{"paper_id":"74a9cfe2-1e9d-4a37-9fad-f6aced89f51d","body_text":"Int. J. Med. Sci. 2020, Vol. 17 \n \n \nhttp://www.medsci.org \n224 \nInternational Journal of Medical Sciences\n \n2020; 17(2): 224-233. doi: 10.7150/ijms.39252 \nResearch Paper \nThe new application of mifepristone in the relief of \nadenomyosis-caused dysmenorrhea \nXuan Che1,2*, Jianzhang Wang1*, Jiayi He1, Xinyue Guo1, Tiantian Li1, Xinmei Zhang1 \n1. Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, P.R. China, 310006 \n2. Jiaxing Maternity and Child Health Care Hospital, Jiaxing, Zhejiang, P.R. China, 314000  \n*These authors contributed equally to this work. \n Corresponding author: Xinmei Zhang, MD, PhD, The Department of Gynecology, Women’s Hospital, School of Medicine, Zhejiang University, 1 Xueshi \nRoad, Hangzhou, Zhejiang, P.R. China, 310006. Phone: 86-571-87061501-2131; Fax: 86-571-87061878; E-mail: zhangxinm@zju.edu.cn \n© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://cr eativecommons.org/licenses/by/4.0/). \nSee http://ivyspring.com/terms for full terms and conditions. \nReceived: 2019.08.12; Accepted: 2019.11.07; Published: 2020.01.14 \nAbstract \nBackground: Adenomyosis is a quite common gynecological disorder and above 30% of patients \nhave typical secondary and progressive dysmenorrhea. Current treatments still have many \ndisadvantages and thereby the novel treatment aiming to relieve dysmenorrhea  still needs to be \nfurther investigated. Mifepristone is a wonderful drug because it is effective, safe and cheap in many \ndiseases including adenomyosis. In this study, we aim to investigate if mifepristone could be used in \nthe treatment of adenomyosis-associated dysmenorrhea. \nMethods: Human primary endometrial epithelial and stromal cells from adenomyosis patients were \nisolated and treated with mifepristone. RNA -sequencing was then performed to detect  the gene \nchanges of pain -related inflammatory mediators.  Meanwhile, the  effect of mifepristone on the \ninfiltration and degranulation of mast cells were investigated in adenomyosis lesions. Additionally, \nthe role of mifepristone on the density of nerve fibers was also studied in the ectopic endometrium. \nAt last, to evaluate the therapeutic efficacy of mifepristone on dysmenorrhea of adenomyosis, \ntwenty participants were included and the visual analog scale ( VAS) score was assessed and \ncompared before and after treatment with mifepristone. \nResults: We demonstrated that mifepristone reduced the secretion of IL -6 and TNF-α  from \nendometrial epithelial and stromal cells, restricted the infiltration and degranulation of mast cells in \neutopic and ectopic endometrium and decreased the density of nerve fibers by inhibiting the \nmigration capacity of nerve cells in adenomyosis. Meanwhile, we found that mifepristone could \nsignificantly relieve dysmenorrhea of adenomyosis. \nConclusion: The findings demonstrated that mifepristone cou ld be applied in the treatment of \ndysmenorrhea for the adenomyosis patients. \nKey words: adenomyosis, dysmenorrhea, mifepristone, inflammation, mast cell, nerve fiber \nIntroduction \nAdenomyosis is defined as invasion of \nendometrial glands and stroma into the myometrium \nand the prevalence of adenomyosis ranges from 8 to \n27% of women in reproductive age [1]. Adenomyosis \ncauses many health problems such as dysmenorrhea, \nhypermenorrhea and subfertility. Above 30% of \npatients have typical secondary and progressive \ndysmenorrhea [2]. Serious dysmenorrhea can affect \nthe qualities of work, eating and sleep and cause \ndepression, which restricts the daily routine of these \npatients and has a tremendous impact on  their \nphysical and mental health [2, 3]. Moreover, \nadenomyosis is diagnosed  in 20 -25% of infertile \nyoung women undergoing assisted  reproductive \ntechnologies [4]. Severe dysmenorrhea is the primary \nreason for patients to choose hysterectomy and lost \n \nIvyspring  \nInternational Publisher \n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n225 \ntheir fertility. Current therapy for adenomyosis - \nassociated pain includes hysterectomy, oral \ncontraceptive drugs and gonadotrophin- releasing \nhormone (GnRH) agonists. However, the  present \nmedical treatment for  adenomyosis-related \ndysmenorrhea is limited for their side effects such as \npremenopausal symptoms, high relapse rate after  \nmedicine withdrawal and high costs [5, 6]. Thus, the \nnovel medical treatment aiming to relieve \ndysmenorrhea should be further investigated. \nThe exact pathogenic mechanism of \nadenomyosis-related dysmenorrhea remain s unclear, \nwhile inflammation and innervation possibly are the \nkey pathogenic factors [7]. Inflammatory mediators, \nincluding IL-6, TNF-α, IL-1β and IL-10, are involved \nin inflammatory pathway and contribute to the \nintense painful symptoms in adenomyosis  [8-10]. \nFurthermore, increasing evidenc e supports that the \nactivated mast cell is regarded as a director of \ncommon inflammatory pathways contributi ng to \nchronic neuropathic pain and may play a role in \npathogenesis of adenomyosis [11-13]. Our previous \nstudy also showed that the recruitment and \ndegranulation of mast cells play  an important role in \nendometriosis-related dysmenorrhea [14]. In addition, \nrecent research found adenomyosis -induced pain \nresembles neuropathic pain [15]. We also proved that \nthe density of nerve fibers in the functional layer \nendometrium of adenomyosis patients was correlated \nwith dysmenorrhea, suggesting the nerve fibers play \nan important role in the mechanisms of pain \ngeneration in adenomyosis [16]. Obviously, drug \ntherapy for adenomyosis can be based on the \nabove-mentioned pathogenesis of adenomyosis. \nMifepristone is the first and one of the most \nwidely used selective progesterone receptor \nmodulators (SPRM) since 1982. Besides mild adverse \neffect and well tolerance in its long -term clinical \napplication, low price of this drug is another great \nadvantage for the patients because adenomyosis \nneeds the long -term medical therapy [17]. In China, \nthe cost of mifepristone is only less than 4 US dollars \nper month while GnRH -a treatment needs more than \n200 US dollars per month in the treatment of \nendometriosis. Currently, we find that mifepristone \nhas more benefits for human health than wh at we \nthought before. Recent studies showed that \nmifepristone strongly decreased the levels of tumor \nnecrosis factor -α (TNF -α), interleukin -1β (IL -1β) and \ninterleukin-6 (IL-6) of paraquat-induced lung injury in \nrats [18]. Treatment of mifepristone significa ntly \ndownregulated the expression of neuronal nitric \noxide synthase (nNOS) and N -methyl-D-aspartate \nreceptor subunit 2B (NR2B) proteins in a rat model of \nradicular pain [19] . Furthermore, Li et al. reported \nthat addition of mifepristone to depot -medroxy-\nprogesterone acetate (DMPA) -exposed endometrium \nsignificantly decreased mast tryptase -positive cells \nand pointed that mifepristone is associated with \ninhibiting the activity of mast cells [20]. Some studies \nin China and we also found that mifepristone could be \napplied in the treatment of adenomyosis. Taken \ntogether, theoretically, mifepristone may be a new \ntherapeutic agent for adenomyosis -related pain. \nHowever, only a few studies were performed to \ninvestigate the role of mifepristone on the \ndysmenorrhea caused by adenomyosis. \nIn this study, human primary endometrial \nepithelial cells and stromal cells from adenomyosis \npatients were isolated and treated with mifepristone. \nRNA-sequencing was then performed to detect  the \ngene changes of inflammatory mediators. Meanwhile, \nwe investigated the  effects of mifepristone on the \ninfiltration and degranulation of mast cells in \nadenomyosis. Additionally, we investigated the role \nand mechanism of mifepristone on the density of \nnerve f ibers in the ectopic endometrium of \nadenomyosis patients. At last, to further study the \ntherapeutic efficacy of mifepristone on dysmenorrhea \nof adenomyosis , twenty participants were included \nand the visual analog scale (VAS)  score was assessed \nand compared  before and after treatment with \nmifepristone. Our study was performed to elucidate \nthe effect of mifepristone on the relief of \ndysmenorrhea, which will provide a solid foundation \nfor the application of mifepristone in the treatment of \nadenomyosis patients with dysmenorrhea. \nMaterials and Methods  \nIsolation and identification of endometrial \nstromal and epithelial cells of adenomyosis \nIsolation of primary endometrial stromal and \nepithelial cells was performed using a previously \nreported method [21] . Briefly,  the tissues were \nwashed with FBS -free medium under aseptic \nconditions and were minced into 1×1× 1 mm3 pieces. \nAfter the minced tissues were digested with 1 mg/mL \ncollagenase type III at 37°C for 60 min, the  \nendometrial epithelial cells and stromal cells were \nseparated by two sequential filtrations of 200 and 70 \nμm cell strainer. Endometrial epithelial cells \nremaining in the cell strainer were collected were \ncultivated in primary epithelial growth medium \n(PriCells, Wuhan, China) and endometrial stromal \ncells were cultivated in Dulbecco's modified Eagle's \nmedium (DMEM)/F12 medium (Thermo Fisher, CA, \nUSA) supplemented with 10% FBS (Sigma -Aldrich, \nMO, USA). Identification of the isolated endometrial \nepithelial cells and stromal cells was assessed with \n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n226 \nanti-cytokeratin and anti -vimentin antibodies by \nimmunohistochemistry as described in our previous \nstudy [22]. \nCell lines \nThe RBL2H3 mast cell line was purchased from \nStem Cell Bank, Chinese Academy of Sciences and \ncultivated in minimum Eagle’s medium (Sigma) \nsupplemented with 10% heat- inactivated fetal calf \nserum (Gibco), 100 U/mL penicillin and 100 μg/mL  \nstreptomycin. PC12 nerve cell line, as a neuronal \nmodel, was purchased from Chinese Academy of \nSciences and cultured in a complete medium \nconsisting of 85% F -12 me dium (Sigma), 10% \nheat-inactivated horse serum (Gibco), and 5% fetal \ncalf serum (Gibco). RBL2H3 Cells and PC12 Cells \nwere treated with or without mifepristone at \nconcentration of 50 μM for 48h in this study. \nThe Cell-counting Kit-8 (CCK-8) Assay \nTo investi gate the suitable concentration of \nmifepristone treatment for the following study, \nprimary endometrial epithelial cells were treated with \nmifepristone in different concentrations (0, 10, 25, 50, \n75, 100 and 200 μM, respectively) for 24h. Then, the \nviability of above cells  was detected by the CCK -8 \nassay (Biosharp, Beijing, China). CCK -8 reagent was \nadded to each well and cells were incubated at 37°C \nfor 2h in accordance with the manufacturer's \ninstructions. The absorbance at 450 nm  (optical \ndensity) was detected to calculate the cell viability.  \nHigh-throughput sequencing \nBased on the result of CCK -8 assay, the viability \nof endometrial epithelial cells was significantly \ndecreased when the concentrations of mifepristone \ntreatment were 75 μM. Subsequently, primary \nendometrial epithelial cells  were treated with \nmifepristone at the concentration of 50 μM for 24 \nhours. The epithelial cells were isolated from four \nindependent samples. Total RNAs were isolated \nusing Trizol reagent (Life Technologies, Grand Island, \nUSA) and subjected to RNA high -throughput \nsequencing by The Beijing Genomics Institute (BGI, \nShenzhen, China).  \nReal-time polymerase chain reaction \nTotal RNA of each endometrium was extracted \nusing Trizol reagent (Takara, Japan) and reverse \ntranscription wa s performed using PrimeScript \nReverse Transcription reagent kit (Takara, Japan). \nRT-PCR was performed using SYBR Premix Ex Taq \nTM Kit (Takara, Japan) with ABI 7500 realtime PCR \nsystem (Thermo, MMAS, USA). The primer  was \ndesigned using Primer 3, and the nuc leotide \nsequences of IL -6 were as follows: sense \n5′-CCTCCAGAACAGATTTGAGAGTAGT-3′; and \nantisense 5′ -GGGTCAGGGGTGGTTATTGC-3′; the \nnucleotide sequences of TNF-A were as follows: sense \n5′-CGAGTGACAAGCCTGTAGCC-3′; and antisense \n5′-TGAAGAGGACCTGGGAGTAGAT-3′. As an \ninternal control, GAPDH was also amplified and the \nnucleotide sequence for the primers were as follows: \nsense 5′ -GCCATCAATGACCCCTTCATT-3′ and \nantisense 5′-TGACGGTGCCATGGAATTT-3. \nMeasurement of mast cell degranulation \nAs our previous study described , RBL 2H3 cell \ndegranulation was measured through the release of \nβ-hexosaminidase [14]. RBL2H3 cells were seeded in \n96-well plates (5 × 10 4 cells /well) with or without \nmifepristone treatment at the concentration of 50 μM \nfor 48h. Then stimulated with DNP -BSA at  the \nconcentrations of 100 ng/ml (A6661, Sigma, USA) and \ndegranulation was detected by the release of hex \naccording to the protocol [23].  \nEnzyme linked immunosorbent assay (ELISA) \nThe endometrial epithelial cells and stromal cells \nwere treated with mifepr istone at different \nconcentrations (0, 50 and 100 μM, respectively) for 48 \nh and the concentration of IL -6 and TNF-α protein in \nendometrial epithelial and stromal cells culture \nsupernatant were detected by ELISA kits of \ninterleukin-6 (IL-6) (ELH-IL6-1, RayBiotech, Peachtree \nCorners, GA, USA) and tumor necrosis factor -α \n(TNF-α) (ELH -TNFa-1, RayBiotech, Peachtree \nCorners, GA, USA) . ELISA was performed according \nto manufacturer’s instructions.  \nImmunohistochemical staining \nAdenomyosis eutopic  endometrium and \ncorresponding ectopic endometrium were collected \nduring surgery. The diagnosis of adenomyosis was \nconfirmed by imaging or histological examination. \nSamples were collected in the proliferative phase of \nthe menstrual cycle. Sections were incubated with \nanti-c-kit antibody (dilution 1:200, ab32363, Abcam, \nCambridge, MA, USA) and anti -PGP9.5 antibody \n(dilution 1:500, Z5116, Dako Cytomation, \nDenmarkA/S). Immunohistochemical assay was \nperformed as previously described [24]. Individual \nnerve fibers were then counted under high power (× \n200) to obtain a nerve count in a defined area. The \naverage nerve count in  five hot spots was calculated \n[25]. \nCell migration assay \nCell migration ability was evaluated by \ntranswell chamber assay using 24 -well plates  with \n8.0-μm pore size membranes (BD Biosciences, CA, \nUSA). To study the effect of mifepristone on the \n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n227 \nmigratory ability of nerve cells, PC12 cells with or \nwithout pre -treatment of mifepristone at the \nconcentration 50 μM were added into the upper \nchamber of the insert in 200 μL of serum-free medium, \nwhile the lower chamber contained growth media \nwith 10% FBS. After 24h incubation, cells in the upper \nchamber were removed with a cotton swab and the \nmigrated cells in the lower chamber were fixed with \nmethanol, stained with crystal violet and counted \nwith a microscope (Olympus, Japan). The PC12 cells \nthat passed through the membrane was defined as \nmigrated cells. \nPatients and clinical evaluation \nThis study was approved by Ethics Committee \nof Women's Hospital, Sc hool of Medicine, Zhejiang \nUniversity and registered in Chinese Clinical Trial \nRegistry (1800015514). Twenty cases of adenomyosis \npatients were included after informed consent. No \nhormone or similar drugs were used for 6 months \nbefore treatment. The patien ts were treated with \nmifepristone by oral administration at 5  mg per day. \nThe visual analog scale (VAS) was used to evaluate \nthe degree of dysmenorrhea before and after \ntreatment of mifepristone for 3 months. The left end of \nthe VAS was scored as 0 to repr esent “no pain” while \nthe right end was scored as 10, representing the “most \nsevere pain imagined” [26]. The VAS score was \nself-assessed by each  patient prior to treatment. On  \nthe other hand, platelet count in serum of \nadenomyosis patients was obtained, an d they were \nanalyzed in the hematology laboratory  of our \nhospital.  \nStatistical Analysis \nSPSS program version 19.0 and Graph Pad Prism \n5 software  were applied for statistical analysis. Data \nare shown as the mean ± Standard Error of Mean \n(SEM). P values wer e determined by the two -tailed \nStudent’s t test or Mann -Whitney U test when \ncomparing two groups and by a one -way ANOVA \nwhen comparing more than two groups. Statistical \ndifference was considered to be significant at a value \nof P< 0.05 (*), highly significa nt at a value of P< 0.01 \n(**) and extremely significant when P< 0.001(***).  \nResults \nMifepristone reduces the secretion of IL-6 and \nTNF-α from endometrial epithelial and \nstromal cells in adenomyosis. \nTo investigate the potential mechanism of \nmifepristone r elieving dysmenorrhea on the \nadenomyosis, RNA -sequencing was performed to \ndetect the changes of gene expression in the primary \nendometrial epithelial cells with or without treatment \nof mifepristone. Firstly, CCK -8 assay was performed \nto determine the effec tive concentrations of \nmifepristone on the primary endometrial epithelial \ncells of adenomyosis. Cells were treated with \nmifepristone at different concentrations (0, 10, 25, 50, \n75, 100 and 200 μM, respectively) for 24h. As shown in \nFig. 1A, the viability o f endometrial epithelial cells \nwas significantly decreased when treated with \nmifepristone at concentrations above 50  μM. The \neffective concentration of mifepristone applied in this \nstudy was similar to that used in treatments of kinds \nof cancers [27]. Based on the result of CCK-8 assay, the \nendometrial epithelial cells were treated with \nmifepristone at the concentration of 50  μM for 24 \nhours (n=4) and gene expression was examined by \nRNA-sequencing. Fig.1B showed that mifepristone \nsignificantly down- regulated the expressions of IL -6 \nand TNF -A in endometrial epithelial cells  when \ncompared to controls, which are the important \npro-inflammatory chemokines closely correlated with \ndysmenorrhea. \nThen, the down-regulations of IL-6 and TNF-α in \nthe mifepristone -treated group were further \nconfirmed by qRT -PCR and ELISA  not only in \nprimary endometrial epithelial cells but also in \nstromal cells. The mRNA expression of IL -6 and \nTNF-A was decreased in both endometrial epithelial \ncells and stromal cells when treated with mifepristone \ntreatment in a dose-dependent manner. Subsequently, \nELISA assay was conducted to detect the \nconcentrations of IL -6 and TNF -α in cell culture \nsupernatants of endometrial epithelial cells and \nstromal cells with and without mifepristone \ntreatment. E ndometrial epithelial cells and stromal \ncells were treated with mifepristone at different \nconcentrations (0, 50 and 100 μM) for 48h. We found \nthat the concentrations of IL -6 and TNF -α in cell \nculture supernatants were significantly decreased in \nboth endome trial epithelial and stromal cells when \ntreated with mifepristone in a dose- dependent \nmanner (Fig. 1C). These results suggested that \nmifepristone reduces the secretion of IL -6 and TNF-α \nfrom endometrial epithelial and stromal cells in \nadenomyosis and therefore may have an effect on the \nrelief of pain for the adenomyosis patients.  \nMifepristone inhibits the infiltration and \nactivity of degranulation of mast cells in \nadenomyosis \nAs well known, mast cells mediate neurogenic \ninflammation and pain [28]. The activated and \ndegranulating mast cells may exert indirect effects on \nthe development of neuropathic pain [29].  \n\nInt. J. Med. Sci. 2020, Vol. 17 \n \n \nhttp://www.medsci.org \n228 \n \nFigure 1. Mifepristone reduces the secretion of IL -6 and TNF -α from endometrial epithelial and stromal cells in adenomyosis. (A) Human primary \nendometrial epithelial cells were treated with mifepristone in different concentrations for 24h, and CCK-8 assay was performed. The viability of endometrial epithelial cells was \nsignificantly decreased when treated wi th mifepristone at 75 μM while there was no significant difference at 50  μM. Concentration at 50 μ M was therefore selected for the \nfollowing RNA-sequencing. (B) Primary endometrial epithelial cells  were treated with mifepristone at the concentration of 50 μM and then subjected to next generation \nsequencing. The endometrial epithelial cells were from four biologically independent samples and the data were shown in quadruplicate. (C) qRT-PCR and ELISA were performed \nto detect the role of mifepristone on the down-regulations of IL-6 and TNF-α in endometrial epithelial and stromal cells in different concentrations. Data were shown as mean \n± SEM. *P<0.05, **P<0.01 and ***P<0.001. \n\n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n229 \n \nFigure 2. Mifepristone decreased the number of mast cells in both eutopic and ectopic endometriums and also inhibited the activity of degranulation. (A) \nImmunohistochemical staining for c-kit was examined in the eutopic and ectopic endometriums with or without mifepristone treatment. The black arrow indicates the mast cells. \nScale bars = 500 μm. Image was captured at 200× magnification. (B) Rate of active degranulation in RBL2H3 mast cells was examined after treatment of 50 μM of mifepristone \nfor 48h. Data were expressed as mean ± SEM. Statistical difference was determined by Mann-Whitney U test. *P<0.05, **P<0.01 and ***P<0.001. \n \nTo study the effect of mifepristone on the mast \ncell-infiltration in adenomyosis, immunohisto -\nchemistry was conducted to detect the number of \nmast cells by staining with c-kit in eutopic and ectopic \nendometriums of adenomyosis. We observed the \nnumbers of mast cells were significantly decreased in \nboth eutopic and ectopic endometriums after \nmifepristone treatment (P < 0.001; Fig. 2A). To further \ndetermine whether mifepristone has effect on the \nactivity of degranulation of mast cells. RBL2H3 mast \ncells were treated with mifepristone at concentration \nof 50 μM for 48h. The rate of degranulation of RBL2H3 \ncells treated with mifepristone was significantly \ndecreased when compared to  mifepristone-untreated \ngroup (p<0.05; Fig.  2B). The above results revealed \nthat mifepristone inhibits the infiltration and the \nactivity of degranulation of mast cells in both eutopic \nand ectopic endometriums of adenomyosis. \nMifepristone decreases the density of nerve \nfibers in both eutopic and ectopic \nendometriums of adenomyosis \nIt is known that afferent sensory fibers are \ncritical for the conduction of adenomyosis -caused \n\n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n230 \npain. Our previous study also found that \ndysmenorrhea was positively correlated with the \ndensity of PGP 9.5 -immunoactive nerve fibers in the \nbasal layer of the endometrium and myometrium [30]. \nTo investigate the effect of mifepristone on the \ninnervation in adenomyosis, immunohistochemistry \nwas conducted to detect PGP 9.5-immunoactive nerve \nfibers in endometrium and myometrium tissue with \nand without mifepristone treatment. Fig. 3A showed \nthat the density of PGP 9.5-immunoactive nerve fibers \nin mifepristone -treated adenomyosis group was \nsignificantly decreased in both eutopic and ectopic \nendometrium when compared to \nmifepristone-untreated group, especially in ectopic \nendometrium of adenomyosis. The findings \nsuggested that mifepristone reduces the density of \nnerve fibers in adenomyosis, which may play an \nimportant role in relieving adenomyosis-caused pain. \n \n \n \nFigure 3. Mifepristone decreases the density of nerve fibers by inhibiting the migratory capacity of nerve cells in adenomyosis. (A) Nerve fibers were stained \nby immunohistochemical staining using PGP9.5 antibody in both eutopic and ectopic endometriums with or without mifepristone treatment. Scale bars = 500 μM. Image was \ncaptured at 200× magnification. (B) Phase-contract images of migrated PC12 nerve cells on the bottom of transwell insert membrane with or without treatment of mifepristone. \nNumber of migrated PC12 nerve cells on the bottom of PET membrane was counted as indicated conditions. Data were expressed as  mean ± SEM. *P<0.05, **P<0.01 and \n***P<0.001.  \n\n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n231 \n \nFigure 4. Mifepristone significantly relieved dysmenorrhea in adenomyosis patients. (A) The visual analog scale (VAS) score was applied for the pain assessment of \ndysmenorrhea in the included patients. The VAS score was significantly decreased after mifepristone treatment for the adenomyosis patients. (B) The platelet count in serum of \nadenomyosis patient was measured before and after three-month treatment with mifepristone. Data were expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001. \n \nMifepristone decreases the density of nerve \nfibers by inhibiting the migration capacity of \nnerve cells in adenomyosis \nTo investigate the potential mechanism of how \nmifepristone decreased the density of nerve fibers in \nadenomyosis, migration assay was performed to \ndetect the effect of mifepristone on the migratory \ncapacity of PC12 nerve cells in adenomyosis. As \nshown in Fig. 3B, the number of migratory PC12 cells \nwas significantly decreased in mifepristone -treated \ngroup when compared to untreated group in a \ndose-dependent manner (p<0.000), demonstrating \nthat the migratory ability of nerve cells was \nsignificantly restricted after treatment with \nmifepristone. The above data indicated mifepristone \ndecreases the density of nerve fib ers by inhibiting the \nmigratory capacity of nerve cells in adenomyosis. \nMifepristone significantly relieves \ndysmenorrhea in adenomyosis patients  \nTo study the therapeutic efficacy of mifepristone \non dysmenorrhea of adenomyosis patients, the VAS  \nscore was a pplied for pain assessment of \ndysmenorrhea. The VAS score was assessed and \ncompared for the same patient before and after \ntreatment with mifepristone. As shown in Fig.  4A, \nmifepristone treatment significantly decreased the \nVAS score of dysmenorrhea for adenomyosis patients \nwhen compared to pre -treatment. In addition, it is \nknown that the platelet count in serum is an \nimportant marker for the development of \nadenomyosis and closely associated with \ndysmenorrhea symptoms. The platelet count in serum \nbefore treatment was 282.66 ± 10.84 10^9/L while the \nmean concentration was 242.95 ± 8.80 10^\n9/L (Fig.4B), \nindicating that the platelet count in serum were \nsignificantly reduced after mifepristone treatment. \nTherefore, we concluded that mifepristone could \nrelieve dys menorrhea symptoms for adenomyosis \npatients. \nDiscussion  \nDysmenorrhea is a common symptom in \nadenomyosis and is one of the main reasons  that \nwomen seek medical treatment. Although medical  \ntherapies such as GnRH -a, medroxyprogesterone \nacetate (MPA) and dana zol have shown certain \nclinical effects for relieving adenomyosis -related \ndysmenorrhea, the potential side effects compromise \nthose clinical applications. Afferent sensory fibers and \nproinflammatory mediators are correlated with \nadenomyosis pain, which can  be considered an \ninflammatory neuropathic pain. Recent studies \nshowed that mifepristone may play important roles in \nthe development of neuropathic pain diseases. \nHowever, the evidence for guiding clinical use of \nmifepristone treatment is insufficient in adenomyosis. \nThe present study will elucidate the feasibility of  this \nold drug for new use in adenomyosis. \nInflammation is a major biological determinant \nin the pathogenesis of adenomyosis and \nproinflammatory/inflammatory cytokines act as \nchemical neurotran smitters to stimulate uterine \ncontraction and cause dysmenorrhea [31] . In the \npresent study, we found that mifepristone reduces the \nsecretion of IL -6 and TNF -α from endometrial \nepithelial and stromal cells in adenomyosis. Similar to \nMPA and danazol treatme nt of adenomyosis [32, 33], \nmifepristone treatment inhibited the secretion of IL -6 \nin endometrial epithelial and stromal cells of \nadenomyosis in our experiments. Yang et al. reported \nthat MPA and danazol have no effect  on the \nsuppression of TNF- α by endometrial and stromal \ncells in a denomyosis [34] while our data showed \nmifepristone significantly decreased the mRNA and \nprotein expression of TNF -α in both endometrial \n\n\nInt. J. Med. Sci. 2020, Vol. 17 \n \nhttp://www.medsci.org \n232 \nepithelial and stromal cells of adenomyosis. Recent \nreports pointed that selective progesterone receptor \nmodulators may be possibly more effectively than \nprogestins in relieving  adenomyosis-associated pain, \nbut the underlying mechanism was still unclear [35, \n36]. Our findings showed that mifepristone \nsignificantly decreased the expressions of IL- 6 and \nTNF-α in both endometrial epithelial and stromal cells \nof adenomyosis, which may be the reason that \nmifepristone is more effectively than progestins in the \nrelief of adenomyosis-associated pain. \nIncreasing evidence supports that activated and \ndegranulating mast cells play an important role in the \ndevelopment of pain, hyperalgesia and dysmenorrhea \n[37, 38]. Our previous study also demonstrate that the \nactivity and degranulation of mast cells play  an \nimportant role i n endometriosis -related \ndysmenorrhea [14]. Moreover, it is reported that mast \ncells contribute to the development of inflammation \nin adenomyosis [10]. Therefore, those drugs that can \ninhibit mast cell -activation and suppress mast \ncell-degranulation may be used as effective \ntherapeutic agents for adenomyosis. As we expected, \nour study showed that the infiltration of mast cells \nwas significantly decreased in both eutopic and \nectopic endometriums after mifepristone treatment. \nMoreover, the rate of degranulatio n of mast cells \ntreated with mifepristone were decreased when \ncompared to mifepristone -untreated group. Hence, \nwe concluded that m ifepristone relieved the \ndysmenorrhea symptom of adenomyosis patients \nthrough inhibiting the infiltration and the activity of \ndegranulation of mast cells in eutopic and ectopic \nendometriums.  \nIt is well known that pain is mediated by sensory \nnerves. Afferent sensory fibers and proinflammatory \nmediators are correlated with adenomyosis pain. Our \nprevious study found that the distri bution of nerve \nfibers in the ectopic endometrium play an important \nrole on the pain symptoms in both endometriosis and \nadenomyosis [16]. T he present study found that \nmifepristone decreases the density of nerve fibers in \nboth eutopic and ectopic endometriu ms of \nadenomyosis. Furthermore, Transwell assay was then \nperformed to confirm that mifepristone decreased the \nmigration of nerve cells in a dose -dependent manner. \nTaken together, our data suggested that the relief of \nadenomyosis-associated dysmenorrhea  by \nmifepristone is related to the decrease of density of \nnerve fibers by inhibiting the migration capacity of \nnerve cells in adenomyosis. \nAt last, the efficacy of mifepristone treatment in \nadenomyosis was further confirmed by comparing \nthe pain assessment of dysmenorrhea in the same \nadenomyosis patient before and after mifepristone \ntreatment. We concluded that mifepristone effectively \nrelieved dysmenorrhea symptoms for adenomyosis \npatients. Furthermor e, it is reported that platelets \nplayed an important role in the development of \nadenomyosis and anti-platelet treatment could reduce \nuterine hyperactivity and improve generalized \nhyperalgesia [39]. Our data showed that mifepristone \nsignificantly decreased platelet count in serum of the \nadenomyosis patients. Therefo re, the clinical results \nfurther proved that mifepristone was efficient in the \ntreatment of adenomyosis -associated dysmenorrhea \nand the effect of treatment in adenomyosis is similar \nto endometriosis [40, 41].  \nConclusion \nWe firstly demonstrated that mifepr istone \nreduced the secretion of IL- 6 and TNF -α from \nendometrial epithelial and stromal cells, restricted the \ninfiltration and degranulation of mast cells in eutopic \nand ectopic endometrium and decreased the density \nof nerve fibers by inhibiting the migratory capacity of \nnerve cells in adenomyosis. Meanwhile, we found \nthat mifepristone could significantly relieve \nadenomyosis-associated dysmenorrhea. The findings \ndemonstrated that mifepristone could be applied in \nthe treatment of dysmenorrhea for the adenomyo sis \npatients. \nAcknowledgments  \nWe deeply appreciate that the study was funded \nby National Key R&D Program of China (Grant \nnumber: 2017YFC1001202) and National Natural \nScience Foundation of China (Grant numbers: \n81671429 and 81802591). \nCompeting Interests \nThe authors have declared that no competing \ninterest exists. \nReferences \n1. Naftalin J, Hoo W, Pateman K, Mavrelos D, Holland T, Jurkovic D. How \ncommon is adenomyosis? A prospective study of prevalence using \ntransvaginal ultrasound in a gynaecology clinic. Hum Reprod. 2012; 27: \n3432-9. \n2. Garcia L, Isaacson K. Adenomyosis: review of the literature. J Minim Invasive \nGynecol. 2011; 18: 428-37. \n3. 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