{"paper_id":"8b19d78f-4691-4da5-a764-d2c8271da9cc","body_text":"Cite as\nKaplan S, Kırıcı P , Türk A. The effects of adalimumab  \non the rat autotransplantation endometriosis model: \nA placebo-controlled randomized study. Adv Clin Exp Med . \n2022;31(4):417–426. doi:10.17219/acem/144369\nDOI\n10.17219/acem/144369\nCopyright\nCopyright by Author(s) \nThis is an article distributed under the terms of the\nCreative Commons Attribution 3.0 Unported (CC BY 3.0)\n(https://creativecommons.org/licenses/by/3.0/)\nAddress for correspondence\nSelçuk Kaplan\nE-mail: kaplan_2384@hotmail.com\nFunding sources\nNone declared\nConflict of i nterest\nNone declared\nAcknowledgements\nThe authors would like to thank Prof. Dr. Mehmet Şimşek \nfrom the Department of Obstetrics and Gynecology, \nAdiyaman University, Turkey, for his contribution.\nReceived on September 16, 2020\nReviewed on August 26, 2021\nAccepted on November 30, 2021\nPublished online on January 18, 2022\nAbstract\nBackground. Endometriosis is  a ch ronic inflammatory pathology that can cause persistent pelvic pain \nand infertility by affecting women of reproductive age. It is defined as the placement of endometrial tissue \noutside the uterine cavity. Hormonal, genetic and immunological factors have an effect on the development \nof e ndometriotic implants. Adalimumab is  a m onoclonal antibody specific for tumor necrosis factor alpha \n(TNF-α), used in the treatment of autoimmune diseases.\nObjectives. To in vestigate the  e ffectiveness of a dalimumab on hi stopathological and biochemical values \nin r ats with experimental endometriosis.\nMaterials and methods. This study is a comparative, prospective, experimental rat study. Wistar albino \nfemale rats were divided into 4 groups. Group 1 was separated as the control group. Endometriotic implants \nwere simultaneously induced in group 2 and group 3. After 4 weeks, developing endometriotic foci were \nmeasured. Adalimumab (5 m g/kg) was simultaneously intraperitoneally (ip.) administered to g roup 3  and \ngroup 4 for 4 weeks. At the end of the study, histopathological scoring and fibrillin-1 scoring were performed. \nTotal antioxidant status (TAS), total oxidant status (TOS) and malondialdehyde (MDA) values were measured. \nFindings in a ll groups were compared.\nResults. When group 1 and group 2 were compared, the histopathological score, as well as MDA and TOS \nlevels increased, while TAS levels decreased in group 2 (p < 0.001). After adalimumab treatment, the average \nendometriotic implant size in group 3 (0.32 ±0.002 mm) decreased compared to group 2 (0.77 ±0.04 mm) \n(p = 0.032). While fibrillin-1 score increased in  group 2 and group  3 compared to  group 1, it decreased \nin group 3 compared to g roup 2 (p < 0. 001). Histopathological score decreased, TAS levels increased and \nMDA levels decreased in group 3 compared to group 2 (p < 0.001).\nConclusions. Adalimumab may play a role in the regression of endometrial implants by showing antioxidant \nand anti-inflammatory effects on hi stopathological damage and fibrosis.\nKey words: endometriosis, rat, adalimumab, antioxidant effect, fibrillin-1\nOriginal papers\nThe effects of adalimumab on the rat autotransplantation \nendometriosis model: A p lacebo-controlled randomized study\nSelçuk KaplanA–F , Pınar KırıcıC– E, Ahmet TürkB,C\nFaculty of Medicine, Adıyaman University, Turkey\nA – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;  \nD – writing the article; E – critical revision of the article; F – final approval of the article\nAdvances in Clinical and Experimental Medicine, ISSN 1899–5276 (print), ISSN 2451–2680 (online)  Adv Clin Exp Med. 2022;31(4):417–426\n\nS. Kaplan, P . Kırıcı, A. Türk. Adalimumab on experimental endometriosis\n418\nBackground\nEndometriosis is a common gynecological pathology af -\nfecting 5–10% of women in the reproductive age. 1 It causes \nsymptoms such as chronic pelvic pain, dysmenorrhea, dys-\npareunia, and infertility.2 It has negative effects on ovarian \nreserve, tubal anatomy, embryo quality, and implantation.3 \nThe pathophysiology of endometriosis is still not entirely clear \nand several theories have been proposed. It has been stated \nthat proinflammatory cytokines play a role in the pathophys-\niology of endometriosis.\n4–6  There are cytokines responsible \nfor inflammatory reactions and tissue neovascularization. \nInterleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) \nhave been previously studied in the pathogenesis of endome-\ntriosis.\n7 The estrogenic microenvironment activates perito-\nneal macrophages with secretion of TNF-α and IL-1, which \nare pro-inflammatory cytokines. The increase in the level \nof IL-6 and TNF-α in the peritoneal fluid of patients with \nabnormal immune cell activity shows the role of cytokines \nin the pathogenesis.\n8–10\nEndometriosis is a pathology caused by the abnormal \nproliferation of endometrial tissue outside the uterine \ncavity.\n1 Transforming growth factor beta (TGF-β) plays \na key role in the pathological growth of many fibrotic \ntissues.\n11 The TGF-β stimulates the expression of extra -\ncellular matrix proteins. 12,13 Its receptors have been de -\ntected in leiomyomas and myometrium, and it has been \nshown that estrogen and TGF-β expression are increased \nin endometriosis, and TGF-β activity mediates the effects \nof estrogen.\n14 This interaction may play a role in the de-\nvelopment of endometriosis. Fibrillin-1 is a protein that \nindicates the activation of TGF-β.\n15\nAdalimumab is a fully human immunoglobulin G (IgG)1 \nneutralizing monoclonal antibody specific for TNF-α. 16,17 \nIt is used in the treatment of many autoimmune diseases, \nsuch as rheumatoid arthritis, Crohn’s disease and psori -\natic arthritis.\n16 In previous experimental studies in rats, \nagents such as etanercept and infliximab have been re -\nported to decrease TNF-α levels in peritoneal fluid in rats \nwith endometriosis.\n18,19 As far as we know, there is no study \nreporting the relationship between histopathological and \nbiochemical changes and adalimumab in endometriosis.\nObjectives\nThe aim of this study is to determine whether adalim -\numab can be an effective medical treatment agent, by ex -\namining its histopathological and biochemical effects \non endometriosis.\nMaterials and methods\nThe experiments in this study were carried out in accor -\ndance with the National Institutes of Health (NIH) animal \nresearch guidelines and were approved by  Adıyaman \nTraining and Research Hospital Ethics Commitee (ap -\nproval No. 2019/062, December 26, 2019).\nAnimals and experimental protocol\nTwenty-eight Wistar albino female rats, 10–12-week \nold, weighing 250–280 g, were divided into 4 groups with \n7 animals in each group. No procedures were performed \nfor 7 days to ensure the adaptation of animals. Rats were \nhoused at 20 ±22°C room temperature during the adap -\ntation and experimental period, in rooms with 12-hour \nlight and 12-hour dark light cycle, with food and water ad \nlibitum. The animals were classified into 4 groups (control \ngroup, endometriosis group, endometriosis + adalimumab \ngroup, and adalimumab group).\nAll rats were anesthetized by intramuscular adminis -\ntration of 60 mg/kg ketamine hydrochloric acid (Ketalar; \nWarner-Lambert, Istanbul, Turkey) and 7 mg/kg xylazine \nhydrochloric acid (Rompun; Bayer, Istanbul, Turkey).\nIn group 1 (control group, n = 7), the pelvic region was \nopened with laparotomy and the adnexa were localized \nas the right and left adnexa. After the adnexa was localized \nwith the right and left uterine horn, the abdominal wall \nwas closed with 4-0 nylon sutures. No action was taken \nuntil the end of the experiment. \nIn group 2 (endometriosis group, n = 7), the autotrans -\nplantation method was used for the induction of endo -\nmetriosis. The reproductive cycles of the animals were \ncontrolled by vaginal smear and the rats in the estrus phase \nwere selected. After general anesthesia, a vertical incision \nwas made to expose the uterus. Both uterine horns were re-\nmoved from the cervix to the point located about 1 cm from \nthe ovaries. Electrocoagulation was used for hemostasis. \nThe uterine horns were divided longitudinally, exposing \nthe endometrium. Without removing the myometrium, en-\ndometrial segment (5 × 5 mm) was implanted into the peri-\ntoneal surface of the right abdominal wall, so that the en -\ndometrium came into contact with the peritoneal surface. \nBoth ends of the implants were fixed to the interior with \n6-0 nonabsorbable polypropylene suture.\n20 All rats were \nallowed to recover for 4 weeks following surgical induction \nof endometriosis. At the end of the 4 weeks, the rats were \noperated to observe the growth of endometriotic implants. \nThe equation: 6 × length × width × height of the implant \nwas used to measure the surface areas of the implants and \ncalculate the endometriotic volume. After the endometri -\notic lesions were photographed, the size of the lesion was \nrecorded and the peritoneal cavity was closed. No proce -\ndure was performed on rats for 4 weeks after the develop -\nment of endometriosis.\nIn group 3 (endometriosis + adalimumab group, n = 7), \nafter the reproductive cycles of the animals were controlled \nby vaginal smear and the rats in the estrous phase were \nselected, the endometriosis induction was performed using \nautotransplantation method. At the end of the four-week \n\nAdv Clin Exp Med . 2022;31(4):417–426\n419\nperiod, after calculating the endometriotic volume and \nphotographing the lesions, 5 mg/kg adalimumab was ad -\nministered intraperitoneally (ip.) for 4 weeks. 21 \nIn group 4 (adalimumab group, n = 7), after the ad -\nnexa was localized with the right and left uterine horn, \nthe abdominal wall was closed with 4-0 nylon sutures and \n5 mg/kg adalimumab was administered ip. every day for \n4 weeks, to start at the same time as group 2.\nHistopathological evaluation\nThe excised endometrial implants were fixed with 10% \nformalin solution upon histopathological examination. \nSections approx. 5-μm thick were taken from a formalin-\nfixed endometriotic implant. Samples were stained with \nhematoxylin and eosin (H&E) and examined under light \nmicroscopy. Histopathological scoring was done\n22 based \non a following rating scale: +3: a well-preserved epithelial \nlayer; +2: a moderately-preserved epithelial layer (leukocyte \ninfiltrated epithelium); +1: a poorly-preserved epithelial \nlayer (sparse epithelial cell); 0: no epithelial cells.\nImmunohistochemical examination\nFor fibrillin-1 antigen retrieval, sections were rehydrated, \nthen boiled in a microwave oven (750 W) 7 times for 5 min \neach in citrate buffer solution (pH 6). Sections were cooled \nat room temperature for 20 min, washed 3 times for 5 min \neach with phosphate-buffered saline (PBS; P4417; Sigma \nChemical Co., St. Louis, USA), then incubated for 5 min \nwith hydrogen peroxide block solution (TA-125-HP; Lab \nVision Corp., San Francisco, USA) to block endogenous \nperoxidase activity. Then, the sections were washed 3 times \nfor 5 min each with PBS. After applying Ultra V Block \n(TA-125-UB; Lab Vision Corp.) for 5 min, sections were \nincubated with primary antibodies for fibrillin-1 (rabbit \npolyclonal bs-1157R; Bioss Antibodies, Woburn, USA) and \ndiluted 1:200 at room temperature for 60 min in a humid \nenvironment. After being washed with PBS 3 times for \n5 min each, the sections were incubated at room tempera -\nture for 30 min in a humid environment with secondary \nantibody (biotinylated goat anti-mouse/rabbit IgG, TP-\n125-BN; Lab Vision Corp.). Sections were washed with PBS \n3 times for 5 min each and incubated at room temperature \nfor 30 min in a humid environment with streptavidin per -\noxidase (TS-125-HR; LabVision Corp.) and then placed \nin PBS. The 3-amino-9-ethylcarbazole (AEC) substrate + \nAEC chromogen (AEC substrate, TA-015 and HAS, AEC \nchromogen, TA-002-HAC; Lab Vision Corp.) solution was \ndripped on the sections. The sections were washed with \nPBS. Sections were counterstained with Mayer’s hematoxy-\nlin, passed through PBS and distilled water and mounted \nwith Large Volume Vision Mount (TA-125-UG; Lab Vision \nCorp.). Sections were evaluated and photographed using \na digital microscope camera (Leica DFC295; Leica Camera \nAG, Wetzlar, Germany). The histoscore, which reflects \nthe prevalence of immunoreactivity of fibrillin-1 on the tis -\nsue, was based on the rating scale: 0.1: < 25%; 0.4: 26−50%; \n0.6: 51−75%; 0.9: 76−100%, and intensity of immunoreac -\ntivity: 0: unstained; 0.5: little staining; 1: some staining; \n2: moderate staining; 3: strong staining. The histoscore \nwas measured using the following equation: \nhistoscore = prevalence × intensity of immunoreactivity.\nDetermination  \nof malondialdehyde (MDA) level\nDetermination of malondialdehyde level was performed \nby applying the Esterbauer method, which is a lipid per -\noxidation measurement method. 23 Malondialdehyde re -\nacting with thiobarbituric acid at 90–95°C forms pink-\ncolored chromogen. Fifteen minutes later, the absorbances \nof the rapidly cooled samples were read spectrophotomet -\nrically at 532 nm. The results are expressed in nmol/g.\nDetermination of total antioxidant status \n(TAS) and total oxidant status (TOS) levels\nTotal antioxidant status and total oxidant status were \nmeasured in serum samples using enzyme-linked immu -\nnoassay (ELISA) method. The TAS (Rat TAS Catalog No. \nYLA3889Ra; YL Biotechnology Co., Ltd, Shanghai, China) \nand TOS (Rat TOS Catalog No. YLA1892Ra; YL Biotech-\nnology Co., Ltd) levels were measured in accordance with \nthe procedures specified in the catalog of kits. The mea -\nsurement range of the Rat TAS ELISA kit was: 1–300 pg/mL, \nintra-assay coefficient of variation (CV) <10%, inter-assay \nCV < 12%, sensitivity 0.54 pg/mL. The measurement range \nof the Rat TOS ELISA kit was 0.02–60 U/mL, intra-assay \nCV < 10%, inter-assay CV < 12%, sensitivity 0.013 U/mL. \nThe  automatic washer BioTek ELx50 (BioTek Instru -\nments, Winooski, USA) was used for plate washing, while \nChroMate Microplate Reader P4300 devices (Awareness \nTechnology, Palm City, USA) were used for absorbance \nreadings. The unit of test results is specified for serum \nsamples in U/mL.\nStatistical analyses\nThe IBM SPSS v. 22 software (IBM Corp., Armonk, USA) \nprogram was used to analyze the data. The Shapiro–Wilk \ntest was used as a normal distribution test. The Shapiro–\nWilk test results for MDA measurement were reported \nas p = 0.830 for group 1, p = 0.898 for group 2, p = 0.881 \nfor group 3, and p = 0.716 for group 4. The Shapiro–Wilk \ntest results for TAS measurement were 0.274 for group 1, \n0.540 for group 2, 0.648 for group 3, and 0.355 for group 4. \nThe Shapiro–Wilk test results for TOS measurement were \nreported as p = 0.707 for group 1, p = 0.598 for group 2, \np = 0.700 for group 3, and p = 0.944 for group 4. The Sha -\npiro–Wilk test results for fibrillin-1 measurement were \n\nS. Kaplan, P . Kırıcı, A. Türk. Adalimumab on experimental endometriosis\n420\nreported as p = 0.812 for group 1, p = 0.652 for group 2, \np = 0.717 for group 3, and p = 0.941 for group 4. Levene’s \nhomogeneity test was performed, in which the data met \nthe assumption of normal distribution for each group, and \nthe results were p = 0.170 for MDA, p = 0.050 for TAS, \np = 0.654 for TOS, and p = 0.191 for fibrillin-1 measure -\nment. It was observed that the variances were homogeneous. \nOne-way analysis of variance (ANOVA) test (post hoc Bon-\nferroni test) was used in the analysis of the data conforming \nto the normal distribution. The score variable does not show \na normal distribution. The p-value of the score variable \nis <0.001. Spearman’s correlation test was used for the val-\nues where the score did not show normal distribution, and \nPearson’s correlation test was used for the others. The value \nof p < 0.05 was considered statistically significant.\nResults\nHistopathological scoring\nThe H&E staining and immunohistochemistry stain -\ning histopathological images were shown in  Fig. 1  and \nFig. 2 . There was a statistically significant difference \nbetween the measurements in different groups. A sta -\ntistical difference was observed between group 2 and \ngroup 3 (p < 0.001) ( Table 1). When group 1 and group 2 \nwere compared, the increase in histopathological damage \nin group 2 was statistically significant (p < 0.001). When \ngroup 3 and group 2 were compared, the histopathologi -\ncal damage score was significantly decreased in group 3 \n(p < 0.001).\nFig. 1. Excised endometrial implants were stained with hematoxylin and eosin (H&E). Accordingly, group 1 (control) (1A) and group 4 (adalimumab) (1C) \nwere of normal appearance. In group 2 (endometriosis) (1B), severe epithelial damage (black arrow) and leukocyte infiltration (black star) were observed. \nIn group 3 (endometriosis + adalimumab) (1D), epithelial damage and leukocyte infiltration were observed to decrease\n\n\nAdv Clin Exp Med . 2022;31(4):417–426\n421\nFig. 2. Examination of immunohistochemical staining for fibrillin-1 immunoreactivity under light microscopy; fibrillin-1 immunoreactivity (black arrow) was \nobserved; 2A. Group 1 (control); 2D. Group 3 (endometriosis + adalimumab); 2B. Group 2 (endometriosis); 2C. Group 4 (adalimumab)\nTable 1. Comparison of biochemical measurements and fibrillin-1 scores by groups*\nGroup Fibrillin-1 MDA TAS TOS\nGroup 1 (n = 7)\nmean 2.31 17.92 1.18 12.98\nSD 0.77 0.99 0.04 1.06\nmedian 0.71 18.01 1.17 13.26\nGroup 2 (n = 7)\nmean 5.51 25.02 0.43 19.75\nSD 2.61 1.61 0.20 1.48\nmedian 3.30 25.30 0.41 20.14\nGroup 3 (n = 7)\nmean 3.64 19.86 0.81 17.68\nSD 1.47 1.77 0.14 1.81\nmedian 1.66 20.10 0.81 17.60\nGroup 4 (n = 7)\nmean 0.93 16.88 1.29 15.55\nSD 0.14 0.94 0.12 1.26\nmedian 0.11 16.78 1.24 15.52\np-value for each group <0.001 <0.001 <0.001 <0.001\ndf (total) for each group 27 27 27 27\nf for each group 12.741 48.104 53.812 28.671\nSD – standard deviation; df – degrees of freedom; MDA – malondialdehyde; TAS – total antioxidant status; TOS – total oxidant status; ADA – adalimumab; \ngroup 1 – control; group 2 – endometriosis; group 3 – endometriosis + ADA; group 4 – ADA; *one-way analysis of variance (ANOVA) test.\n\n\nS. Kaplan, P . Kırıcı, A. Türk. Adalimumab on experimental endometriosis\n422\nMacroscopic examination\nThe mean size of rats endometriotic implants in group 2 \nwas 0.77 ±0.04 mm 2. After adalimumab treatment, it mea-\nsured 0.32 ±0.002 mm 2. This decrease was statistically \nsignificant (p = 0.032) ( Fig. 3).\nBiochemical analysis\nPost hoc one-way ANOVA results for biochemical mea-\nsurements are given in Table 2 .\nMDA level\nThe MDA levels significantly increased in group 2 com -\npared with group 1 (p < 0.001). The MDA values   w ere \nsignificantly decreased in group 3 compared with group 2 \n(p < 0.001). There was no statistically significant difference \nbetween group 1 and group 4 (p > 0.05) ( Table 1, Table 2).\nTAS level\nThe TAS levels were significantly decreased in group 2 \ncompared with group 1 (p < 0.001). The TAS values   i n-\ncreased in group 3 compared to group 2 (p < 0.001). There \nwas no statistically significant difference in TAS values   \nbetween group 1 and group 4 (p > 0.05) ( Table 1, Table 2).\nTOS level\nThe distribution of scores in respective groups is given \nin  Table 3. The TOS levels were significantly increased \nin group 2 compared with group 1 (p < 0.001). The TOS \nvalues   i n group 2 were significantly decreased compared \nwith group 4 (p < 0.001). However, there was no sta -\ntistical difference in TOS values   b etween group 2 and \ngroup 3 (p > 0.05). The TOS values   w ere significantly \nincreased in group 4 compared with group 1 (p = 0.016) \n(Table 1, Table 2).\nWhen histopathological scores and biochemical values \nwere compared, there was a strong negative correlation \nbetween MDA value and TOS value and a strong positive \ncorrelation between MDA value and TAS value (p < 0.001) \n(Table 3).\nImmunohistochemistry examination score\nIn the immunohistochemistry examination, fibrillin-1 \nscores differed significantly for 4 groups. According to \nthe post hoc one-way ANOVA analysis between groups, \nfibrillin-1 activity increased in groups 2 and 3 compared \nto group 1 (p < 0.001). Moreover, fibrillin-1 immune reac -\ntivity decreased in group 3 compared to group 2 (p < 0.001) \n(Table 3).\nFig. 3. A,B. Endometriotic foci developed \nas a result of autotransplantation; \nC,D. After adalimumab treatment, the size \nof the endometriotic foci decreased\n\n\nAdv Clin Exp Med . 2022;31(4):417–426\n423\nDiscussion\nIn this study, the effect of adalimumab on histopatho -\nlogical changes in endometriotic implants and its antioxi -\ndant effects were investigated. The size of endometriotic \nimplants decreased after adalimumab treatment, as shown \nwith macroscopic examination. Histopathological damage \nscore increased in rats with experimental endometriosis \nand decreased with adalimumab administration. Fibrillin-1 \nscore was increased in endometriotic implants, but fibril -\nlin-1 score decreased in endometriotic implants treated \nwith adalimumab. In addition, adalimumab decreased \nMDA levels and increased TAS levels in endometriotic \nimplants. These findings show the  histopathological \nTable 2. Post hoc one-way analysis of variance (ANOVA) of biochemical values and fibrillin-1 scores by groups\nDependent variable Group (I) Group (J) Average difference (I–J) p-value\nMDA\ngroup 1 \ngroup 2\ngroup 3\ngroup 4 \n–7.10*\n–1.94\n1.03\n<0.001\n0.087\n1.000\ngroup 2\ngroup 1 \ngroup 3\ngroup 4 \n7.10*\n5.15*\n8.13*\n<0.001\n<0.001\n<0.001\ngroup 3\ngroup 1 \ngroup 2\ngroup 4 \n1.94\n–5.15*\n2.97*\n0.087\n<0.001\n0.003\ngroup 4\ngroup 1 \ngroup 2\ngroup 3\n–1.03\n–8.13*\n–2.97*\n1.000\n<0.001\n0.003\nTAS\ngroup 1 \ngroup 2\ngroup 3\ngroup 4 \n0.74*\n0.36*\n–0.11\n<0.001\n<0.001\n0.907\ngroup 2\ngroup 1 \ngroup 3\ngroup 4\n–0.74*\n–0.38*\n–0.86*\n<0.001\n<0.001\n<0.001\ngroup 3\ngroup 1\ngroup 2\ngroup 4\n–0.36*\n0.38*\n–0.48*\n<0.001\n<0.001\n<0.001\ngroup 4\ngroup 1\ngroup 2\ngroup 3\n0.11\n0.86*\n0.48*\n0.907\n<0.001\n<0.001\nTOS\ngroup 1 \ngroup 2\ngroup 3\ngroup 4\n–6.77*\n–4.70*\n–2.56*\n<0.001\n<0.001\n0.016\ngroup 2\ngroup 1\ngroup 3\ngroup 4 \n6.77*\n2.07\n4.20*\n<0.001\n0.075\n<0.001\ngroup 3\ngroup 1\ngroup 2\ngroup 4\n4.70*\n–2.07\n2.13\n<0.001\n0.075\n0.062\ngroup 4 \ngroup 1 \ngroup 2\ngroup 3\n2.56*\n–4.20*\n–2.13\n0.016\n<0.001\n0.062\nFibrillin-1 \ngroup 1\n group 2\ngroup 3\ngroup 4\n–1.28*\n–1.15*\n0.37\n<0.001\n<0.001\n0.083\ngroup 2\ngroup 1\ngroup 3\ngroup 4\n1.28*\n0.12\n1.65*\n<0.001\n1.000\n<0.001\ngroup 3\ngroup 1\ngroup 2\ngroup 4\n1.15*\n–0.12\n1.53*\n<0.001\n1.000\n<0.001\ngroup 4\ngroup 1\ngroup 2\ngroup 3\n–0.37\n–1.65*\n–1.53*\n0.083\n<0.001\n<0.001\nMDA – malondialdehyde; TAS – total antioxidant status; TOS – total oxidant status; ADA – adalimumab; group 1 – control; group 2 – endometriosis; \ngroup 3 – endometriosis + ADA; group 4 – ADA; I – group designated for comparison; J – other groups compared; * groups with statistically significant \ndifferences between them. Values in bold show statistically significant differences between groups.\n\nS. Kaplan, P . Kırıcı, A. Türk. Adalimumab on experimental endometriosis\n424\nimprovement and antioxidant activity caused by adalim -\numab treatment in endometriotic implants.\nOxidative stress and inflammation play an important \nrole in the pathogenesis of endometriosis. 24–26  Moreover, \nstudies have shown that erythrocytes, apoptotic endo -\nmetrial tissue, cell debris in the peritoneal cavity, and \nmacrophages induce oxidative stress and inflammation, \nand cause endometriosis.\n26 It is known that the balance \nbetween reactive oxygen species (ROS) and antioxidants \nis lost in the development of endometriosis.\n27 The pres -\nence of ROS in the environment affects gene expression, \ncausing protein dysfunction and cellular damage.\n26–28  \nThis oxidative stress can be both a cause and a conse -\nquence of endometriosis. 26 In addition, the role of mi -\ntophagy and autophagy in the pathophysiology of endo -\nmetriosis supports the role of oxidative damage. 28 It has \nbeen hypothesized that eliminating oxidative stress may \nreduce the histopathological grade in endometrial im -\nplants and many studies have been conducted on this \nsubject.\n27,29,30\nProinflammatory cytokines such as IL-1, IL-6, IL-8, and \nTNF-α have also been shown to play a role in the pathol -\nogy of  endometriosis. 31 It  has been demonstrated that \nthere is an increase in TNF-α levels in the peritoneal fluids \nof women with endometriosis.\n32 It is thought that endo -\nmetrial cell proliferation increases and endometriotic le -\nsions develop by inducing IL-8 secretion with the increase \nof TNF-α.\n33,34 In previous studies using etanercept, which \nhas an anti-TNF-α activity, it was reported that endome -\ntriotic implants were reduced and histopathological scores \ndecreased.\n35 In another study, etanercept was reported to de-\ncrease MDA levels in endometriotic focus. 36 In our study, \nadalimumab treatment has decreased endometriotic implant \ndimensions, histopathological damage score and fibrillin-1 \nscore, which is a fibrosis marker.\nFibrillin-1 has been associated with heart and liver fi -\nbrosis, and has been shown to cause the expression of ex -\ntracellular matrix proteins in various studies in rats.\n37,38 \nIn addition, it has been previously observed that topical \napplication of estrogen increases the activity of fibrillin-1 \nand other extracellular matrix proteins.\n39 Moreover, it was \ndemonstrated that fibrillin-1 activity increased in propor -\ntion to the size in leiomyomas. 40 In our study, the increase \nin fibrillin-1 score in endometriotic implants may be re -\nlated to the role of the estrogenic microenvironment in en -\ndometrial implant development. Since estrogen levels were \nnot measured in our study, large studies can be conducted \nto investigate this hypothesis.\nAdalimumab is a drug that is effective both as mono -\ntherapy and in combination with disease-modifying anti -\nrheumatic drugs in the treatment of many chronic, inflam -\nmatory, immune-mediated diseases.\n16 It has been reported \nthat adalimumab decreases cell proliferation and increases \nthe function of natural immune pathways by decreased \nIL-8 levels.\n41 Considering the role of proinflammatory \ncytokines in the pathogenesis of endometriosis, it is not \nsurprising that endometriotic implants and histopatho -\nlogical score decreased along with adalimumab treatment. \nMoreover, the immunomodulatory activity of adalimumab \nmay demonstrate decreased fibrillin-1 activation in endo -\nmetriotic implants.\nLimitations\nThis study has some limitations. The fibrillin-1 score \nis  established primarily with immunohistochemistry \nstaining, but there were no polymerase chain reaction \n(PCR) tests measuring the fibrillin-1 protein expressions. \nSecond, the study was experimentally performed in rats. \nIn order to generalize the results to society, studies should \nbe carried out primarily with large human populations.\nConclusions\nAdalimumab, through its antioxidant and anti-inflam -\nmatory effects, plays an important role in the treatment \nof histopathological damage and fibrosis in endometri -\notic implants developed experimentally. It can be used \nas a nonhormonal agent in the treatment of endometriotic \nimplants. However, experiments with different parameters \nare needed in large groups of animals and humans.\nTable 3. Correlations between biochemical measurements*\nVariables Score MDA TAS TOS\nScore\nr 1 −0.834 0.840 −0.680\np-value – <0.001 <0.001 <0.001\nMDA\nr – 1 −0.863 0.740\np-value – – <0.001 <0.001\nTAS\nr – – 1 −0.766\np-value – – – <0.001\nTOS\nr – – – 1\np-value – – – –\nMDA – malondialdehyde; TAS – total antioxidant status; TOS – total oxidant status; * Spearman’s and Pearson’s correlation test. Since the score did not show \nnormal distribution, the values in bold were obtained with the Spearman’s test, and the other results were obtained with the Pearson’s correlation test.\n\nAdv Clin Exp Med . 2022;31(4):417–426\n425\nORCID iDs\nSelçuk Kaplan  https://orcid.org/0000-0002-2887-6165\nPınar Kırıcı  https://orcid.org/0000-0001-7616-4181\nAhmet Türk  https://orcid.org/0000-0003-0903-3522\nReferences\n1. L iu Y, Zhang Z, Lu X, Meng J, Qin X, Jiang J. Anti-nociceptive and \nanti-inflammatory effects of sulforaphane on sciatic endometriosis \nin a rat model. Neurosci Lett . 2020;723:134858. doi:10.1016/j.neulet. \n2020.134858\n2. M atarese G, De Placido G, Nikas Y, Alviggi C. Pathogenesis of endo -\nmetriosis: Natural immunity dysfunction or autoimmune disease? \nTrends Mol Med . 2003;9(5):223–228. doi:10.1016/s1471-4914(03)  \n00051-0\n3. G auché-Cazalis C, Koskas M, Cohen Scali S, Luton D, Yazbeck C. Endo -\nmetriosis and implantation: Myths and facts. Middle East Fertil Soc J. \n2012;17:79e81. doi:10.1016/j.mefs.2012.04.002\n4. C alhaz-Jorge C, Costa AP , Barata M, Santos MC, Melo A, Palma-Carlos ML.  \nTumour necrosis factor alpha concentrations in the peritoneal fluid \nof infertile women with minimal or mild endometriosis are lower \nin patients with red lesions only than in patients without red lesions. \nHum Reprod. 2000;15(6):1256 –1260. doi:10.1093/humrep/15.6.1256\n5. G azvani R, Templeton A. Peritoneal environment, cytokines and angio -\ngenesis in the pathophysiology of endometriosis. Reproduction. 2002; \n123(2):217–226. doi:10.1530/rep.0.1230217\n6. T askin MI, Gungor AC, Adali E, Yay A, Onder GO, Inceboz U. A human -\nized anti-interleukin 6 receptor monoclonal antibody, tocilizumab,  \nfor the treatment of endometriosis in a rat model. Reprod Sci. 2016; \n23(5):662–669. doi:10.1 177/19337191 15612134\n7. L aganà AS, Vitale SG, Salmeri FM, et al. Unus pro omnibus, omnes \npro uno: A novel, evidence-based, unifying theory for the pathogen-\nesis of endometriosis. Med Hypotheses. 2017;103:10–20. doi:10.1016/j.\nmehy.2017.03.032\n8. K alu E, Sumar N, Giannopoulos T, et al. Cytokine profiles in serum and \nperitoneal fluid from infertile women with and without endometri -\nosis. J Obstet Gynaecol Res . 2007;33(4):490–495. doi:10.1111/j.1447-\n0756.2007.00569.x\n9. M ahnke JL, Dawood MY, Huang JC. Vascular endothelial growth fac -\ntor and interleukin-6 in peritoneal fluid of women with endometrio -\nsis. Fertil Steril. 2000;73(1):166–170. doi:10.1016/s0015-0282(99)00466-5\n10. O thman Eel-D, Hornung D, Salem HT, Khalifa EA, El-Metwally TH, \nAl-Hendy A. Serum cytokines as biomarkers for nonsurgical predic -\ntion of endometriosis. Eur J Obstet Gynecol Reprod Biol. 2008;137(2): \n240–246. doi:10.1016/j.ejogrb.2007.05.001\n11. B ranton MH, Kopp JB. TGF-beta and fibrosis. Microbes Infect . 1999; \n1(15):1349–1365. doi:10.1016/s1286-4579(99)00250-6\n12. S ozen I, Arici A. Interactions of cytokines, growth factors, and the \nextracellular matrix in the cellular biology of uterine leiomyomata. \nFertil Steril. 2002;78(1):1–12. doi:10.1016/s0015-0282(02)03154-0\n13. Lu o X, Ding L, Xu J, Chegini N. Gene expression profiling of leio -\nmyoma and myometrial smooth muscle cells in response to trans -\nforming growth factor-beta. Endocrinology . 2005;146(3):1097–1118. \ndoi:10.1210/en.2004-1377\n14. C hegini N, Ma C, Tang XM, Williams RS. Effects of GnRH analogues, \n‘add-back’ steroid therapy, antiestrogen and antiprogestins on leio-\nmyoma and myometrial smooth muscle cell growth and transform-\ning growth factor-beta expression. Mol Hum Reprod . 2002;8(12):  \n1071–1078. doi:10.1093/molehr/8.12.1071\n15. K aartinen V, Warburton D. Fibrillin controls TGF-beta activation.  \nNat Genet. 2003;33(3):331–332. doi:10.1038/ng0303-331\n16. M atsumoto T, Motoya S, Watanabe K, et al. Adalimumab monother -\napy and a combination with azathioprine for Crohn’s disease: A pro-\nspective, randomized trial. J Crohns Colitis . 2016;10(11):1259–1266. \ndoi:10.1093/ecco-jcc/jjw152\n17. B urmester GR, Landewé R, Genovese MC, et al. Adalimumab long-\nterm safety: Infections, vaccination response and pregnancy out -\ncomes in patients with rheumatoid arthritis. Ann Rheum Dis . 2017; \n76(2):414–417. doi:10.1136/annrheumdis-2016-209322\n18. B arrier BF, Bates GW, Leland MM, Leach DA, Robinson RD, Propst \nAM. Efficacy of anti-tumor necrosis factor therapy in the treatment \nof spontaneous endometriosis in baboons. Fertil Steril. 2004;81(Suppl 1): \n775–779. doi:10.1016/j.fertnstert.2003.09.034\n19. F alconer H, Mwenda JM, Chai DC, et al. Treatment with anti-TNF \nmonoclonal antibody (c5N) reduces the extent of induced endome -\ntriosis in the baboon. Hum Reprod. 2006;21(7):1856–1862. doi:10.1093/\nhumrep/del044\n20. P elch KE, Sharpe-Timms KL, Nagel SC. Mouse model of surgically-\ninduced endometriosis by auto-transplantation of uterine tissue. \nJ Vis Exp. 2012;59:e3396. doi:10.3791/3396\n21. Y ilmaz M, Tekekoglu S, Herek O, Ozmen O, Sahinduran Ş, Buyukoglu T.  \nAmeliorative effect of adalimumab on experimentally induced acute \npancreatitis in rats. Pancreas. 2010;39(8):1238–1242. doi:10.1097/MPA. \n0b013e3181dec1a6\n22. Yavuz E, Oktem M, Esinler I, Toru SA, Zeyneloglu HB. Genistein causes \nregression of endometriotic implants in the rat model. Fertil Steril. \n2007;88(4 Suppl):1129–1134. doi:10.1016/j.fertnstert.2007.01.010\n23. E sterbauer H, Cheeseman KH. Determination of aldehydic lipid per -\noxidation products: Malonaldehyde and 4-hydroxynonenal. Methods  \nEnzymol. 1990;186:407–421. doi:10.1016/0076-6879(90)86134-h\n24. Y un BH, Chon SJ, Choi YS, Cho S, Lee BS, Seo SK. Pathophysiology \nof endometriosis: Role of high mobility group box-1 and toll-like \nreceptor 4 developing inflammation in endometrium. PLoS One. 2016; \n11(2):e0148165. doi:10.1371/journal.pone.0148165\n25. S antanam N, Kavtaradze N, Murphy A, Dominguez C, Parthasara -\nthy S. Antioxidant supplementation reduces endometriosis-related \npelvic pain in humans. Transl Res . 2013;161(3):189–195. doi:10.1016/j.\ntrsl.2012.05.001\n26. Donnez J, Binda MM, Donnez O, Dolmans MM. Oxidative stress in \nthe pelvic cavity and its role in the pathogenesis of endometriosis. \nFertil Steril. 2016;106(5):1011–1017. doi:10.1016/j.fertnstert.2016.07.1075\n27. H arlev A, Gupta S, Agarwal A. Targeting oxidative stress to treat endo -\nmetriosis. Expert Opin Ther Targets. 2015;19(11):1447–1464. doi:10.1517/ \n14728222.2015.1077226\n28. S iracusa R, D’Amico R, Impellizzeri D, et al. Autophagy and mitoph -\nagy promotion in a rat model of endometriosis. Int J Mol Sci. 2021; \n22(10):5074. doi:10.3390/ijms22105074\n29. C ordaro M, Trovato Salinaro A, Siracusa R, et al. Hidrox® and endo -\nmetriosis: Biochemical evaluation of oxidative stress and pain.  \nAntioxidants (Basel). 2021;10(5):720. doi:10.3390/antiox10050720\n30. S iracusa R, D’Amico R, Cordaro M, et al. The methyl ester of 2-cyano-\n3,12-dioxooleana-1,9-dien-28-oic acid reduces endometrial lesions \ndevelopment by modulating the NFkB and Nrf2 pathways. Int J Mol Sci.  \n2021;22(8):3991. doi:10.3390/ijms22083991\n31. G enovese T, Siracusa R, D’Amico R, et al. Regulation of inflamma -\ntory and proliferative pathways by fotemustine and dexametha -\nsone in endometriosis. Int J Mol Sci. 2021;22(11):5998. doi:10.3390/\nijms22115998\n32. Dmowski PW, Braun DP. Immunology of endometriosis. Best Pract \nRes Clin Obstet Gynaecol. 2004;18(2):245–263. doi:10.1016/j.bpobgyn. \n2004.02.001\n33. K yama CM, Overbergh L, Debrock S, et al. Increased peritoneal and \nendometrial gene expression of biologically relevant cytokines and \ngrowth factors during the menstrual phase in women with endo -\nmetriosis. Fertil Steril. 2006;85(6):1667–1675. doi:10.1016/j.fertnstert. \n20 05.11.0 6 0\n34. A rici A, Oral E, Attar E, Tazuke SI, Olive DL. Monocyte chemotactic \nprotein-1 concentration in peritoneal fluid of women with endo -\nmetriosis and its modulation of expression in mesothelial cells. Fer-\ntil Steril. 1997;67(6):1065–1072. doi:10.1016/s0015-0282(97)81440-9\n35. A rici A. Local cytokines in endometrial tissue: The role of interleu -\nkin-8 in the pathogenesis of endometriosis. Ann N Y Acad Sci. 2002; \n955:101– 4 0 6 . doi:10.1111/j.1749 - 6 632 . 20 02 .tb 02770. x\n36. E raldemir FC, Keleş CD, Kum T, Vural B, Baydemir C. The effect of etan -\nercept treatment on serum malonaldialdehyde levels in experi -\nmental endometriosis rat model. KOU Sag Bil Derg . 2015;1(1):26–29. \ndoi:10.30934/kusbed.349539\n37. L orena D, Darby IA, Reinhardt DP , Sapin V, Rosenbaum J, Desmoulière A.  \nFibrillin-1 expression in normal and fibrotic rat liver and in cultured \nhepatic fibroblastic cells: Modulation by mechanical stress and role \nin cell adhesion. Lab Invest. 2004;84(2):203–212. doi:10.1038/labinvest. \n3700023\n38. B ouzeghrane F, Reinhardt DP , Reudelhuber TL, Thibault G. Enhanced \nexpression of fibrillin-1, a constituent of the myocardial extracel -\nlular matrix in fibrosis. Am J Physiol Heart Circ Physiol . 2005;289(3): \nH982–H991. doi:10.1152/ajpheart.00151.2005\n\nS. Kaplan, P . Kırıcı, A. Türk. Adalimumab on experimental endometriosis\n426\n39. S on ED, Lee JY, Lee S, et al. Topical application of 17beta-estradiol \nincreases extracellular matrix protein synthesis by stimulating TGF-\nbeta signaling in aged human skin in vivo. J Invest Dermatol . 2005; \n124(6):1149 –1161. doi:10.1111/j.0 022-202 X . 20 05. 23736 . x\n40. Z hao Y, Wen Y, Polan ML, Qiao J, Chen BH. Increased expression of \nlatent TGF-beta binding protein-1 and fibrillin-1 in human uterine leio-\nmyomata. Mol Hum Reprod. 2007;13(5):343–349. doi:10.1093/molehr/ \ngam007\n41. H endriks AG, van der Velden HM, Wolberink EA, et al. The effect \nof adalimumab on key drivers in the pathogenesis of psoriasis.  \nBr J Dermatol. 2014;170(3):571–580. doi:10.1111/bjd.12705","source_license":"CC0","license_restricted":false}