{"paper_id":"0bc9c1a3-f378-4cad-bca8-b92ec020531e","body_text":"Abstract\nObjectives: This study aimed to observe the difference in the area of endometriosis lesions and the histopathology of inflammatory \ncells and granuloma masses in an endometriosis mouse model treated with endometrial cell implants, endometrioma capsules, and \nadenomyosis tissue. \nMaterials and Methods: This is an experimental study with posttest-only research design which was conducted with the control \ngroup. Thirty-two mice (Mus musculus) were injected with 0.2 mL/mice cyclosporin A and then were divided into three groups which \nwere injected with endometrial tissue from the uterine cavity (group A), endometriosis from endometrioma capsule (group B), and \nendometriosis from adenomyosis (group C). The injection was done slowly into the peritoneal cavity, 0.1 mL each, and followed \nby intramuscularly Ethinyl estradiol, 0.2 μG/mice. On the 15th days, mice were dissected to observe the peritoneal endometriosis \nimplant and microscopic examination with hematoxylin-eosin (HE) staining to determine the inflammatory cell infiltration and \nmass granuloma presence. Data were analyzed using SPSS, version 19. \nResults: The study obtained that the area of implanted endometriosis lesions in group C covered a larger area of endometriosis \nimplants than other groups (P < 0.05). The peritoneal damage in group C was the most severe based on the Klopfleisch method ( P \n< 0.05), with mass granuloma and massive infiltration of inflammatory cells and fibrous connective tissue formation occurring in \nmuscle tissue. \nConclusions: The implantation of adenomyosis cell tissue is the best method to develop mice model of endometriosis based on its \ninflammatory infiltration, the extent of lesion implant, and granuloma mass.\nKeywords: Endometriosis, Granuloma mass, Implant area, Peritoneal damage\nThe Effect of Implant Origin Differences on Peritoneal \nEndometriosis in an Endometriosis Mouse Model  \nSutrisno Sutrisno1* ID , Sri Andarini1, I Wayan Arsana Wiyasa1, Umi Kulsum1, Noerhamdani \nNoerhamdani1, Hidayat Suyuti1, Hendy Hendarto1\nOpen Access                                                                                              Original Article\nInternational Journal of Women’s Health and Reproduction Sciences \nVol. 7, No. 1, January 2019, 34–40\nhttp://www.ijwhr.net doi 10.15296/ijwhr .2019.06\nISSN 2330- 4456\nReceived 6 March 2018, Accepted 14 August 2018, Available online 29 August 2018\n1Department of Obstetrics and Gynecology, Faculty of Medicine, Brawijaya University, Malang 65145, East Java, Indonesia.\n*Corresponding Author: Sutrisno Sutrisno, Tel: +62-341-569117, Fax: +62-341-564755, Email: snospogk@gmail.com\n \nIntroduction \nThroughout the history of the world, the ones who had \nconfronted the bitterest face of poverty and war had al -\nways been the women. As known poverty and war affects \nhuman health either directly or indirectly, the effects of \nthis condition on health and status of women in the so-\nciety should not be ignored. This study intends to cast \nlight on the effects of war and poverty on the reproductive \nhealth of women. For this purpose, the face of war affect -\ning the women, the problem of immigration, inequalities \nin distribution of income based on gender and the effects \nof all these on the reproductive health of women will be \naddressed.\nWar and Women’s Health\nFamine, synonymous with war and poverty, is clearer for \nwomen; war means deep disadvantages such as full de -\nstruction, loss of future and uncertainty for women. Wars \nare conflicts that destroy families, societies and cultures \nthat negatively affect the health of community and cause \nviolation of human rights. According to the data of World \nHealth Organization (WHO) and World Bank, in 2002 \nwars had been among the first ten reasons which killed \nthe most and caused disabilities. Civil losses are at the rate \nof 90% within all losses (1).\nWar has many negative effects on human health. One of \nthese is its effect of shortening the average human life. \nAccording to the data of WHO, the average human life is \n68.1 years for males and 72.7 years for females. It is being \nthought that severe military conflicts in Africa shorten \nthe expected lifetime for more than 2 years. In general, \nWHO had calculated that 269 thousand people had died \nin 1999 due to the effect of wars and that loss of 8.44 mil-\nlion healthy years of life had occurred (2,3).\nWars negatively affect the provision of health services. \nHealth institutions such as hospitals, laboratories and \nhealth centers are direct targets of war. Moreover, the wars \ncause the migration of qualified health employees, and \nthus the health services hitches. Assessments made indi -\ncate that the effect of destruction in the infrastructure of \nhealth continues for 5-10 years even after the finalization \nof conflicts (3). Due to resource requirements in the re-\nstructuring investments after war, the share allocated to \nhealth has decreased (1).\nMortalities and Morbidities\nThe ones who are most affected from wars are women and \nchildren. While deaths depending on direct violence af -\nfect the male population, the indirect deaths kill children, \nwomen and elders more. In Iraq between 1990-1994, in -\nfant deaths had shown this reality in its more bare form \nwith an increase of 600% (4). The war taking five years \nincreases the child deaths under age of 5 by 13%. Also 47% \nof all the refugees in the world and 50% of asylum seekers \nand displaced people are women and girls and 44% ref -\nugees and asylum seekers are children under the age of \n18 (5).\nAs the result of wars and armed conflicts, women are \nAbstract\nWar and poverty are ‘extraordinary conditions created by human intervention’ and ‘preventable public health problems. ’ War and \npoverty have many negative effects on human health, especially women’s health. Health problems arising due to war and poverty are \nbeing observed as sexual abuse and rape, all kinds of violence and subsequent gynecologic and obstetrics problems with physiological \nand psychological courses, and pregnancies as the result of undesired but forced or obliged marriages and even rapes. Certainly, \nunjust treatment such as being unable to gain footing on the land it is lived (asylum seeker, refugee, etc.) and being deprived of \nsocial security, citizenship rights and human rights brings about the deprivation of access to health services and of provision of \nservice intended for gynecology and obstetrics. The purpose of this article is to address effects of war and poverty on the health of \nreproduction of women and to offer scientific contribution and solutions.\nKeywords: Poverty, Reproductive health, War\nWomen on the Other Side of War and Poverty: Its Effect \non the Health of Reproduction\nAyse Cevirme1, Y asemin Hamlaci2*, Kevser Ozdemir2\nOpen Access                                                                                                          Review\nInternational  Journal of Women’s Health and Reproduction Sciences \nVol. 3, No. 3, July 2015, 126–131\nReceived 12 December 2014, Accepted 25 April 2015, Available online 1 July 2015 \n1Department of Nursing, Sakarya University, Sakarya, Turkey. 2Department of Midwifery, Sakarya University, Sakarya, Turkey.\n*Corresponding author: Y asemin Hamlaci, Department of Midwifery, Sakarya University, Sakarya, Turkey. Tel: +905556080628, \nEmail: yaseminhamlaci@gmail.com\nhttp://www.ijwhr.net doi 10.15296/ijwhr .2015.27\nISSN 2330- 4456\nIntroduction\nEndometriosis is a disease that can occur in humans and \nsome other primates. The pathophysiology of this disease \nhas not been clearly understood although many theories \nare evolving and the existing research is continuously \ndemonstrating contradictory results. Complaints, \ndiagnostic processes, therapies, progressiveness, and \nrecurrence are always associated with laparoscopic action \nleading to separate issues like financing, safety, and \nethics. Therefore, research about endometriosis should be \nconducted on endometriosis animal subjects in order to \nminimize the cost, ethical, safety and legal issues (1).\nRegarding the human subjects, research must be \nperformed by laparoscopy and continually observe the \ncondition of disease and the results of therapy and monitor \nits recurrence and to find the technical, financial, and legal \nconstraints. In addition, new therapeutic explorations \nare unethical if directly tested on humans before being \ntested on experimental animals (2). Many studies used \nboth primate and non-primate endometriosis models. \nNon-primate animals such as mice do not experience \nspontaneous endometriosis, but it can be induced by using \neither autologous uterine or human endometrial tissues \n(3). However, primates may spontaneously experience \nendometriosis. Meanwhile, it is more challenging to \ncontinually observe endometriosis in the apes or baboons \n(4,5).\nAlthough non-human primates are the most appropriate \nmodels for studying endometriosis, the procedure takes \na long time and requires human-like diagnostic tools. \nTherefore, non-primate animals such as rats and mice \nare selected as alternatives to solve these constraints since \nthey are more practical models of endometriosis ( 6,7). \nThis study observed the ideal mice implants regarding \nendometriosis research. The model is already developed \nbut no model could produce a high rate of success as \nendometriosis mice model. Using adenomyosis is the \nrational reason to increase the rate of success because \nadenomyosis is specific endometriosis that produces a \ncomplicated problem in a human setting. No design model \nof endometriosis mice used adenomyosis as the origin of \nthe implant. The present study sought to demonstrate the \nsignificant differences in endometriosis lesion implants \nand histopathological features of inflammatory cells and \n\nSutrisno et al\nInternational  Journal of Women’s Health and Reproduction Sciences, Vol. 7, No. 1, January 2019\n35\ngranuloma masses between endometriosis model mice \nreceiving endometrial cells from the uterine cavity (A), \nendometrioma capsules (B), and endometriosis from \nadenomyosis (C). Further, it was attempted to investigate \nthe best model for designing endometriosis model in \nmice.\nMaterials and Methods\nExperimental Design\nThis experimental study used a posttest research design \nonly with the control group. Thirty-two female mice (Mus \nmusculus) weighing 20-30 g and aged 2-3 months were \nobtained from the Laboratory of Reproductive Physiology \nEmbryology, Faculty of Veterinary Medicine, Airlangga \nUniversity, Surabaya out of which 30 were used for the \npurpose of the study. \nThe Sampling of Endometrial Tissue\nThe endometrial cells from the uterine cavity, \nendometrioma capsules, and uterine adenomyosis were \ncollected by the following steps: The endometrium \ncell was obtained by scraping the uterine cavity wall \nwith a curette spoon to obtain the viable endometrial \ntissue. A wall biopsy/capsule (2x2 cm) was performed \non endometrioma. Adenomyosis tissue (2x2 cm) was \nobtained from the female uterus with adenomyosis. Then, \nthe tissue was further inserted into the tube containing the \nlabeled formaldehyde solution and stored at a temperature \nof 20-250°C.\nExperimental Treatments\nAfter the mice underwent an adaptation process in the cage \nby receiving the same feed for 1 week, they were injected \nwith 0.2 cc/mice cyclosporin A (8). The cyclosporine \ninjection was used to suppress the immune status of the \nmice in order to facilitate the growth of endometriosis \nimplant in the mice peritoneal cavity. Furthermore, the \nmice were classified into 3 groups each containing 10 \nmice employing the following experimental treatments: \nMice in group A were injected with endometrial cells from \nthe endometrial uterine cavity, those of group B recieved \nendometriosis from the capsule of endometrioma, and \nfinally mice in group C were injected with endometriosis \nfrom adenomyosis tissue. The differences of this cell \norigin are based on the pathophysiology of endometriosis \nin which these three endometriosis forms differ in the \ncomplaint, clinical finding, diagnostic, and therapeutic \nprocess and recurrences.\nThe endometrial tissue of the uterine cavity, the \nendometriosis tissue from the capsule of endometrioma, \nand adenomyosis tissue were stored in phosphate buffer \nsaline (PBS) and then centrifuged twice (2500 rpm). \nThe pellet was removed, and then PBS was added along \nwith 200 μG/mL of streptomycin and 200 IU/mL of \npenicillin (8). Each mouse was slowly injected with \n0.1 mL of supernatant through the peritoneal cavity \nover 60 seconds. Then, the mice were injected with an \nethinyl estradiol dose of 0.2 μGR/mice intramuscularly \non the thighs using a disposable 1 mL syringe. On the \nday 15, the mice were dissected to calculate the extent \nof endometriosis implantation in the peritoneum and \nunderwent histopathological examination employing \nhematoxylin-eosin (HE) staining.\nSampling Endometriosis Model Mice\nSamples were collected immediately after the mice \nwere euthanized. The abdominal wall and peritoneum \nwere separated, then the peritoneum was excised and \nstretched on millimeter paper and documented using a \nphoto to observe the extent of endometriosis. Next, the \nhistopathological examination was prepared. Afterward, \nthe result was recorded on the data collection sheets and \nanalyzed statistically. The preparation for anatomical \npathology examination was performed applying the \nreddest peritoneal tissue taken for preparation, which was \nthen preserved with 10% formalin.\nExtensive Examination of the Peritoneum\nThe peritoneum was examined using a Nikon H600L \nmicroscope equipped with a Fi2 300-megapixel DS digital \ncamera and the Nikon image processing software (Nikon \nCorporation). The area of the endometriosis implant was \nmacroscopically assessed in the area of hyperemia which \nwas then confirmed by taking the samples in the most \nhyperemic areas to be examined for any endometriosis \nlesion. Measurements of the implantation of endometriosis \nlesions were made by calculating the red area lesion on \nthe peritoneal wall by mm2 units calculated using Motic \nImage software, which is specific computer software for \ncomputing the certain area.\n Histopathological Examination\nThe level of damage to the peritoneum was determined \nby examining the inflammatory cell infiltration and the \npresence of granuloma mass. Additionally, the degree of \nperitoneal damage was assessed using a scoring system \naccording to the modified Klopfleisch method where \nthe damage level was computed by summing up all the \nscores of the lesions (9). The scoring system contained \ntwo assessments based on inflammatory cell infiltration \n(Table 1) and granuloma mass (Table 2).\nThe staining used in this study included HE streptavidin \nand biotin (labeled as streptavidin-biotin-method/LSAB). \nEndometriosis spots in peritoneum were embedded in \nparaffin, then cut 4-6 μM. The tissue was deparaffinized \nin xylol two times (5 minutes each). Then, it was \nconsecutively soaked in ethanol absolute (2 times for 3 \nminutes), ethanol 95% (two times 3 minutes each), and \nethanol 70% (for 3 minutes). The tissue was washed with \naquabides (2H20) and then sprayed with proteinase K \nsolution for 5 minutes. Afterward, it was double washed \nwith PBSsprayed with hydrogen peroxidase 3% (H2O2) \n\nSutrisno et al\nInternational  Journal of Women’s Health and Reproduction Sciences, Vol. 7, No. 1, January 2019\n36\nfor 5 minutes, and then double washed using PBS 2 times.\nData Analysis\nData were analyzed using the following steps: conducting \ndata normality test employing the Shapiro-Wilk test, \ncomparative test using the independent sample t test \n(normally distributed data) or Mann-Whitney (when \nnot normally distributed), and one-way ANOV A test \n(F-test) (if the data were normally distributed) or Kruskal \nWallis test (if the data were not normally distributed). All \ncalculations were performed using the statistical package \nfor the social sciences (SPSS) software, version 19.\nResults\nArea of Implantation on the Peritoneum\nBased on the size of the implanted endometriosis spots, it \nappears that group C had larger peritoneal endometriosis \nimplants (P < 0.05) of 42.75 ± 3.28 mm2 compared to other \ngroups while group B had endometriosis with an implant \narea that was 10.68 ± 1.41 mm 2 smaller than that of the \ngroup A (Figure 1).\nMacroscopic Overview Wide Implantation of Endometriosis\nMacroscopically, group C had more hyperemic features, \nwhich indicated better hypervascularization/implantation \ncompared to groups A and B (Figure 2).\nHistopathology Degree of Peritoneal Damage\nThis analysis aimed at examining the level of damage \nto the peritoneum. Based on the calculation using the \nKlopfleisch scoring method, group C was found to have \na higher score (9.1 ± 3.28; P < 0.05) than other groups \nTable 1. Scoring Peritoneal Damage Level Based on Inflammatory \nCell Infiltration (8)\nLesion Score  Information\nInflammation cell \ninfiltration\n0  Not detected\n1  < 10 in 5 lp (400x) \n2  Between 11-50 in 5 lp (400x) \n3  Between 51-100 in 5 lp (400x) \n4  > 100 in 5 lp (400x) \nTable 2.  Scoring Degree of Peritoneal Damage Based on Mass \nGranuloma (8)\nLesion Score Information\nGranuloma \n0 Granuloma mass was not detected \n2 Granuloma mass was detected\n4 Granuloma mass was detected with abscess \n6 Granuloma mass was detected with abscess \nand muscle tissue necrosis \n8 Granuloma mass was detected with abscess, \nmuscle tissue necrosis, and fibrosis \nNote: Damage level is the total of the 2 above lesions which are \nbetween 0–12 intervals.\nFigure 1.  The calculation of hypervascularization/endometriosis \nimplantation in endometriosis mice.\nFigure 2. Measurement of Hypervascularization Area in Endometriosis \nModel of Mice.\n(group A: 0.9 ± 0.88; group B: 1.7 ± 1.42), the differences \nof which are displayed in Figure 3 (9).\nHistopathology Lesions Endometriosis\nIn the histopathological examination of endometriosis \nlesions formed in each group, it was observed that mice \nin groups A and B had inflammation. Generally, the \ninflammation score was between 1 and 5. Additionally, \ngroup C mice had severe inflammation which was \nfollowed by myocyte cell death and fibrous connective \ntissue formation in some cases. The comparison of \n\nSutrisno et al\nInternational  Journal of Women’s Health and Reproduction Sciences, Vol. 7, No. 1, January 2019\n37\nhistopathological examination is presented in Figure 4. In \ngroup C model mice, granuloma mass, inflammatory cell \nmass infiltration, and fibrous connective tissue formation \noccurred in muscle tissue (Figure 5).\nDiscussion\nEndometriosis is defined as the endometrial tissue \nwhich is present outside the uterine cavity. The most \nnormally affected areas are pelvic or peritoneal organs \nalthough other areas may either have the possibility to be \naffected. Clinical manifestations may be the lesions that \nare typically acquired on the peritoneal surface of the \nreproductive organs, but they may occur anywhere in the \nfemale organs. The size of the lesions varies considerably \nfrom microscopic to large invasive masses that erode the \ninside of the organ and cause extensive adhesion (10).\nFollowing the macroscopic analysis, it was found that \nthe implant tissue was the rounded nodules with varying \nsizes which were strongly attached to the peritoneal tissue \nFigure 3. The Differences in Peritoneal Degradation Level.\nFigure 4. Histopathology Level of Peritoneal Damage. In A and B models, the inflammatory group scored between 1 and 3 whereas in group C \n(the treatment group) there was severe inflammation some of which were followed by the death of myocyte cells and fibrous connective tissue \nformation. Inflammation is illustrated by the arrows (M = 100x).\nunderneath. The damage level in endometriosis nodules \noccurred as a result of experimental treatment mice varied \nbetween the groups. The damage level was determined by \nassessing the extent of the area of endometriosis implants \nformed in the peritoneum mice model. As shown in Figure \n1, group C had a larger area of implanted endometriosis \ncompared to groups A and B. Therefore, supernatant \ninjection of the adenomyosis should be used to obtain \nthe most endemic mouse model of the endometriosis. \nEven the heterologous model of a mouse model of \nendometriosis which was reported as a good alternative \nto make peritoneal endometriosis in mice for research \npurposes had a specific limitation (11)\nIn this study, histopathological examination was used \nto determine the damage level to the peritoneum based \non inflammatory cell infiltration and the appearance \nof granuloma mass. The scoring method of the damage \nlevel uses a scoring system with the modified Klopfleisch \nmethod (9). Based on microscopic observation, group \nC had a mean value infiltration of inflammatory cells \nof about >100 cells in 5 viewing fields (M = 400x). In \nother words, group C had severe inflammation some of \nwhich was followed by myocyte cell death and fibrous \nconnective tissue formation. In groups A and B, however, \ninflammation was present at the scores between 1 and 3. \nGreaves et al declared that the implantation of human \nendometrium tissue to peritoneal mice would produce \nsimilar characteristics with original tissue in a human \nsetting which is suitable for endometriosis research \npurposes (12). Measurement size of the lesion proved \nthat adenomyosis tissue could induce an inflammatory \nenvironment more severely than either endometrium or \nendometrioma capsule. In addition, worsen inflammation \n\nSutrisno et al\nInternational  Journal of Women’s Health and Reproduction Sciences, Vol. 7, No. 1, January 2019\n38\nstate of the intraperitoneal cavity would induce \nendometriosis more severely and vice versa (10).\nBased on histopathological examination of the \nendometriosis lesions in the peritoneum, group C had \nthe most severe damage level. The granuloma mass, \nmassive infiltration of inflammatory cells, and fibrous \nconnective tissue formation occurred in muscle tissue in \nthis group. Conversely, mice injected with the supernatant \nof endometrial tissue did not develop granuloma masses \n(Figure 5). Further, no granuloma masses were detected \nin group A while it was observed only in one of the mice \nof group B. However, a granuloma mass was noted in \nalmost all the mice of group C. That is, mouse model C \nwas the best one through which inflammatory infiltration \nand granuloma mass simulation were obtained among \nthe three treatment groups. Inflammatory mediators \nsuch as TNF-α and IL-6 up-regulated vascular endothelial \ngrowth factors and led to increased angiogenesis and \ninflammation reactions and stimulated the growth of \nnodule/ endometriosis spot in the gut, the muscle of \nthe abdomen wall, liver, and adipose tissue surrounding \nabdominal organ (12). \nThe immune system which involved in the development \nof endometriosis includes humoral and cellular immunity. \nIn patients with endometriosis, the occurrence of immune \nsystem disorders is characterized by the reduced T cells \nand a natural killer cell response (13). Furthermore, \nthe disease indicates an increase in humoral immune \nresponse and macrophage activity (13). Endometriosis \nlesions secrete haptoglobin which affects the normal \nfunction of the macrophages. Moreover, the inflammatory \nmediator that can stimulate the cascade reaction with \nthe end product includes increasing endometriosis cell \nproliferation, showing less response to apoptosis stimulus, \nincreasing the formation of the new vascular vessel, and \naggravating the development of endometriosis lesion (10). \nThe deficient immune system in mice for a heterologous \nmodel of endometriosis is useful for studying the immune \nmodulating drug in endometriosis (12,14)\nAccording to the immunological theory, the adhesion \nof endometrial cells released onto the peritoneal surface \nand invasion of the subperitoneal involves the appearance \nof extracellular membrane adhesion molecules (ECAM) \nmolecules and their co-receptor. Endometrial fragments \nmay accumulate in certain places within the pelvic \ncavity and adhere to the peritory surface. A microscopic \ndefect causes the endometrial cells to come into direct \ncontact with the submesothelium matrix, which then \nproliferate, spread, grow, and sometimes invade down \nto the subperitoneal layer. Endometriosis is often found \nin women with low cellular immunity due to its inability \nto degrade the tissue of endometriosis that enters the \nperitoneum (10).\nMacrophages and monocytes in the peritoneal fluid \nare vital elements of the immune system contained \nin the peritoneal fluid. The macrophage is the most \ncommon type of cell which is found in peritoneal fluids \nand is involved in the pathogenesis of endometriosis. \nAdditionally, peritoneal macrophages and monocytes of \nthe endometriosis have an increased effect of cytokine \nproduction, growth and angiogenic factors, and other \nsubstances that stimulate ectopic endometrial proliferation \nand decrease apoptosis. Increased cytokine production \nmediates a number of endometriosis symptoms such as \ninfertility and pain in women of reproductive age (15,16).\nThe growth of the ectopic endometrium, facilitation \nof infiltration by the immune cells, and the increased \nproduction of pro-inflammatory cytokines as well as \nangiogenesis and growth factors are considered the \nentire picture of the inflammatory response detected \nin endometriotic implants. This condition leads to the \nmobilization of fibroblasts and the proliferation of the \nconnective tissue as a homeostatic mechanism to isolate \nand cure the injury site. The emergence of fibroblasts \nand connective tissues plays an important role in the \npathogenesis of this disease. However, it remains unclear \nwhether these immunological abnormalities are the cause \nor consequence of endometriosis (15).\nEctopic endometrium growth stimulates excessive \nmacrophage production, proinflammatory cytokine \nFigure 5. Description of Granuloma (GR) Mass and Infiltration of Inflammatory Cells and Connective Tissues in Muscle Tissue of Mice \nReceiving an Injection of Adenomyosis Tissue (group C). Note. (A) The form of granuloma and massive infiltration of inflammatory cells in \nmuscle tissue (arrows). (B) The formation of fibrous connective tissue (arrows) between the muscle cells  (M = 200x).\n\nSutrisno et al\nInternational  Journal of Women’s Health and Reproduction Sciences, Vol. 7, No. 1, January 2019\n39\nproducts, and growth factors in peritoneal fluid, leading to \nfurther growth. The major pro-inflammatory cytokines, \nnamely, TNF-αand IL-1β are released from peritoneal \nmacrophages and endometriosis cells which subsequently \nactivate transcription factors such as nuclear factor-\nkappa B (NF-kB) and protein activator 1 (AP-1). Active \ntranscription factors bind to endometriotic cell DNA and \nstimulate subsequent gene transcription activity (13).\nOur knowledge about the etiology of peritoneal \nendometriosis is limited. The most broadly accepted \nexplanation is the “Sampson hypothesis” which suggests \nthat peritoneal endometriosis occurs due to retrograde \nmenstruation when the endometrial tissues pass through \nthe fallopian tubes into the peritoneal cavity where the \ntissue undergoes implantation (17). Nevertheless, this \nmechanism cannot justify why endometriosis happens \nonly in some women if retrograde menstruation is about \nto occur in about 90% women (18). Metaplasia of the colon \nas another usual hypothesis indicates that the epithelium \ncan be converted into endometrium by metaplasia. \nHowever, this theory cannot account for the extreme \nrarity of endometriosis in men, its common localization \nin the abdominal cavity, and lack of increase with age as \ncompared to other metaplasia (19).\nThe implantation theory offers that the endometriosis \nformation in the peritoneal cavity needs the endometrial or \ncell tissue in order to complete the adhesion, invasion, and \nproliferation process. Several studies examined whether \npelvic peritoneum was involved in the endometriosis \nformation and maintenance or contained these changes \nin women with endometriosis. Some potential roles in \nthe pathophysiology of peritoneal endometriosis were \ndiscussed and considered which include providing the \nof ectopic endometrium cell attachment sites, facilitation \nof endometrial cell invasion, transcendental epithelial-\nmesenchymal potential, changes in immune cell \nactivation or recruitment, and the differential expression \nof inflammatory cytokines.\nThere are epithelium, stromal, and endometrial \nglands in endometriotic implants, the histology picture \nof which is similar to the eutopic endometrium (18,20). \nMicroscopic analysis demonstrated that endometriosis \nencompass endometrial glands and stroma which are \nsometimes found in smooth muscle fibers and respond \nto hormonal circulation as was reported for eutopic \nand ectopic endometrium (21). Injecting endometriosis \nfrom human origin into mice peritoneal cavity increases \nthe inflammatory reaction in peritoneal cavity organ \nand develops more nodule growth and adhesion \nintraperitoneally. In addition, it is a more practical method \nto develop the mice model of endometriosis (12,14).\nConclusions\nThe size of the implanted endometriosis lesions which \nwere injected with supernatant from adenomyosis into the \nmice (group C) caused larger   endometriosis implant areas \nand most severe damage to the p eritoneum, granuloma \nmass, and massive infiltration of inflammatory cells and \nthe formation of fibrous connective tissue in the muscle \ntissue. Generally, based on the results, implantation of the \nadenomyosis cell tissue is regarded as the best method for \ndeveloping the mice model of endometriosis.\nEthical Issues\nAll the methods were approved by the Ethical Committee \nof Medicine Faculty, Brawijaya University with the ethical \nclearance No.197/EC/KEPK-S3/05/2017.\nConflict of Interests\nAuthors declare that they have no conflict of interests.\nFinancial Support\nNone. \nAcknowledgments\nThe authors would thank Brawijaya University for \nproviding the required research facility. \nReferences\n1. Nowak N. Development and Evaluation of Two Mice \nModels for Endometriosis Focused on the Involvement \nof the Immune System in Endometriosis Establishment. \nDissertation. Eingereicht im Fachbereich Biologie. Berlin: \nChemie, Pharmazie der Freien Universität Berlin; 2008.\n2. Grümmer R. Animal models in endometriosis research. \nHum Reprod Update. 2006;12:641. doi: 10.1093/humupd/ \ndml026.\n3. Vernon MW , Wilson EA. 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