{"paper_id":"439c8779-f455-4451-9e17-09f376a06d26","body_text":"R E S E A R C H Open Access\nAnti-platelet therapy holds promises in\ntreating adenomyosis: experimental\nevidence\nBo Zhu 1†, Yumei Chen 1†, Xiaolu Shen 1, Xishi Liu 2,3 and Sun-Wei Guo 2,3*\nAbstract\nBackground: Recently emerging evidence indicates that endometriotic lesions are wounds undergoing repeated\ntissue injury and repair (ReTIAR), and platelets induce epithelial-mesenchymal transition (EMT), fibroblast-to-\nmyofibroblast transdifferentiation (FMT), leading ultimately to fibrosis. Due to the commonality of cyclic bleeding as\nin endometriosis, adenomyotic lesions are also wounds that undergo ReTIAR, and we have recently provided\nevidence corroborating platelet-induced EMT, FMT and fibrogenesis in adenomyosis. This study sought to evaluate\nthe effect of antiplatelet therapy in a mouse model of adenomyosis.\nMethods: Adenomyosis was induced in 57 female ICR mice with neonatal dosing of tamoxifen, while another 12\n(group C) were dosed with solvent only, serving as a blank control. Starting from 4 weeks after birth, hotplate test\nwas administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly\ndivided into 6 groups: untreated, low- and high-dose Ozagrel, low- and high-dose anti-mouse GPIb α polyclonal IgG\nantibody to deplete platelets, and isotype-matched inert IgG non-immune antibody. Group C received no\ntreatment. After 3 weeks of treatment, they were hotplate tested again, their uterine horns and brains were\nharvested, and a blood sample was taken to measure the plasma corticosterone level by ELISA. The left uterine\nhorn was used for immunohistochemistry analysis. The brainstem nucleus raphe magnus (NRM) sections were\nsubjected to immunofluorescence staining for GAD65. The depth of myometrial infiltration and uterine contractility\nwere evaluated.\nResults: We found that both Ozagrel treatment and platelet depletion dose-dependently suppressed myometrial\ninfiltration, improved generalized hyperalgesia, reduced uterine contractility, and lowered plasma corticosterone\nlevels, improved the expression of some proteins known to be involved in adenomyosis and slowed down the\nprocess of fibrogenesis. It also elevated the number of GAD65-expressing neurons in the brainstem NRM, possibly\nboosting the GABAergic inhibition of pain due to adenomyosis.\nConclusion: This study further provides evidence that platelets play important roles in the development of\nadenomyosis. Anti-platelet treatment is efficacious in suppression of myometrial infiltration, improving generalized\nhyperalgesia, reducing uterine hyperactivity and systemic corticosterone levels. Collectively, these results\ndemonstrate that anti-platelet therapy seems to be promising for treating adenomyosis.\nKeywords: Adenomyosis, Generalized hyperalgesia, Hotplate latency, Mouse, Ozagrel, Platelet, Uterine contractility\n* Correspondence: hoxa10@outlook.com\n†Equal contributors\n2Shanghai Key Laboratory of Female Reproductive Endocrine-Related\nDiseases, Shanghai 200011, China\n3Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419\nFangxie Road, Shanghai 200011, China\nFull list of author information is available at the end of the article\n© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 \nDOI 10.1186/s12958-016-0198-1\n\nBackground\nAdenomyosis is a common gynecologic disorder with a\npoorly understood pathogenesis [1]. As in endometriosis,\nit is characterized by the ectopic deposition and growth\nof endometrial glands and stroma deep and haphazardly\ninto the myometrium [1]. It shares with endometriosis\nmany similarities in terms of estrogen-dependency, pro-\ngesterone resistance, symptomology, and many molecu-\nlar aberrations but differs in risk factors, age at onset\nand, possibly, etiology [2]. Similar to endometriosis, our\ncurrent knowledge of its pathophysiology is still woefully\ninadequate. Consequently, treatment of adenomyosis has\nbeen a challenge [3], with hysterectomy being the treat-\nment of choice for severe symptomatic adenomyosis.\nThus, medical treatment of adenomyosisis still an unmet\nmedical need.\nAdenomyosis is first and foremost viewed as an\nestrogen-dependent disease, featuring increased local\nproduction of estrogen [4]. It also displays signs of\ninflammation, characterized by the constitutive activa-\ntion of NF-kB [5], increased macrophage infiltration\n[6], and elevated expression of COX-2, a rate-limiting\nenzyme in catalyzing prostaglandin (PG) E2 (PGE 2)\n[7], and increased production of proinflammatory cy-\ntokines and chemokines [8]. All existing therapeutics\nfor adenomyosis are hormonal drugs.\nAs with endometriosis, the ectopic endometrium in ade-\nnomyosis also experience cyclic bleeding. Yet bleeding, an\nindication of vascular injury, is a cardinal hallmark of a\nwound or tissue damage. Cons equently, a physiological\nprocess, called wound healing o r tissue repair, ensues. As\nsuch, platelets must be involved, as shown recently for\nendometriosis [9]. In fact, based on serial immunohisto-\nchemistry analyses of ectopic endometrium in a mouse\nmodel of adenomyosis, we recently report that activated\nplatelets coincide with TGF-β1 release and the induction of\nTGF-β/Smad signaling pathway in adenomyosis, as well as\nevidence of epithelial-mesenchymal transition (EMT)\nand fibroblast-to-myofibrobl ast transdifferentiation\n(FMT), resulting ultimately in fibrosis [10] and also\nsmooth muscle metaplasia (Shen et al., unpublished\ndata). These observations are confirmed in human\nadenomyosis [11]. Therefore, due to the commonality\nshared with endometriosis, i.e., cyclic bleeding, adeno-\nmyotic lesions behave just like endometriotic lesions,\nwhich are essentially wounds that undergo repeated\ntissue injury and repair (ReTIAR) [9, 12, 13].\nIn light of the important roles that platelets play in the\ndevelopment of endometriosis [9, 14, 15] and adenomyosis\n[10, 11], one may wonder as whether anti-platelet therapy\nmay have any potential in treating adenomyosis. This study\nwas undertaken to test the hypo thesis that anti-platelet\ntreatment, by either platelet de pletion or administration of\nOzagrel, indeed has potential for therapeutic purposes.\nMethods\nChemicals\nOzagrel, a thrmboxane A2 (TXA 2) synthese inhibitor\n[16], was purchased from YaoDa Pharmacology Industry\nCompany (Shenyang, China) and was dissolved in 0.9 %\nnormal saline for intraperitoneal administration. The rat\nanti-mouse GPIb α polyclonal IgG antibody and its\nisotype-matched non-immune rat anti-mouse IgG anti-\nbody were purchased from Emfret Analytics (Eibelstadt,\nGermany). Tamoxifen citrate was purchased from Fudan\nForward Pharmaceutical Company (Shanghai, China).\nAll other chemicals were purchased from Sigma unless\nstated otherwise.\nAnimals and the procedure for induction of adenomyosis\nFour pregnant ICR mice with a gestational age of 15 –16\ndays were purchased from Shanghai Laboratory Animal\nCorporation (Shanghai, China) and each of them was\nhoused in a single cage during the rest of the gestation\nperiod and the ensuing birth and nursing period. Their\npups (1 day after birth) were sexed and the female pups\nwere selected for use in this study. The same litter of\npups and the dam were housed in the same cage until\nweaned. All mice were housed in an animal care facility\nunder controlled conditions (20 °C, 12:12 light/dark\ncycle with lights on at 6:00 AM) and had free access to\nchow and fresh water.\nFollowing Parrott et al. [17, 18], and as reported previ-\nously [2, 19, 20], adenomyosis was induced by orally\ndosing female neonatal mice with 1 mg/kg tamoxifen\nsuspended in peanut oil/lecithin/condensed milk mix-\nture (2:0.2:3, by volume) at a dose volume of 5 μl/g\nbodyweight from day 2 to day 5 after birth. Female con-\ntrol neonatal mice, selected randomly, were fed similarly\nwith the same amount of solvent, without tamoxifen.\nWhen these female mice reached 3 weeks of age, they\nwere weaned and separated from the dams.\nAll experiments were performed under the guidelines\nof the National Research Council ’s Guide for the Care\nand Use of Laboratory Animals [21] and approved by\nthe institutional experimental animals review board of\nShanghai OB/GYN Hospital, Fudan University.\nExperimental protocol\nThis experiment was conducted side-by-side with another\nexperiment evaluating the efficacy of epigallocatechin-3-\ngallate (EGCG) in treating adenomyosis in mice, as re-\nported in [20]. Fifty-six female neonatal pups were orally\ndosed with tamoxifen from day 2 to day 5 after birth,\nwhile another 12 were dosed in similar fashion with the\nsolvent only (control group, or group C). Starting from\n4 weeks after birth, hotplate test was administered to all\nmice every 4 weeks, as described previously [2, 19] (see\nAdditional file 1 for full description). At the 16th week\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 2 of 16\n\nafter birth, all mice dosed with tamoxifen were randomly\ndivided into6 groups of roughly equal size, Group U ( n =\n9), or the untreated group, received the vehicle only.\nAnother two groups received, for 3 weeks, daily intraperi-\ntoneal (i.p.) administration of either low-dose (12.5 μg/g)\nOzagrel (Group L, n = 10) or of high-dose (25 μg/g)\nOzagrel (Group H, n = 10). The Ozagrel doses were deter-\nmined based on the dosage given to an adult female and\nthen converted from human to mouse based on body sur-\nface area and further adjusted based on each mouse ’s\nbodyweight measured every day before Ozagrel adminis-\ntration. The mice in group C received no treatment at all\nand served as a blank control. The other three groups\nwere intravenously (i.v.) administrated with different doses\nof rat anti-mouse GPIb α polyclonal IgG and non-immune\nrat anti-mouse IgG: Group LD ( n = 10) received a low-\ndose (1 μg/g) rat anti-mouse GPIb α polyclonal IgG treat-\nment; Group HD ( n = 9) received a high-dose (2 μg/g) rat\nanti-mouse GPIbα polyclonal IgG treatment; Group NI ( n\n= 8) received 1 μg/g non-immune (NI) rat anti-mouse IgG\nisotope-matched with the anti-GPIb α antibody; The mice\nin group C were treated the same as above. The dosages\nof rat anti-mouse GPIb α polyclonal IgG were determined\nbased on instructions provided by Emfret Analytics.\nAfter the 3-week-long treatment period (at the 19th\nweek), the final hotplate test was administered to all the\nmice with or without induced adenomyosis, after body-\nweight measurement. A 0.5 ml blood sample was taken\nbetween 9:00 and 15:00 of the dayfrom each mouse, and\nwas used for the measurement of plasma corticosterone\nlevels by enzyme linked immunosorbent assay (ELISA,\nsee Additional file 1 for more details). After the blood\nsamples were taken, all mice were sacrificed by perfusing\nthe heart with formalin. For each mouse, both uterine\nhorns were harvested and the uterine weight was re-\ncorded. The left uterine horn was used for uterine con-\ntractility measurement (described in Additional file 1),\nand the right one was fixed in 4 % paraformaldehyde im-\nmediately after collection and then embedded in paraf-\nfin. The brains of all mice were harvested and analyzed\n(described below). The experiment design is shown\nschematically in Fig. 1.\nWe evaluated the depth of myometrial infiltration of ec-\ntopic endometrium following the criteria of Bird et al.\n[22], as reported previously [2]. Briefly, Grade 1 was de-\nfined to be the case where penetration of the ectopic\nendometrium into superficial myometrium; Grade 2,\npenetration into mid-myometrium; and Grade 3, penetra-\ntion beyond mid-myometrium. For ease of statistical ana-\nlysis, Grade 0 was recorded when there was a complete\nabsence of any ectopic endometrium in the myometrium.\nFor histological examination, serial 4- μm sections were\nobtained from each paraffin-embedded tissue block, and\nthen 3 randomly selected sections were chosen for H&E\nstaining to confirm pathologic diagnosis, as described\npreviously [2, 19]. If endometrial glands and stroma were\nseen to be infiltrated into in myometrium, the diagnosis\nof adenomyosis was made.\nHistochemistry and immunohistochemistry analyses\nAs described above, the right horn of uterus was fixed in\n4 % formalin, and then embedded in paraffin. From each\ntissue block, serial 4- μm sections were obtained and\nsubjected to H&E staining to confirm pathologic diagno-\nsis of adenomyosis, which was characterized by the pres-\nence of endometrial glands and stroma that are\ncompletely enveloped by myometrium and discontinu-\nous with the endometrial cavity [23, 24].\nThe rabbit polyclonal antibodies against progester-\none receptor isoform B (PR-B, ab2765; Abcam, Hong\nKong, China), phosphorylated-p65 (ab30623; Abcam),\ncyclooxygenase 2 (COX-2, #4842; CST, Boston, USA),\noxytocin receptor (OTR, ab115664; Abcam), transient\nreceptor potential cation channel, subfamily V, mem-\nber 1 (TRPV1, ab31895;Abcam), collagen I (ab292;\nAbcam), collagen IV (ab6586; Abcam), and the rat\nmonoclonal antibody against F4/80 (MCA497G; AbD-\nSerotec, Cambridge, England), CD41(ab33661), diluted\nto1:50,1:80,1:200, 1:100, 1: 1000,1:100, 1:500, 1:200 and\n1:100, respectively, were used as primary antibodies.\nSerial 4- μm sections were made from paraffin-\nembedded tissue blocks. After routine deparaffinization\nand dehydration, some sections were randomly selected\nto be heat-retrieved with Tis-EDTA buffer (0.5 mol/L\nPH 9.0) over 98 °C for a total of 30 min for immuno-\nstaining for PR-B, p-p65, COX-2, OTR and TRPV1, and\nthe others were heat-retrieved with citric acid (0.01 mol/\nL pH 6.0) over 98 °C for a total of 30 min for immuno-\nhistochemistry analysis of collagen I, collagen IV, and\nF4/80. Then all sections were cooled naturally at room\ntemperature, and then incubated with the primary anti-\nbodies at 4 °C overnight. After the sections were rinsed\nwith PBS, they were incubated with the secondary anti-\nbody (Sunpoly-HII, BioSunTechnclogy, Shanghai, China)\nfor half an hour, or, for F4/80, with the goat anti-rat anti-\nbody (AbDSerotec) for 1 hour. The bound antibody\ncomplexes were stained for 3 –5 minor until appropriate\nfor microscopic examination with diaminobenzidine\n(DAB) (BioSunTechnclogy Co., Ltd) and then counter-\nstained with hematoxylin and mounted.\nImages were obtained with the microscope (Olympus\nBX51, Olympus, Tokyo, Japan) fitted with a digital camera\n(Olympus DP70, Olympus). Five randomly selected im-\nages from 10 to 12 images on 2 –4 sections of each mouse\nwere taken for each immunostaining marker to obtain a\nmean optional density value by Image Pro-Plus 6.0 (Media\nCybernetics, Inc., Bethesda, MD, USA) as reported in [25].\nStaining was defined via color intensity, and a color mask\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 3 of 16\n\nwas made. The mask was then applied equally to all im-\nages, and measurement readings were obtained. Immuno-\nhistochemical parameters assessed in the area detected\nincluded (a) integrated optical density (IOD); (b) total\nstained area (S); and (c) mean optical density (MOD),\nwhich is defined as MOD = IOD/S, equivalent to the mean\nintensity of stain in all positive cells.\nFor F4/80, we counted the number of F4/80-positive\nmacrophages from five randomly selected images and\ncalculated their average. Myometrial OTR staining\nscores were calculated by multiplying the percentage of\npositive OTR cells per section (0 –100 %) by a semi-\nquantitative classifier for immunohistochemical staining\nintensity, which was scored as 0 if there was a complete\nabsence of any staining, 1 for weak staining, 2 for mod-\nerate staining, and 3 for strong staining. Consequently,\nthe staining scores ranged from a minimum of 0 to a\nmaximum of 300. We used the mean score averaged\nover 5 randomly selected images.\nFor all markers, the staining levels were scored on ec-\ntopic endometrium in mice with induced adenomyosis.\nFor mice in the control group or in mice without ectopic\nendometrium due to treatment, they were scored in the\nendometrium. We counted the number of macrophages\n(F4/80-positive) infiltrated into the ectopic endometrium\nin the entire focal field for mice with adenomyosis. For\nthe mice in control group or mice that had none such\nlesion due to treatment, the number of F4/80-positive\nmacrophages in endometrium was counted.\nThe mouse spleen tissue was used for the positive im-\nmunostaining of macrophages, while breast cancer tissue\nsections were used for positive control for other\nmarkers, Negative control sections were processed simi-\nlarly, but using a non-immune rabbit or rat IgG instead\nof the primary antibody, or by omitting the primary anti-\nbody from the incubation medium. No positive reaction\nwas observed under these conditions. All sections were\ninspected a single investigator (BZ) without the know-\nledge of the group identity. The positive and negative\ncontrols are shown in Additional file 1: Figure S2 of\nSupplemental Information.\nGAD65 immunofluorescence staining of neurons in\nbrainstem nucleus raphe magnus\nThe procedure has been reported previously in [26].\nBriefly, the mouse brains containing the nucleus raphe\nmagnus (NRM) were harvested and immediately embed-\nded in O.C. T. compound in liquid phlegm after the\nmice were sacrificed. The NRM sections were between\n5.68 and 6.48 mm to the bregma and the NRM was lo-\ncated 1.72 –2.68 mm interaurally, as described previously\n[26]. Serial 6- μm sections were performed on a cryostat\nfor each block and stored at the temperature below\n−20 °C until use. The sections were incubated in goat\nanti mouse serum (BioSunTechnclogy) for 10 min and\nthen incubated in mouse anti primary antibody against\nGAD65 (ab26113, Abcam; 1:1,000) at 4 °C overnight.\nGAD65 is expressed in the cytoplasm of presynaptic\nneurons. The sections were rinsed with PBS (pH 7.4)\nand incubated in secondary antibody mixed with\nDyLight649 (E032610, Earthox, San Francisco, USA) for\none hour, and then rinsed with PBS (pH 7.4) again and\nmounted.\nFig. 1 Schematic illustration of the experiment design of this study. Untreated: mice that received no treatment; Lo-dose Ozagrel (Oza): mice\ntreated with 12.5 μg/g Oza (low-dose); Hi-dose Oza: mice treated with 25μg/g Oza (high-dose). Lo-dose PD: mice treated with 1μg/g rat anti-mouse\nGPIbα polyclonal IgG antibody for platelet depletion (PD); Hi-dose PD: mice treated with 2μg/g rat anti-mouse GPIbα polyclonal IgG antibody; Non-\nimmune: received 1μg/g non-immune (NI) rat anti-mouse IgGisotope-matched with the anti-GPIbα antibody See text on experiment protocol for\nmore details\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 4 of 16\n\nImages were obtained with a microscope (Olympus\nBX51) fitted with a digital camera (Olympus DP70). Five\nrandomly selected images out of 6 –7 sections of each\nmouse were taken for each immunostained parameter to\ncount the numbers of GAD65-positive (red) cells in the\nNRM, located between 1.72 mm and 2.68 mm interau-\nrally, and the mean was calculated.\nStatistical analysis\nComparison of the distributions among two or more\ngroups of continuous variables was made using the Wil-\ncoxon and Kruskal-Wallis tests, respectively, and the\npaired Wilcoxon test was used when the before-after\ncomparison was made for the same group of subjects.\nPearson’s or Spearman ’s rank correlation coefficient was\nused when evaluating correlations between two variables\nwhen both variables were continuous or when at least\none variable was ordinal. To see whether Ozagrel treat-\nment or platelet depletion and other possible factors\nwere responsible for the change in hotplate latency be-\nfore and after the treatment, a multiple linear regression\nmodel was used. To see whether there is trend in immu-\nnostaining levels as a function of the depth of myome-\ntrial infiltration, Jonckheere trend was used.\nTo determine correlates of depth of myometrial infil-\ntration, we used the Cox proportional odds logistic re-\ngression model. This model assumes, implicitly, that the\ndata were ordered categorical data, with an implicit\nunderlying order (scale of severity) in the data [27], with\n4 categories corresponding to Grade 0, I, II, and III\ninfiltration.\nP values of less than 0.05 were considered statistically\nsignificant. All computations were made with R statistics\nsoftware system version 3.3.1 [28].\nResults\nConsistent with Parrott et al. [17, 18] and as previously\nreported [2, 19], we found that adenomyosis was suc-\ncessfully induced in all (100 %) mice dosed with tamoxi-\nfen but none in un-dosed mice.\nOzagrel was well-tolerated, as no mice in either LO or\nHO group died, and we found nothing unusual in these\nmice. In HD group, however, 1 mouse died after it re-\nceived the 4th injection of the depletion antibody, and 2\nappeared to be lethargic. In the LD group, no mice died\nand nothing appeared unusual. There was no difference\nin platelet counts between the mice in groups UT, NI,\nLO, and HO at the end of the experiment. However, the\nplatelet count in mice in both LD and HD groups was\nreduced by 99.6 and 99.7 % as compared with those in\nthe NI group, demonstrating the effectiveness of platelet\ndepletion in these two groups.\nTreatment effect on the depth of myometrial infiltration,\nhotplate latency, and uterine and bodyweight\nWe first evaluated the effect of Ozagrel treatment or plate-\nlet depletion on the depth of myometrial infiltration. We\nfound that, compared with un treated mice, mice treated\nwith either low- or high-dose O zagrel had significantly less\ninfiltration (both p-values <0.001; Fig. 2a). Compared with\nNI mice, mice in either LD or HD group also had signifi-\ncantly less infiltration (both p-values <0.001; Fig. 2a). Mice\nin HO and HD groups appeared to have less infiltration\nthan those in the LO or LD group (Fig. 2a).\nThe multiple linear regression analysis suggested that\nboth Ozagrel treatment and platelet depletion significantly\nand dose-dependently reduced the depth of myometrial\ninfiltration (regression coefficient β = −0.956, p =3 . 2 × 1 0−7,\nand β = −0.627, p =1 . 5 × 1 0-6, respectively, R2 =0 . 6 2 ) , b u t\nNI mice had deeper infiltration ( β =0 . 8 5 6 ,p =0 . 0 1 5 ) . T h e\nCox regression analysis yielded similar results (all three p-\nvalues <0.028).\nWe found that there is a significant difference in uterine\nweight vs. bodyweight ratio among the 7 groups of mice\n(p < 0.001; Fig. 2b). Using the ratio as a dependent variable\nand the bodyweight after treatment, the induction of ade-\nnomyosis, dose of Ozagrel, the non-immune IgG injection\nor not, and the dose of antibody used in platelet depletion\nas covariates, we found, via a linear multiple regression,\nthat the non-immune IgG was positively associated with\nthe ratio ( p <0 . 0 1 ;R2 = 0.60; Fig. 2b) while both Ozagrel\nand anti-platelet doses were negatively associated with the\nratio (both p-values <0.001). Similar results were obtained\nfor the uterine weight (data not shown).\nThe induction of adenomyosis was significantly asso-\nciated with reduced latency just 4 weeks after birth or\n23 days after the completion of tamoxifen dosing ( p <\n0.05; Fig. 2c). At 8 weeks after birth, the difference in\nhotplate latency amount the four groups of mice be-\ncame very pronounced ( p < 0.001; Fig. 2c), with the\nmice with induced adenomyosis all having reduced la-\ntency ( p < 0.001). At 12 and 16 weeks after birth, the\nmice with induced adenomyosis had further progres-\nsively reduced hotplate latency (both p-values <0.001;\nFig. 2c). In all mice with induced adenomyosis, the la-\ntency evaluated at week 8, 12 and 16 was all\nsignificantly decreased as compared with the previously\nevaluated latency (all p-values <0.001; Fig. 2c).\nAfter platelet depletion or treatment with Ozagrel\nfor 3 weeks, however, the hotplate latency was signifi-\ncantly improved in a dose-dependent fashion ( β =\n4.008, p < 0.001, and β = 1.792, p < 0.001, respectively,\nin a multiple linear regression analysis, R2 = 0.73;\nFig. 2c). In contrast, the presence of adenomyosis and\nthe injection of the dummy antibody were associated\nwith decrease in hotplate latency ( p <0 . 0 0 1 a n dp <\n0.01, respectively).\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 5 of 16\n\nWhile there was no difference in bodyweight among\nthe 7 groups of mice at 4 and 8 weeks after birth (both\np-values >0.05; Fig. 2d), the difference became statisti-\ncally and progressive significant starting from week 12\n(p <0 . 0 5 a n dp < 0.01), and was significant at the end of\nthe experiment (p < 0.001; Fig. 2d). The multiple linear re-\ngression analysis using pre-treatment bodyweight, pres-\nence of adenomyosis, and the dose of Ozagrel, the non-\nimmune IgG injection or not, and the dose of antibody\nused in platelet depletion as covariates, we found that only\nthe induction of adenomyosis was negatively associated\nwith the bodyweight ( p <0 . 0 0 1 ;R2 = 0.66) while the pre-\ntreatment bodyweight was positively associated with the\nbodyweight ( p < 0.001). In other words, neither Ozagrel\ntreatment nor platelet depletion had any impact on body-\nweight, but the induction of adenomyosis had a negative\nimpact due, possibly, to adenomyosis-associated pain and/\nor pain-induced suppression of appetite.\nTreatment effect on uterine contractility\nThere was a significant difference in the amplitude of uter-\nine contractility after drug treatment among the 7 groups\n(p < 0.001; Fig. 3a). In particular, both untreated and NI\nmice had a significantly higher amplitude as compared with\nthe mice without adenomyosis (both p-values < 0.001;\nFig. 3A). Regressing the amplitude on the Ozagrel\ndose, the presence of adenomyosis, the non-immune\nIgG injection or not, and the dose of antibody used in\nplatelet depletion indicated that, while Ozagrel treat-\nment and platelet depletion were both negatively asso-\nciated with the amplitude in a dose-dependent fashion\n(both p-values < 0.001), the induction of adenomyosis\nand the IgG injection were positively and significantly\nassociated with increased amplitude ( p < 0.001, and\np <0 . 0 1 , r e s p e c t i v e l y ;R2 = 0.60).\nSimilarly, there was a significant difference in the fre-\nquency of uterine contractility after drug treatment ( p <\nFig. 2 Some summary results of the experiment. a Boxplot of the depth of myometrial infiltration among different groups of mice with induced\nadenomyosis. b Boxplot of uterine vs. body weight ratio at the end of 3-week-long RSV treatment among different groups of mice. c Kinetic\nchanges in average hotplate latency among different treatment groups. d Kinetics of mean bodyweight among different treatment groups. UT:\nUntreated group; CT: Blank control group; LO: low-dose Ozagrel group; HO: high-dose Ozagrel group; LD: platelet depletion using low-dose anti-\nbody; HD: platelet depletion group using high-dose antibody; NI: mock depletion using non-immune antibody; Tx: Treatment; Exp ’t: Experiment.\nThe arrows showing different tests are the administrated hotplate tests. Theblue line in (c) and (d) indicates the duration of the treatment. In (a)a n d(b),\nthe statistical significance of the difference between the testing group and the comparison group was indicated, and“***” means that the p-value is less\nthan 0.001. In (d), the statistical significance was referring to the difference among the 7 groups of mice. *:p < 0.05; **: p < 0.01; ***: p < 0.001\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 6 of 16\n\n0.01; Fig. 3B). Regressing the frequency (log-transformed\nto enhance normality) on the uterine weight vs. body-\nweight ratio, Ozagrel dose, the presence of adenomyosis,\nthe IgG injection or not, and the dose of antibody used\nin platelet depletion indicated that the induction of ade-\nnomyosis was positively associated with the contractile\nfrequency ( p < 0.01; R2 = 0.30) while Ozagrel treatment\nand platelet depletion were both negatively associated\nwith the frequency ( p < 0.001 and p < 0.01, respectively).\nThe contractile amplitude correlated positively with\nthe contractile frequency ( r = 0.51, p < 0.001). Both the\namplitude and frequency were found to correlate posi-\ntively with the uterine vs. bodyweight ratio ( r = 0.79, p <\n0.001, and r = 0.52, p < 0.001).\nTreatment effect on plasma level of CORT\nWe found that there is a significant difference in plasma\nCORT levels among the 7 groups of mice ( p < 0.001;\nFig. 3c). In particular, the untreated mice had a signifi-\ncantly elevated CORT levels as compared with mice\nwithout adenomyosis, so did the NI mice (both p-values\n<0.001; Fig. 3c). Regressing the plasma CORT level (log-\ntransformed to enhance normality) on the Ozagrel dose,\nthe presence of adenomyosis, the non-immune IgG in-\njection or not, and the dose of antibody used in platelet\ndepletion indicated that both the induction of adeno-\nmyosis and the injection of the dummy antibody were\npositively associated with the CORT levels ( p < 0.001\nand p < 0.05, respectively) while both Ozagrel treatment\nand platelet depletion were dose-dependently and nega-\ntively associated with the CORT levels (both p-values\n<0.001; R2 = 0.59). We also found that the CORT levels\ncorrelated negatively with the hotplate latency ( r = −0.90,\np < 0.001; Fig. 3d), suggesting that pain severity may be\npositively associated with the severity of stress.\nEffect of antiplatelet treatment on platelet aggregation,\nmacrophage infiltration, and select markers in ectopic\nendometrium\nWe evaluated the immunoreactivity results for all mice.\nFigure 4 shows the extent of platelet aggregation and of\nmacrophage infiltration and p-p65, PR-B, COX-2, and\nFig. 3 Summary results on uterine contractility and plasma corticosterone levels. a Boxplot of the amplitude of uterine contractility among\ndifferent groups of mice with induced adenomyosis. b Boxplot of the frequency of uterine contractility among different groups of mice. c\nBoxplot of the plasma corticosterone levels among different groups of mice. d Scatter plot showing the relationship between hotplate latency\nand the plasma corticosterone levels. In ( a), (b) and ( c), the statistical significance of the difference between the testing group and the\ncomparison group was indicated. *: p < 0.05; **: p < 0.01; ***: p < 0.001. In ( d), each letter represents one mouse, and the alphabet indicates the\ngroup identity, which is the same as used in Fig. 2c, d. The correlation coefficient, with its statistical significance level, is shown in the figure\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 7 of 16\n\nTRPV1 immunostaining in ectopic endometrium among\ndifferent groups. For COX-2, the staining was predomin-\nantly localized in the cytoplasm of glandular epithelial\ncells in ectopic and eutopic endometrium. Both PR-B\nand p-p65 staining was localized primarily in the nuclei\nof glandular epithelial cells of eutopic and ectopic endo-\nmetrium while TRPV1 staining was seen mainly in the\ncytoplasm and cell membranes of glandular epithelial\ncells;\nWe found that there was a significant difference in im-\nmunoreactivity to PR-B, p-p65, COX-2, and TRPV1 in\nectopic/eutopic endometrium and to OTR in myome-\ntrium among different groups (all p-values <0.01; Figs. 5\nand 6, and Table 1). In particular, multiple linear regres-\nsion analyses (all immunoreactivity levels were square-\nroot transformed to improve normality unless stated\notherwise) indicated that while adenomyosis induction\nwas associated with the increase (decrease for PR-B)\nwhile Ozagrel treatment or platelet depletion was associ-\nated, in a dose-dependent manner, with a significant re-\nduction (increase for PR-B) of immunoreactivity to all\nthese proteins or the extent of platelet aggregation/\nmacrophage infiltration (all p-values <0.01, with R2 ran-\nging from 0.31 to 0.76; Table 1 and Fig. 5).\nIn addition to these markers, we also performed im-\nmunostaining of lesional OTR, a marker of SMM, and\ncollagen I and IV, markers of fibrosis, in adenomyotic\nlesions, as well as OTR in myometrium, which was likely\nresponsible for uterine hyperactivity. OTR staining was\nlocalized in both the cell membrane and the cytoplasm\nof glandular epithelial and stromal cells as well as myo-\nmetrial smooth muscle cells (Fig. 6). We scored OTR\nstaining levels in epithelial/stromal cells and myometrial\nmuscle cells separately. No difference in OTR staining\nlevels in the epithelial component was found (data not\nshown), and hence only the data in the stromal compo-\nnent were demonstrated. Both collagen I and collagen IV\nstaining was seen nearly uniformly in extracellular matrix\nof the ectopic endometrial stromal tissues, irrespective of\nthe proximity to the glandular epithelial cells or not.\nWe found that for all these markers, the presence\nof adenomyosis and, in the case of lesional and myo-\nmetrial OTR staining, the injection of non-immune\nantibodies were positively associated with the staining\nlevels while Ozagrel treatment and platelet depletion\nwere associated, in a dose-dependent manner, with a\nsignificant reduction of immunoreactivity to all these\nproteins (all p-values <0.001; Fig. 5; Table 1).\nWe found that the immunostaining levels of these pro-\nteins were all highly correlated, with the positive correlation\ncoefficients ranged from 0.69 to 0.95 (PR-B excluded; all p-\nvalues <0.001) and the negative correlation coefficients\nranged from −0.72 to −0.93 (all p-values < 0.001 for PR-B\nvs. others).\nFig. 4 Representative photomicrographs of indicated immunostaining in eutopic (for blank control group) or ectopic (all other groups)\nendometrium among different treatment groups, which are arranged in different columns. CD41: CD41-labeled platelets; F4/80: F4/80-labled macrophages\n(indicated by yellow arrows); p-p65: phosphorylated form of NF-kB p65 subunit; PR-B: progesterone receptor isoform B; COX-2: Cyclooxygenase 2; TRPV1:\ntransient receptor potential cation channel, subfamily V, member 1. Blank: blank control endometrium; Hi-Oza: high-dose Ozagrel group; Lo-Oza: low-dose\nOzagrel group; Hi-Dep: platelet depletion with high-dose antibody; Lo-Dep: platelet depletion withlow-dose antibody; IgG: non-immune mock antibody.\nScale bar = 125μm\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 8 of 16\n\nFig. 5 Summary of immunohistochemistry results. Boxplot of immunoreactivity against CD41 ( a), the number of F4/80+ positive macrophages\n(b), p-p65 c, PR-B ( d), COX-2 ( e), TRPV1 ( f),OTR (g), myometrial OTR ( h), Collagen I ( i), and Collagen IV ( j) in ectopic/eutopic endometrium. The\ngroup labels are the same as used in Fig. 2\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 9 of 16\n\nWe found that the extent of platelet aggregation and\nof the macrophage infiltration as well as the immunore-\nactivity to PR-B, p-p65, COX-2 and TRPV1 were all\nhighly correlated with the depth of myometrial infiltra-\ntion (all positive except PR-B, which was negative, and\nthe Spearman ’s correlation coefficients ranged from 0.69\nto 0.88 ( −0.87 for PR-B), all p-values <0.001). The Jonc-\nkheere trend test indicated that all these immunostain-\ning levels were significantly associated with the depth of\nmyometrial infiltration (all p-values <0.001; Fig. 7). A\nmultiple linear regression analysis indicated that the\nOTR and PR-B staining levels in ectopic endometrium\nwere the only 2 co-variables that are associated with the\ndepth of myometrial infiltration (OTR, positive associ-\nation, p = 6.6×10−15, PR-B, negative association, and p =\n0.0020, respectively; R2 = 0.83).\nEffect of treatment on the number of GAD65-positive\nneurons in the brainstem nucleus raphe magnus (NRM)\nTo see whether Ozagrel treatment and platelet depletion\nhad any effect on the GABAergic inhibition system in the\nNRM, we performed an immunofluorescent staining of\nGAD65 in the NRM (Fig. 8a) and counted the number of\nGAD65-positive and synapsin I-positive neurons in the\nNRM. This number would be a measure of the number of\nGAD65-expressing neurons in the NRM.\nWe found that there is a significant difference in\nthe number of GAD65-positive neurons in the NRM\namong the seven groups ( p < 0 . 0 0 1 ;F i g .8 b ) .Am u l -\ntiple linear regression analysis (the number of cells\nwas square-root transformed to improve normality)\nindicated that while adenomyosis induction was asso-\nciated with the reduction in the number of GAD65-\npositive neurons in the NRM ( p < 0.001) as previously\nreported [26] while both Ozagrel treatment and plate-\nlet depletion were associated dose-dependently with a\nsignificant increase of the number of GAD65-positive\nneurons (both p-values <0.001; R2 = 0.92).\nThe number of GAD65-positive neurons (log-trans-\nformed) in the NRM was found to be positively corre-\nlated with the hotplate latency after treatment ( r = 0.88,\np < 0.001; Fig. 8c). It also was found to be negatively cor-\nrelated with the plasma CORT levels ( r = −0.86, p <\n0.001; Fig. 8D).\nFig. 6 Representative immunohistochemisty staining of markers of smooth muscle metaplasia and fibrosis in ectopic and eutopic endometrium.\nDifferent rows indicate different proteins in different groups (arranged in different columns) with different doses of Ozagrel, different doses of rat\nanti-mouse GPIbα polyclonal IgG and non-immune rat anti-mouse IgG. For oxytocin receptor (OTR), M stands for staining in the myometrium,\nstromal and gland epithelium was separately evaluated. Magnification in all figures: ×400. Scale bar = 125 μm\nTable 1 Results from early/later platelet depletion experiment.\nAll results were based on multiple regression analyses with the\nindependent variable square-root transformed and dummy vari-\nables indicating the presence or absence of adenomyosis, non-\nimmune IgG antibody injection or not, the dosage of antibody\nto deplete platelets and the dosage of Ozagrel as co-variables\nName Induction of\nadenomyosis\nOzagrel\ntreatment\nPlatelet\ndepletion\nR2\nExtent of CD41+ platelet\naggregation\n↑\n***\n↓\n***\n↓\n***\n0.54\nNumber of infiltrated F4/\n80+ macrophages\n↑\n***\n↓\n**\n↓\n*\n0.30\nPhosph-p65 ↑\n***\n↓\n**\n↓\n**\n0.31\nPR-B ↓\n***\n↑\n***\n↑\n***\n0.73\nCOX-2 ↑\n***\n↓\n***\n↓\n***\n0.48\nTRPV1 ↑\n***\n↓\n***\n↓\n***\n0.47\nOTR ↑\n***\n↓\n***\n↓\n***\n0.58\nCollagen I ↑\n***\n↓\n***\n↓\n*\n0.29\nCollagen IV ↑\n***\n↓\n**\n↓\n***\n0.33\nMyometrial OTR ↑\n***\n↓\n***\n↓\n***\n0.77\n↓: Denotes that the immunoreactivity to this protein at hand was significantly\ndecreased based on multiple linear regression analysis; ↑Denotes that the\nimmunoreactivity to this protein of interest was significantly increased based\non multiple linear regression analysis. The R2 value of the corresponding\nregression model is shown at the right-most column. Symbols of statistical sig-\nnificance levels: *: p<0.05; **: p<0.01; ***: p<0.001\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 10 of 16\n\nFig. 7 (See legend on next page.)\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 11 of 16\n\nFactors associated with the uterine contractility\nBoth contractile amplitude and frequency correlated posi-\ntively with the myometrial OTR staining levels ( r =0 . 8 2 ,p\n< 0.001, and r = 0.35, p < 0.01, respectively; Additional file 1:\nFigure S3A, B of Supplemental Information) and also with\nthe lesional OTR staining levels in the ectopic endomet-\nrium (r =0 . 9 4 ,p < 0.001, and r =0 . 5 0 ,p <0 . 0 0 1 ; A d d i t i o n a l\nfile 1: Figure S3 c, d of Supplemental Information).\nFor contractile amplitude, the multiple linear regres-\nsion incorporating uterine weight/bodyweight ratio,\n(See figure on previous page.)\nFig. 7 Summary results on immunohistochemistry measures as a function of the depth of myometrial infiltration. Boxplot of the extent of platelet\naggregation (a), extent of macrophage infiltration (b), immunoreactivity against p-p65 (c), PR-B (d), COX-2 (e), TRPV1 (f), OTR (g), myometrial OTR (h),\ncollagen I (i), and collagen IV (j) in ectopic endometrium as a function of the depth of myometrial infiltration of endometrial tissues. The p-value shown\nin each figure is the statistical significance of the Jonckheere trend test\nFig. 8 a Micrographs of immunofluorescent staining of GAD65 in the nucleus raphe magnus (NRM) in different groups of mice. Both GAD65- and\nSynapsin I-positive neurons were identified, as indicated by white arrows. To see the picture more closely, the area of interested was amplified three\ntimes. The original amplification:x400. The scale bar represents 125μm. b Boxplot showing the number of GAD65+ neurons in the NRM among different\ntreatment groups. The dashed line represents the median value of all mice. Blank: blank control endometrium; Hi-Oza: high-dose Ozagrel group; Lo-Oza:\nlow-dose Ozagrel group; Hi-Dep: platelet depletion with high-dose antibody; Lo-Dep: platelet depletion with low-dose antibody; IgG: non-immune mock\nantibody.c Scatter plot of hotplate latency vs. the log-transformed number of GAD65-positive neurons in the NRM for all groups of mice;d Scatter plot of\nplasma corticosterone levels vs. the log-transformed number of GAD65-positive neurons in the NRM for all groups of mice. Each alphabet in the figure\nrepresents one experimental observation, and thealphabets are the abbreviations of different treatment groups. C: Blank control; U: Untreated; o:Low-\ndose Ozagrel; O: High-dose Ozagrel; d: Platelet depletion using low-dose antibody; D: Platelet depletion using high-dose antibody; N: Non-immune IgG.\nThe correlation coefficient and its statistical significance levels are shown in (c)a n d(d). ***: p < 0.001\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 12 of 16\n\ndepth of myometrial infiltration (0 if no adenomyosis),\nextent of platelet aggregation and macrophage infiltra-\ntion and all immunostaining measurements in ectopic\nendometrium identified the depth of myometrial infiltra-\ntion ( p < 0.01), uterine weight/bodyweight ratio ( p <\n0.001), the extent of platelet aggregation ( p < 0.001), and\nthe number of infiltrating macrophages ( p < 0.01) as four\ncovariates that were associated with the contractile amp-\nlitude ( R2 = 0.94). For contractile frequency, we found,\nthrough multiple linear regression analysis, that only the\nuterine weight/bodyweight ratio ( p < 0.001), lesional and\nmyometrial OTR staining levels (both p < 0.05), and PR-\nB staining levels ( p < 0.05) were associated with the con-\ntractile frequency ( R2 = 0.42).\nDeterminants of thermal response latency after treatment\nWe carried out a multiple linear regression analysis to\nidentify which factors potentially determine the change\nin thermal response latency before and after drug treat-\nment using the pre-treatment latency, bodyweight, depth\nof myometrial infiltration (grade = 0 if no adenomyosis),\nuterine weight vs. bodyweight ratio, amplitude and fre-\nquency of uterine contraction, and presence of adeno-\nmyosis as covariates. We found that the uterine weight\nvs. bodyweight ratio ( p < 0.01), contractile amplitude ( p\n< 0.01), the presence of adenomyosis ( p < 0.001) and the\ndepth of myometrial infiltration ( p < 0.001) were all\nnegatively associate with the change in before-after hot-\nplate latency ( R2 = 0.90).\nDiscussion\nWe have provided evidence that anti-platelet treatment,\nthrough either platelet depletion or Ozagrel treatment,\nresulted in the suppression of myometrial infiltration,\nimproved generalized hyperalgesia, reduced uterine\nweight vs. bodyweight ratio and stress level, and reduced\namplitude and frequency of uterine contraction in mice\nwith induced adenomyosis. The anti-platelet treatment\nalso improved the expression of some proteins known to\nbe involved in adenomyosis and reduced the number of\ninfiltrating macrophages. In particular, it reduced the\nlesional expression of OTR, a SMM marker [29], and of\ncollagen I and IV, markers of extracellular matrix de-\nposits and thus fibrosis. Moreover, it increased the num-\nber of GAD65-expressing neurons in the brainstem\nNRM, thus likely boosting the GABAergic inhibition of\npain due to adenomyosis, which in turn helps pain relief\nand reduces the stress level.\nOur data are consistent with our finding that increased\nplatelet aggregation and the extent of fibrosis in both\nmouse and human adenomyosis [10, 11]. They are also\nconsistent with our previous report that anti-platelet\ntherapy is effective in treating endometriosis in mouse\n[9, 14, 30] and that the expression of tissue factor in\nadenomyosis is elevated [5, 31]. Tissue factor plays a\ncritical role in the initiation of platelet activation and co-\nagulation [32]. In addition, considerable experimental\ndata [33, 34] and limited clinical data [35] support the\ninvolvement of hyperprolactinemia in adenomyosis, yet\nprolactin is a potent cofactor for platelet aggregation\n[36, 37]. These data, taken together, seem to suggest that\npatients with adenomyosis may be in a hypercoagulable\nstate as those with endometriosis [38]. This may explain\nthe report of cerebral infarcts associated with adeno-\nmyosis [39] and increased mean platelet volume in\nwomen with adenomyosis [40].\nOur data are also consistent with our previous reports\nthat PR-B expression in adenomyosis is reduced [41] due\npossibly to PR-B promoter hypermethylation [42]. In\naddition, they are consistent with reported constitutive acti-\nvation of NF- κB [5], increased expression of COX-2 [7],\nTRPV1 [43] and OTR in adenomyosis [29, 43, 44]. The in-\ncreased uterine contractility, and, in particular, its close as-\nsociation with the OTR expression and with the reduced\nhotplate latency as reported in this study are consistent\nwith what we reported in human adenomyosis [44]. In\nother words, the mouse model used in this study recapitu-\nlates several important features of human adenomyosis, i.e.,\ninflammation and angiogenesis as displayed by the consti-\ntutive activation of NF- κB and increased COX-2 but de-\ncreased PR-B expression in adenomyotic lesions, increased\nuterine weight and presumably enlarged uterus, increased\ngeneralized hyperalgesia, and elevated uterine contractility,\ndue possibly to elevated myometrial OTR expression. Re-\nmarkably, anti-platelet treatment either reversed or abro-\ngated these changes.\nWe note that the anti-platelet treatment achieved the\ntherapeutic effects very similar to EGCG [20, 26, 45] and\nresveratrol [46, 47] as we reported earlier. However, it is\nperhaps no coincidence that EGCG is anti-platelet [48]\nand so is resveratrol [46]. In fact, some compounds that\nare reported to be promising in treating adenomyosis in\npreclinical and clinical studies, such as andrographolide\n[2, 5, 49], valproic acid [19, 50, 51], and statins [52, 53], all\nturn out to be anti-platelet [54 –57]. Even danazol, a once-\npopular, FDA-approved drug for treating endometriosis,\nhas long been reported to have anti-platelet effect [58, 59].\nThat said, we should emphasize that, despite promis-\ning results of anti-platelet treatment by either platelet\ndepletion or Ozagrel treatment as shown here, we are\nnot advocating their use in clinical setting per se , even\nthough Ozagrel is a prescription drug as of now. Adeno-\nmyosis is a benign disease and certainly not life-\nthreatening. As such, it places higher premium on drug\nsafety as compared with other life-threatening diseases\nsuch as cancer. While Ozagrel is generally safe and holds\npromises in treating adenomyosis, the hemorrhage risk\nit entails deserves caution. This study was meant to be a\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 13 of 16\n\nproof-of-concept study, demonstrating the therapeutic\npotential of anti-platelet therapy for adenomyosis. It is\nnot intended to advocate Ozagrel per se for the treat-\nment of human adenomyosis. More research is needed\nto determine which anti-platelet compound has the de-\nsirable benefit-to-risk ratio in treating adenomyosis.\nWhile the exact mechanisms of action for anti-\nplatelet therapy remain to be investigated, it is possible\nthat anti-platelet treatment suppresses the activation of\nthe TGF- β1/Smad3 signaling pathway and the expres-\nsion of ER- β, both of which can be induced by activated\nplatelets [15, 60]. In addition, the treatment suppresses\nthe activation of NF- κB, which also can be induced by\nplatelets (Zhang et al., unpublished data). Moreover, ac-\ntivated platelets express P-selectin (CD62P/GMP-140)\non their cell surface [61 –63], which binds to its ligand,\nP-selectin glycoprotein ligand-1 (PSGL-1), that is\nexpressed on the cell surface of most leukocytes, such\nas neutrophils, monocytes, Th1 lymphocytes, eosino-\nphils, and basophils, and facilitates inflammation,\nhemostasis, thrombosis, and the growth and metastasis\nof cancer [62, 64]. P-selectin interacts with PSGL-1, a\ntransmembraine homodimer, to mediate the rolling of\nleukocytes on stimulated endothelial cells and the het-\nerotypic aggregation of activated platelets and leuko-\ncytes [65], and activate mitogen-activated protein\nkinases (MAPKs) [66] and β2 integrins [67]. Hence,\nanti-platelet treatment should abrogate or attenuate in-\nflammation caused by adenomyosis, as seen in reduced\np - p 6 5e x p r e s s i o ni nt h i ss t u d y .\nAnti-platelet therapy can also suppress neurite outgrowth\nand thus hyperinnervation in adenomyosis [68, 69] since\nectopic endometrial stromal cells secrete platelet inducers\nsuch as thrombin and thromboxane A2 (TXA2)[ 7 0 ] .T X A2\nhas been reported to stimulate neurite outgrowth in cere-\nbral cortical neurons [71], and we have also found that it\ncan do so in dorsal ganglia root neurons [72]. Since TXA 2,\nPGH2,a n dP G I2 have been reported to be potent inducers\nof uterine contractility [73] and uterine contractility is doc-\numented to be correlated with the severity of dysmenor-\nrhea in adenomyosis [44], the suppression of platelet\nactivation and the resultant COX-2 down-regulation may\nsuppress hyperinnervation and uterine hyperactivity, thus\nresponsible for improved gene ralized hyperalgesia and re-\nduced plasma CORT levels.\nThe reduced plasma CORT levels in mice with anti-\nplatelet treatment is likely to result from the modulation\nof chronic stress (adenomyosis-induced pain) response\nthrough GABA receptors as in a chick model of acute\nstress [74]. Alternatively, adenomyosis-induced pain or\nhyperalgesia may result in synaptic dysfunction, for ex-\nample, HDAC-mediated impairment of GABA synaptic\ninhibition in the brainstem NRM [75]. However, whether\nsuppression of platelet activation may restore the GABA\nsynaptic inhibition in NRM through the reduction of\nHDAC activity remains to be clarified.\nConclusions\nThis study further provides evidence that platelets play\nimportant roles in the development of adenomyosis. In\naddition, this study demonstrates that anti-platelet treat-\nment is efficacious in suppressing myometrial infiltra-\ntion, improving generalized hyperalgesia, reducing\nboth uterine hyperactivity and systemic CORT levels in\nmice with induced adenomyosis. Collectively, these re-\nsults demonstrate that anti-platelet therapy holds prom-\nises as a non-hormonal treatment for treating\nadenomyosis.\nAdditional file\nAdditional file 1: Supplemental materials [76 –80]. (PDF 358 kb)\nAbbreviations\nCORT: Corticosterone; COX-2: Cyclooxygenase-2; EGCG: Epigallocatechin-3-\ngallate; EMT: Epithelial-mesenchymal transition; ER- β: Estrogen receptor β;\nFDA: FMT: fibroblast-to-myofibroblast transdifferentiation; GABA: γ-\naminobutyric acid; GAD65: Glutamic acid decarboxylase 65;\nH&E: Hematoxylin and eosin; HD: Platelet depletion by high-dose rat anti-\nmouse GPIb α polyclonal IgG treatment; HDAC: Histone deacetylase;\nHO: High-dose Ozagrel treatment; LD: Platelet depletion by low-dose rat\nanti-mouse GPIb α polyclonal IgG treatment; LO: Low-dose Ozagrel\ntreatment; MAPK: Mitogen-activated protein kinase; NI: Non-immune rat anti-\nmouse IgG isotope-matched with the anti-GPIb α antibody; NRM: Nucleus\nraphe magnus; OTR: Oxytocin receptor; PGH 2: Prostaglandin H2, PGI 2\nprostaglandin I2; p-p65: Phosphorylated p65 subunit; PR-B: Progesterone\nreceptor isoform B; PSGL-1: P-selectin glycoprotein ligand-1;\nReTIAR: Repeated tissue injury and repair; SMM: Smooth muscle metaplasia;\nTGF-β1: Transforming growth factor β1; TRPV1: Transient receptor potential\ncation channel, subfamily V, member 1; TXA 2: Thromboxane A2\nAcknowledgment\nThe authors would like to thank the Administration of Wenzhou People ’s\nHospital for its support and encouragement, and funding agencies for their\nfinancial support.\nThis paper has been presented orally at the First Congress of the Society of\nEndometriosis and Uterine Disorders (SEUD) held in Paris on May 9, 2015.\nFunding\nThis work was supported in part by grant Y14H040004 (YMC) from the\nNational Science Foundation of Zhejiang Province, grants 81471434 (SWG),\n81270676 (SWG), 81530040 (SWG), and 81370695 (XSL) from the National\nScience Foundation of China. None of the funders, however, has any role in\nthe design of the study, and the collection, analysis, and interpretation of\ndata and in writing the manuscript.\nAvailability of data and materials\nThe data used in this study are available upon request.\nAuthors' contributions\nSWG conceived and designed the study, performed data analysis and data\ninterpretation, and drafted the manuscript. BZ and YMC carried out most of\nthe experiment, XLS and XSL provided assistance in immunohistochemistry\nanalysis. All participated in writing up the manuscript. All authors read and\napproved the final manuscript.\nCompeting interests\nThe authors declare that they have no competing interests.\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 14 of 16\n\nConsent for publication\nNot applicable.\nEthics approval\nThis study was approved by the institutional experimental animals review\nboard of Shanghai OB/GYN Hospital, Fudan University.\nAcknowledgment of financial support\nThis research was supported in part by grant Y14H040004 (YMC) from the\nScience Foundation of Zhejiang Province, grants 81471434 (SWG), 81270676\n(SWG), 81530040 (SWG), 81370695 (XSL) and 81671436 (XSL) from the\nNational Natural Science Foundation of China.\nAuthor details\n1Department of Obstetrics and Gynecology, The People ’s Hospital, Wenzhou,\nZhejiang 325800, China. 2Shanghai Key Laboratory of Female Reproductive\nEndocrine-Related Diseases, Shanghai 200011, China. 3Shanghai Obstetrics\nand Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai\n200011, China.\nReceived: 17 August 2016 Accepted: 23 September 2016\nReferences\n1. Benagiano G, Habiba M, Brosens I. The pathophysiology of uterine\nadenomyosis: an update. Fertil Steril. 2012;98:572 –9.\n2. Mao X, Wang Y, Carter AV, Zhen X, Guo SW. The retardation of myometrial\ninfiltration, reduction of uterine contractility, and alleviation of generalized\nhyperalgesia in mice with induced adenomyosis by levo-tetrahydropalmatine\n(l-THP) and andrographolide. Reprod Sci. 2011;18:1025–37.\n3. Wood C. Adenomyosis: difficult to diagnose, and difficult to treat. Diagn\nTher Endosc. 2001;7:89 –95.\n4. Kitawaki J. Adenomyosis: the pathophysiology of an oestrogen-dependent\ndisease. Best Pract Res Clin Obstet Gynaecol. 2006;20:493 –502.\n5. Li B, Chen M, Liu X, Guo SW. Constitutive and tumor necrosis factor-alpha-\ninduced activation of nuclear factor-kappaB in adenomyosis and its\ninhibition by andrographolide. Fertil Steril. 2013;100:568 –77.\n6. Khan KN, Kitajima M, Hiraki K, Fujishita A, Sekine I, Ishimaru T, et al. Changes\nin tissue inflammation, angiogenesis and apoptosis in endometriosis,\nadenomyosis and uterine myoma after GnRH agonist therapy. Hum Reprod.\n2010;25:642–53.\n7. Ota H, Igarashi S, Sasaki M, Tanaka T. Distribution of cyclooxygenase-2 in\neutopic and ectopic endometrium in endometriosis and adenomyosis. Hum\nReprod. 2001;16:561–6.\n8. Ulukus EC, Ulukus M, Seval Y, Zheng W, Arici A. Expression of interleukin-8\nand monocyte chemotactic protein-1 in adenomyosis. Hum Reprod. 2005;\n20:2958–63.\n9. Ding D, Liu X, Duan J, Guo SW. Platelets are an unindicted culprit in the\ndevelopment of endometriosis: clinical and experimental evidence. Hum\nReprod. 2015;30:812–32.\n10. Shen M, Liu X, Zhang H, Guo SW. Transforming Growth Factor β1 Signaling\nCoincides with -Mediated Epithelial-Mesenchymal Transition and Fibroblast-\nto-Myofibroblast Transdifferentiation in Drive the Development of\nAdenomyosis in Mice. Hum Reprod 2016; In press.\n11. Liu X, Shen S, Qi Q, Zhang H, Guo S-W. Corroborating Evidence for\nPlatelet-Induced Epithelial-Mesench ymal Transition and Fibroblast-to-\nMyofibroblast Transdifferentiationin the Development of Adenomyosis.\nHum Reprod 2016; In press.\n12. Guo SW, Ding D, Shen M, Liu X. Dating endometriotic ovarian cysts based\non the content of cyst fluid and its potential clinical implications. Reprod\nSci. 2015;22:873 –83.\n13. Zhang Q, Duan J, Olson M, Fazleabas A, Guo SW. Cellular changes\nconsistent with epithelial-mesenchymal transition and fibroblast-to-\nmyofibroblast transdifferentiation in the progression of experimental\nendometriosis in baboons. Reprod Sci 2016; In press.\n14. Guo SW, Ding D, Geng JG, Wang L, Liu X. P-selectin as a potential\ntherapeutic target for endometriosis. Fertil Steril. 2015;103:990 –1000. e8.\n15. Zhang Q, Ding D, Liu X, Guo SW. Activated platelets induce estrogen\nreceptor beta expression in endometriotic stromal cells. Gynecol Obstet\nInvest. 2015;80(3):187 –92.\n16. Loo MH, Egan D, Vaughan Jr ED, Marion D, Felsen D, Weisman S. The\neffect of the thromboxane A2 synthesis inhibitor OKY-046 on renal\nfunction in rabbits following release of unilateral ureteral obstruction. J Urol.\n1987;137:571–6.\n17. Green AR, Styles JA, Parrott EL, Gray D, Edwards RE, Smith AG, et al.\nNeonatal tamoxifen treatment of mice leads to adenomyosis but not\nuterine cancer. Exp Toxicol Pathol. 2005;56:255 –63.\n18. Parrott E, Butterworth M, Green A, White IN, Greaves P. Adenomyosis –a\nresult of disordered stromal differentiation. Am J Pathol.\n2001;159:623–30.\n19. Liu X, Guo SW. Valproic acid alleviates generalized hyperalgesia in mice with\ninduced adenomyosis. J Obstet Gynaecol Res. 2011;37:696 –708.\n20. Chen Y, Zhu B, Zhang H, Liu X, Guo SW. Epigallocatechin-3-gallate reduces\nmyometrial infiltration, uterine hyperactivity, and stress levels and alleviates\ngeneralized hyperalgesia in mice induced with adenomyosis. Reprod Sci.\n2013;20(12):1478–91.\n21. Council NR. Guide for the Care and use of Laboratory Animals. Washington:\nNational Academies Press; 1996.\n22. Bird CC, McElin TW, Manalo-Estrella P. The elusive adenomyosis of the\nuterus–revisited. Am J Obstet Gynecol. 1972;112:583 –93.\n23. Barrier BF, Malinowski MJ, Dick Jr EJ, Hubbard GB, Bates GW. Adenomyosis\nin the baboon is associated with primary infertility. Fertil Steril. 2004;82\nSuppl 3:1091–4.\n24. Vercellini P, Ragni G, Trespidi L, Oldani S, Panazza S, Crosignani PG.\nAdenomyosis: a deja vu? Obstet Gynecol Surv. 1993;48:789 –94.\n25. Wang-Tilz Y, Tilz C, Wang B, Tilz GP, Stefan H. Influence of lamotrigine and\ntopiramate on MDR1 expression in difficult-to-treat temporal lobe epilepsy.\nEpilepsia. 2006;47:233–9.\n26. Chen Y, Zhu B, Zhang H, Ding D, Liu X, Guo SW. Possible Loss of GABAergic\nInhibition in Mice With Induced Adenomyosis and Treatment With\nEpigallocatechin-3-Gallate Attenuates the Loss With Improved Hyperalgesia.\nReprod Sci. 2014;21(7):869 –82.\n27. Liu X, Guo SW. Dysmenorrhea: risk factors in women with endometriosis.\nWomens Health (Lond Engl). 2008;4:399 –411.\n28. R Core Team. R: A Language and Environment for Statistical Computing.\nVienna: R Foundation for Statistical Computing; 2016.\n29. Mechsner S, Grum B, Gericke C, Loddenkemper C, Dudenhausen JW, Ebert\nAD. Possible roles of oxytocin receptor and vasopressin-1alpha receptor in\nthe pathomechanism of dysperistalsis and dysmenorrhea in patients with\nadenomyosis uteri. Fertil Steril. 2010;94:2541 –6.\n30. Guo SW, Ding D, Liu X. Anti-platelet therapy is efficacious in treating\nendometriosis induced in mouse..Reprod Biomed Online 2016; In press.\n31. Liu X, Nie J, Guo SW. Elevated immunoreactivity to tissue factor and its\nassociation with dysmenorrhea severity and the amount of menses in\nadenomyosis. Hum Reprod. 2011;26:337 –45.\n32. Engelmann B, Luther T, Muller I. Intravascular tissue factor pathway –a model\nfor rapid initiation of coagulation within the blood vessel. Thromb Haemost.\n2003;89:3–8.\n33. Mori T, Singtripop T, Kawashima S. Animal model of uterine adenomyosis: is\nprolactin a potent inducer of adenomyosis in mice? Am J Obstet Gynecol.\n1991;165:232–4.\n34. Ficicioglu C, Tekin HI, Arioglu PF, Okar I. A murine model of adenomyosis:\nthe effects of hyperprolactinemia induced by fluoxetine hydrochloride, a\nselective serotonin reuptake inhibitor, on adenomyosis induction in Wistar\nalbino rats. Acta Eur Fertil. 1995;26:75 –9.\n35. Yu Y, Jing Z, Zhi-Yu H, Xia M, Yan-Li H, Chang-Tao X, et al. Ultrasound-\nguided percutaneous microwave ablation for adenomyosis: efficacy of\ntreatment and effect on ovarian function. Sci Rep. 2015;5:10034.\n36. Wallaschofski H, Donne M, Eigenthaler M, Hentschel B, Faber R, Stepan H, et\nal. PRL as a novel potent cofactor for platelet aggregation. J Clin Endocrinol\nMetab. 2001;86:5912–9.\n37. Urban A, Masopust J, Maly R, Hosak L, Kalnicka D. Prolactin as a factor for\nincreased platelet aggregation. Neuro Endocrinol Lett. 2007;28:518 –23.\n38. Wu Q, Ding D, Liu X, Guo SW. Evidence for a hypercoagulable state in\nwomen with ovarian endometriomas. Reprod Sci. 2015;22:1107 –14.\n39. Yamashiro K, Tanaka R, Nishioka K, Ueno Y, Shimura H, Okuma Y, et al.\nCerebral infarcts associated with adenomyosis among middle-aged women.\nJ Stroke Cerebrovasc Dis. 2012;21:910. e1-5.\n40. Bodur S, Gun I, Alpaslan Babayigit M. The significance of mean platelet\nvolume on diagnosis and management of adenomyosis. Med Glas (Zenica).\n2013;10:59–62.\nZhu et al. Reproductive Biology and Endocrinology  (2016) 14:66 Page 15 of 16\n\n41. Nie J, Lu Y, Liu X, Guo SW. Immunoreactivity of progesterone receptor\nisoform B, nuclear factor kappaB, and IkappaBalpha in adenomyosis. Fertil\nSteril. 2009;92:886 –9.\n42. Jichan N, Xishi L, Guo SW. Promoter hypermethylation of progesterone receptor\nisoform B (PR-B) in adenomyosis and its rectification by a histone deacetylase\ninhibitor and a demethylation agent. Reprod Sci. 2010;17:995–1005.\n43. Nie J, Liu X, Guo SW. Immunoreactivity of oxytocin receptor and transient\nreceptor potential vanilloid type 1 and its correlation with dysmenorrhea in\nadenomyosis. Am J Obstet Gynecol. 2010;202:346. e1-8.\n44. Guo SW, Mao X, Ma Q, Liu X. Dysmenorrhea and its severity are associated\nwith increased uterine contractility and overexpression of oxytocin receptor\n(OTR) in women with symptomatic adenomyosis. Fertil Steril.\n2013;99:231–40.\n45. Jin YR, Im JH, Park ES, Cho MR, Han XH, Lee JJ, et al. Antiplatelet activity of\nepigallocatechin gallate is mediated by the inhibition of PLCgamma2\nphosphorylation, elevation of PGD2 production, and maintaining calcium-\nATPase activity. J Cardiovasc Pharmacol. 2008;51:45 –54.\n46. Zhu B, Chen Y, Zhang H, Liu X, Guo SW. Resveratrol reduces myometrial\ninfiltration, uterine hyperactivity, and stress levels and alleviates generalized\nhyperalgesia in mice with induced adenomyosis. Reprod Sci.\n2015;22(11):1336–49.\n47. Shen MY, Hsiao G, Liu CL, Fong TH, Lin KH, Chou DS, et al. Inhibitory\nmechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK\nand NO/cyclic GMP. Br J Haematol. 2007;139:475 –85.\n48. Ok WJ, Cho HJ, Kim HH, Lee DH, Kang HY, Kwon HW, et al.\nEpigallocatechin-3-gallate has an anti-platelet effect in a cyclic AMP-\ndependent manner. J Atheroscler Thromb. 2012;19:337 –48.\n49. Liu X, Yu S, Guo SW. A pilot study on the use of andrographolide to treat\nsymtomatic adenomyosis. Gynecol Minim Invas Ther. 2014;3:119 –26.\n50. Liu X, Guo SW. A pilot study on the off-label use of valproic acid to treat\nadenomyosis. Fertil Steril. 2008;89:246 –50.\n51. Liu X, Lei Y, Guo SW. Valproic acid as a therapy for adenomyosis: a\ncomparative case series. Reprod Sci. 2010;17:904 –12.\n52. Piotrowski PC, Kwintkiewicz J, Rzepczynska IJ, Seval Y, Cakmak H, Arici A, et\nal. Statins inhibit growth of human endometrial stromal cells independently\nof cholesterol availability. Biol Reprod. 2006;75:107 –11.\n53. Esfandiari N, Khazaei M, Ai J, Bielecki R, Gotlieb L, Ryan E, et al. Effect of a\nstatin on an in vitro model of endometriosis. Fertil Steril.\n2007;87:257–62.\n54. Lien LM, Su CC, Hsu WH, Lu WJ, Chung CL, Yen TL, et al. Mechanisms of\nandrographolide-induced platelet apoptosis in human platelets: regulatory\nroles of the extrinsic apoptotic pathway. Phytother Res. 2013;27:1671 –7.\n55. Lu WJ, Lee JJ, Chou DS, Jayakumar T, Fong TH, Hsiao G, et al. A novel role\nof andrographolide, an NF-kappa B inhibitor, on inhibition of platelet\nactivation: the pivotal mechanisms of endothelial nitric oxide synthase/\ncyclic GMP. J Mol Med (Berl). 2011;89:1261 –73.\n56. Davidson DC, Hirschman MP, Spinelli SL, Morrell CN, Schifitto G, Phipps RP, et al.\nAntiplatelet activity of valproic acid contributes to decreased soluble CD40 ligand\nproduction in HIV type 1-infected individuals. J Immunol. 2011;186:584–91.\n57. Cai A, Zhou Y, Li L. Rho-GTPase and atherosclerosis: pleiotropic effects of\nstatins. J Am Heart Assoc. 2015;4.\n58. Fraser IS, Burridge J. Danazol treatment and platelet function. Med J Aust.\n1980;1:313–4.\n59. Arrowsmith JB, Dreis M. Thrombocytopenia after treatment with danazol. N\nEngl J Med. 1986;315:585.\n60. Zhang Q, Duan J, Liu X, Guo SW. Platelets drive smooth muscle metaplasia\nand fibrogenesis in endometriosis through epithelial-mesenchymal\ntransition and fibroblast-to-myofibroblast transdifferentiation. Mol Cell\nEndocrinol 2016; In press.\n61. Vestweber D, Blanks JE. Mechanisms that regulate the function of the\nselectins and their ligands. Physiol Rev. 1999;79:181 –213.\n62. Chen M, Geng JG. P-selectin mediates adhesion of leukocytes, platelets, and\ncancer cells in inflammation, thrombosis, and cancer growth and metastasis.\nArch Immunol Ther Exp (Warsz). 2006;54:75 –84.\n63. Furie B, Furie BC, Flaumenhaft R. A journey with platelet P-selectin: the\nmolecular basis of granule secretion, signalling and cell adhesion. Thromb\nHaemost. 2001;86:214–21.\n64. Kim YJ, Borsig L, Varki NM, Varki A. P-selectin deficiency attenuates tumor\ngrowth and metastasis. Proc Natl Acad Sci U S A. 1998;95:9325 –30.\n65. McEver RP, Zhu C. Rolling cell adhesion. Annu Rev Cell Dev Biol. 2010;26:363–96.\n66. Hidari KI, Weyrich AS, Zimmerman GA, McEver RP. Engagement of P-selectin\nglycoprotein ligand-1 enhances tyrosine phosphorylation and activates\nmitogen-activated protein kinases in human neutrophils. J Biol Chem.\n1997;272:28750–6.\n67. Ma YQ, Plow EF, Geng JG. P-selectin binding to P-selectin glycoprotein\nligand-1 induces an intermediate state of alphaMbeta2 activation and acts\ncooperatively with extracellular stimuli to support maximal adhesion of\nhuman neutrophils. Blood. 2004;104:2549 –56.\n68. Zhang X, Lu B, Huang X, Xu H, Zhou C, Lin J. Endometrial nerve fibers in\nwomen with endometriosis, adenomyosis, and uterine fibroids. Fertil Steril.\n2009;92:1799–801.\n69. Zhang X, Lu B, Huang X, Xu H, Zhou C, Lin J. Innervation of endometrium\nand myometrium in women with painful adenomyosis and uterine fibroids.\nFertil Steril. 2010;94:730 –7.\n70. Guo S-W, Du Y, Liu X. Endometriosis-Derivedtic Stromal Cells Secrete\nThrombin and Thromboxane A2, Inducing Platelet Activation..Reprod Sci\n2016;In press.\n71. Sumimoto S, Muramatsu R, Yamashita T. Thromboxane A2 stimulates\nneurite outgrowth in cerebral cortical neurons via mitogen activated\nprotein kinase signaling. Brain Res. 2015;1594:46 –51.\n72. Yan D, Liu X, Guo SW. Endometriosis-Derived Thromboxane A2 Induces\nNeurite Outgrowth. Reprod Sci 2016; In press.\n73. Wilhelmsson L, Wikland M, Wiqvist N. PGH2, TxA2 and PGI2 have potent\nand differentiated actions on human uterine contractility. Prostaglandins.\n1981;21:277–86.\n74. Adachi N, Tomonaga S, Tachibana T, Denbow DM, Furuse M.\n(−)-Epigallocatechin gallate attenuates acute stress responses through\nGABAergic system in the brain. Eur J Pharmacol. 2006;531:171 –5.\n75. Zhang Z, Cai YQ, Zou F, Bie B, Pan ZZ. Epigenetic suppression of GAD65\nexpression mediates persistent pain. Nat Med. 2011;17:1448 –55.\n76. Le Bars D, Gozariu M, Cadden SW. Animal models of nociception.\nPharmacol Rev. 2001;53:597 –652.\n77. Bannon AW, Malmberg AB. Models of nociception: hot-plate, tail-flick, and\nformalin tests in rodents. Curr Protoc Neurosci. 2007;Chapter 8:Unit 8:9. doi:\n10.1002/0471142301.ns.0809s41.\n78. Wang HW, Wu CC. Effects of oxymetazoline on isolated rat ’s tracheal\nsmooth muscle. Eur Arch Otorhinolaryngol. 2008;265:695 –8.\n79. Calixto JB, Yunes RA. Antagonism of kinin-induced contraction of isolated\nrat uterus by the crude hydroalcoholic extract from Mandevilla illustris. Gen\nPharmacol. 1991;22:99–101.\n80. Hernandez-Magro PM, Villanueva Saenz E, Alvarez-Tostado Fernandez F, Luis\nRocha Ramirez J, Valdes Ovalle M. Endoanal sonography in the assessment\nof perianal endometriosis with external anal sphincter involvement. J Clin\nUltrasound. 2002;30:245–8.\n•  We accept pre-submission inquiries \n  Our selector tool helps you to find the most relevant journal\n  We provide round the clock customer support \n  Convenient online submission\n  Thorough peer review\n  Inclusion in PubMed and all major indexing services \n  Maximum visibility for your research\nSubmit your manuscript at\nwww.biomedcentral.com/submit\nSubmit your next manuscript to BioMed Central \nand we will help you at every step:\nZhu et al. 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