of the model was \nconﬁ rmed by investigating the effect of the clinically-used \nGnRH agonist, leuprolide.\nFirst, this study investigated the potential in ﬂ uence of the \nhormonal cycle on the growth of endometriosis lesions. Some \nauthors used uterine tissues at the proestrus/estrus phase \n[12,23] since the endometrium is thicker compared to other \nstages providing enough tissue for suturing in line with our \nobservations. However, a correlation between the volume of \nendometriosis lesions and the estrous cycle has been described \n[24] while others reported that lesion size is independent of \nthe estrous stages [25]. Thus, the ﬁ rst aim of this study was to \nexamine the inﬂ uence of the estrous stage and to set the stage \nto be used in subsequent experiments.\nEndometriosis is an estrogen-dependent and dynamic \ndisorder. Indeed, endometriosis lesions’ appearance and \nprogression/regression are dependent on hormonal levels. \nIt has been reported that endometriosis lesions growth is \ntriggered by high estrogen levels while low estrogen levels lead \nto lesions regression [26]. Besides, it is known that estrogen \nlevel starts to decrease at late proestrus corresponding to the \nfollicular phase in women that is characterized by the release \nof luteinizing hormone and follicular-stimulating hormone \n[27]. The increase in follicular stimulating hormone induces \novulation matching the estrus stage of the cycle in rodents \nduring which the estrogen levels decrease and prolactin levels \npeak. During the metestrus/diestrus stage, progesterone \nlevels increase, corresponding to the luteal phase in women, \nfollowed by a peak in estrogen levels during the late luteal \nphase, a decline in progesterone levels, and regression of the \ncorpus luteum, leading to menstruation in women. The late \nluteal phase and the regression of the corpus luteum phase \ncorrespond to the diestrus stage in rodents [28,29].\nTaken together, it is plausible to suggest that the diestrus \nstage in rodents is comparable to the menstrual phase in women \nand the changes in estrogen/progesterone levels during the \nestrous cycle may inﬂ uence the endometriosis lesions growth. \nAccordingly, this study is to our best knowledge among the few \nstudies showing by direct comparison that the estrous cycle \nsigniﬁ cantly affected the size and the macroscopic growth of \nendometriosis lesions using an autologous endometriosis rat \nmodel without ovariectomy. Indeed, signi ﬁ cant growth of \nendometriosis lesions was observed when the preparation of \nthe lesions and the induction of the surgery was done when the \nrats were in the diestrus stage but not when in the proestrus/\nestrus stage.\nIf the main characteristic of endometriosis is the presence of \ntissue lesions containing stromal, epithelial and inﬂ ammatory \ncells, the main clinical sign of endometriosis is pain, even though \nasymptomatic endometriosis has been reported [4,6]. Women \nsuffering from endometriosis described different intensities \nand types of pains ranging from severe dysmenorrhea to \nchronic pelvic pain accompanied or not with other comorbid \npain conditions leading to a signi ﬁ cant impact on the quality \nof life of patients [30]. Recent studies have illustrated that \nwomen with endometriosis develop signi ﬁ cant sensitivity \nto any stimuli as a consequence of central sensitization [31]. \nThus, the development of new and effective therapies is a \nresearch priority that requires the development of appropriate \npre-clinical models of pain-associated endometriosis and \nvalidated tools for pain assessment which can advance our \nunderstanding of this condition of central sensitization. \nThe ﬁ rst publications describing surgically induced rodent \nmodels of endometriosis focused mainly on lesion description \n[12,16,22,32]. More recently, investigators developing rodent \nmodels of endometriosis models have given more focus on pain \nendpoint but not speciﬁ cally pelvic pain and rather generalized \npain by performing paw withdrawal behavioral tests as an \nindicator of stimulus-evoked pain [21,33,34]. Indeed, only a \nminority of studies measured pelvic pain [20,35-37]. Thus, we \naimed in this study to set up a speci ﬁ c pelvic-pain-associated \nendometriosis model to allow in the future to understand the \nrelated mechanisms. Thus, we investigated both phenotypes \nof pain, the stimulus evoked pelvic pain response and the \nspontaneous pain as indicators of chronic intermittent pain \nreported by patients suffering from endometriosis.\nIndeed, this study showed a signi ﬁ cant increase in pain \nperception in rats suffering from endometriosis. Measurement \nof pain was performed by evaluation of behavioural responses \nstimulus- dependent using Von Frey ﬁ laments. Furthermore, \nrats suffering from endometriosis showed exacerbated \nspontaneous pain behavior compared to sham rats, even \nthough the design of this study did not allow to reach a \nstatistical signi ﬁ cance in the number of times the rat licked \nthe abdominal region, and thus it needs to be ﬁ ne-tuned in \nthe future experiments. In fact, very few authors reported \nboth phenotypes of pain in a rodent model. Indeed, pelvic and \nspontaneous pain was recently reported in a non-surgical mice \nmodel [36] but not yet in a rat model of endometriosis.\nIt is now well established that the association between the \nseverity of pelvic pain and the endometriosis lesions growth is \ninconsistent [38]. Stratton and Berkley [39] have shown that \na major contributing factor to endometriosis-associated pain \nis not the ectopic lesions growth but rather the development \nof nerve supply creating a direct interaction between lesions \nand the central nervous system. Another feature that has \nbeen associated with the severity of endometriosis pain is the \nvascularity of the lesions and the development of new blood \nvessels. This coordinated growth of blood vessels and nerves \nhas been termed “neuroangiogenesis” [40]. In line with these \nsuggestions, the model described in this study mimics pain-\nassociated endometriosis and the non-correlation between the \nseverity of pelvic pain and the growth of the endometriosis \nlesions.\nThe validation of the translational value of this model \nwas con ﬁ rmed by testing one of the most clinically-used \ncompounds, leuprolide, a GnRH agonist. Subcutaneous \nleuprolide administered in a dosage regimen similar to its \nclinical use signi ﬁ cantly decreased pain sensation in rats \nsuffering from endometriosis as well as the endometriosis \n\n041\nhttps://www.peertechzpublications.com/journals/journal-of-gynecological-research-and-obstetrics\nCitation: Assaly R, Compagnie S, Allimonnier L, Bracconi M, Giuliano F, et al. (2022) Establishment and validation of a rodent model of e ndometriosis to evaluate the \neffect of new therapeutic strategies. J Gynecol Res Obstet 8(3): 036-042. DOI: https://dx.doi.org/10.17352/jgro.000114\nlesions growth. Indeed, in clinical practice, leuprolide and \nmore broadly the GnRH agonist are prescribed as second-line \ntherapy in endometriosis-related pain with a satisfactory result \non pain and on decreasing lesions volume when the ﬁ rst-line \nsuch as combined oral contraceptives and progestin fail or due \nto intolerance or contraindications which represent 1/4 to 1/3 of \npatients [41]. However, the GnRH agonists induce an arti ﬁ cial \nmenopause state and thus add-on therapy is needed to limit \nsome related risks such as premature bone mass density loss. \nMoreover, this type of treatment can only be prescribed to \npatients who do not wish to become pregnant [4].\nIn conclusion, there is a need to develop more effective \ntherapeutic and curative strategies with fewer unwanted \neffects. To this end, it is necessary to establish a reliable and \nstandardized animal model of endometriosis. In this study, we \nhave established a model of endometriosis-associated pain \nthat responds to clinically active drugs and can, therefore, be \nused to identify novel therapies. It should be noted, however; \nno model is expected to mimic all aspects of the women’s \ndisorder and it is probable that the new therapies have to be \ntested in several models, each replicating some features of the \ndisease to improve the transitional value and to increase the \nsuccess rate in clinical trials.\nAcknowledgement\nThis work was supported by a restricted grant from the « \nPlan France 2030 » National Program.\nReferences\n1. Parasar P, Ozcan P, Terry KL. Endometriosis: Epidemiology, Diagnosis and \nClinical Management. Curr Obstet Gynecol Rep. 2017 Mar;6(1):34-41. doi: \n10.1007/s13669-017-0187-1. Epub 2017 Jan 27. PMID: 29276652; PMCID: \nPMC5737931. \n2. Bulletti C, Coccia ME, Battistoni S, Borini A. Endometriosis and infertility. 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