{"paper_id":"b65eecf4-1fa1-4be9-b02e-a642cb20e07c","body_text":"Original Article  | JOGCR. 2025; 10(7): 532-536 \n     Volume 10, July 2025       Journal of Obstetrics, Gynecology and Cancer Research \n Journal of Obstetrics, Gynecology and Cancer Research | ISSN: 2476-5848 \n \nInduction of Fresh Chocolate Cyst Pulp into Endometriosis Mice Model  \n \nIvanna Beru Brahmana1* , Soetrisno Soetrisno2, Mulyoto Pangestu3, Rita Cempaka4, Ika Puspitasari5 \n \n1. Department of Obstetrics and Gynecology, Medical Study Program, Faculty of Medicine and Health Sciences, \nUniversitas Muhammadiyah Yogyakarta, Indonesia \n2. Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia \n3. Education Program in Reproduction and Development, Department Obstetrics and Gynecology, Monash University, \nAustralia \n4. Department of Pathology Anatomy, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia \n5. Faculty of Pharmacy, Academic Hospital, Universitas Gadjah Mada, Yogyakarta, Indonesia \nArticle Info  ABSTRACT \n  \n  10.24200/jogcr.10.7.532 \n \n \n \nBackground & Objective: A chocolate cyst is a type of endometriosis in addition to \nthe appearance of endometrial lesions. The limitations of obtaining these samples led to \ntrying to induce fresh chocolate cyst pulp to become a model of endometriosis mice. \nChocolate cyst pulpy can be obtained through cyst puncture with no need to perform \nlaparoscopy or laparotomy. This study was conducted with the aim of proving that \ninduced mice with fresh chocolate cyst pulp samples succeeded in becoming \nendometriosis models of mice. \nMaterials & Methods: Fresh chocolate cyst slurry was injected as much as 5.10-3 mL/g \nof mice weight intraperitoneally on day zero. Mice of Mus musculus strain Balb/c were \ninjected with cyclosporine 1.4.10-2 mg/g of the weight of mice intraperitoneally daily \nfor 14 days. On the 1st and 5th day, the mice were injected with estradiol 2.6.10-4 mL/g \nof the weight of the mice, intramuscularly. Induction research on the endometriosis \nmice model was conducted at the Pharmacology Laboratory of the Universitas Gadjah \nMada, Yogyakarta, Indonesia. On the 15 th day, the mice were necropsied for \nhistopathological examination at the Anatomical Pathology Laboratory, Faculty of \nMedicine, Public Health & Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, \nwith Hematoxylin-Eosin staining.  \nResults: A total of 6 mice induced with fresh chocolate cyst pulp histopathologically \nshowed signs of endometriosis. Histopathologically readings showed external \nendometriosis cyst (1 mouse), endometriosis tissue (1 mouse), hemosiderosis (1 \nmouse), and stromatosis (3 mice). \nConclusion: The six mice were induced with fresh chocolate cyst slurry were 100% \nsuccessful in becoming endometriosis mice models, as a novelty in endometriosis \nstudies using experimental animals. \nKeywords: Anatomical pathology examination, Endometriosis mice model, Fresh \nChocolate cyst pulp \n \nReceived: 2025/01/18 \nAccepted: 2025/04/26 \nPublished Online: 30 Jun. 2025 \n \n \n \n \n \n \n \n \n \n \n \nCorresponding Information:  \nIvanna Beru Brahmana, \nDepartment of Obstetrics and Gynecology, \nMedical Study Program, Faculty of Medicine \nand Health Sciences, Universitas \nMuhammadiyah Yogyakarta, Indonesia \n \nEmail: ivanna@umy.ac.id \n \n \nCopyright © 2025, This is an original open -access article distributed under the terms of the Creative Commons Attribution-noncommercial \n4.0 International License which permits copy and redistribution of the material just in noncommercial usages with proper citation . \n \n \n1. Introduction\nEndometriosis is defined as a chronic gynecological \ndisease in which endometrial tissue is found outside the \nuterine cavity (1), especially in the pelvic and ovarian \ntissue (2,3). About 10% of women complain of \nendometriosis worldwide (4,5). The two main \ncomplaints of endometriosis are pain and infertility (5–\n9). Pain complaints are found in around 10-15%, while \ninfertility complaints are found in 50% of cases (10), \nor both are found together in 35-50% of cases (11). \nEndometriosis therapy takes a relatively long time, \naveraging about 3 -6 months. It creates a social and \neconomic burden due to a significant reduction in \nproductivity and health costs during treatment \n(5,12,13). It is concerning that endometriosis therapy \nshows a high recurrence rate, reaching 21.5% after two \nyears of therapy and 50% after five years of therapy \n(14). On the other hand, the effectiveness of \nendometriosis treatment is still limited (5).  \nAn effective endometriosis treatment continues to be \nfound. Therefore, more research continues to be carried \nout. Research begins by using experimental animals. \nExperimental animal models of endometriosis in the \nform of mice have been carried out by implan ting \nmyometrial and endometrial tissues of patients with \nadenomyosis through surgery (15). \n\n\n533 Induction of Fresh Chocolate Cyst Pulp \n      Volume 10, July 2025       Journal of Obstetrics, Gynecology and Cancer Research \nEndometriosis has three clinical forms: \nEndometriotic implants on the peritoneal surface of the \npelvis and ovaries or in the subperitoneal fat tissue, \nOvarian cysts (endometriomas), and Rectovaginal \nnodules (3).  \nChocolate cyst as a form of endometriosis is possible \nwith a simpler operation. This action is in the form of \na cyst puncture, namely by puncturing the contents of \nthe chocolate cyst so that a chocolate cyst pulp is \nobtained. The chocolate cyst slurry was tested to be \ninduced in  experimental animals by selecting Mus \nmusculus mice to become endometriosis mice models. \nInduction of chocolate cyst pulp in experimental \nanimals was published in 1992 but has not shown the \nresults of endometriosis formation (16). The large \nnumber of chocolate cyst cases has given rise to the \nidea of trying to induce fresh chocolate cyst pulp \nobtained from chocolate cyst puncture surgery. The \nfresh chocolate cyst gruel was induced in Mus \nmusculus mice due to the need to conduct rese arch \nusing endometriosis mice models. The present study \nwas conducted with aim to prove that mice induced \nwith fresh chocolate cyst pulp samples succeeded in \nbecoming endometriosis models of mice.  \n \n2. Materials and Methods \nThis experimental study was conducted by inducing \nfemale mice Mus musculus sp strain Balb/c with fresh \nchocolate cyst pulp.  \nFresh chocolate cyst pulp \nChocolate cyst pulp was obtained from chocolate \ncyst puncture surgery on chocolate cyst sufferers. The \nchocolate cyst slurry from the puncture results was \ntaken to the experimental animal laboratory to be \nprepared and induced in the experimental animal.  \nMice models of endometriosis \nFemale Mus musculus sp. strain Balb/c mice as \nresearch subjects aged 1.5 -2 months, weighing about  \n20-40 grams, were placed in cages with a minimum \nheight of 12 cm. The cage was well -ventilated and \nquiet and had lighting (12 hours of dark and light), \nroom temperature (20-24oC), and relative humidity 45-\n65%. They were given food and drank standard BR1, \nand were in good health, had no physical disability, and \nhad normal activities. The six induced mice were \nobtained from livestock facilities and infrastructure  at \nthe Pharmacology and Toxicology Laboratory, Faculty \nof Pharmacy, Universitas Gadjah Mada, Yogyakarta, \nIndonesia, which was also the place for animal testing. \nFresh chocolate cyst pulp was washed with \nPhosphate-Buffered Saline (BPS) twice at 3000 rpm at \n4oC. The supernatant was removed, and then PBS was \nadded again. On Day Zero (D0), induction was started \nby injecting fresh chocolate cyst pulp intraperitoneally \nat a dose of 0.1 mL/20 g or 5.10-3 mL/g weight of mice. \nFor 14 days, mice have been conditioned to \nexperience immunodeficiency by giving \nintraperitoneal injections of cyclosporine at a dose of \n0.28 mg/20 g or 1.4.10 -2 mg/g weight of mice. \nEndometriosis is an estrogen -dependent disease, and \nthis condition was made by injecting estradiol on the \nFirst Day (D1) and the Fifth Day (D5) intramuscularly \nat a dose of 0.0052 ml/20 g or 2.6.10 -4 mL/g weight of \nmice. On Day 15 (D15), the mice were sacrificed by \ncervical dislocation, which was previously \nadministered intraperitoneally with 0.1 mL of ketamine \nanesthesia. After the mice were immobile, the surgery \nwas performed to remove the uterine organs, ovaries, \nand fatty tissue around the uterus and ovaries. Samples \nwere fixed with 10% buffered formalin, sent to the \nAnatomical Pathology Laboratory for Hematoxillin -\neosin (HE) examination, and read  \nmicroscopically with an Olympus BX51 microscope \nwith 40x and 100x magnification.  \nThis research has received ethical approval from the \nMedical and Health Research Ethics Committee \n(MHREC) Faculty of Medicine, Public Health and \nNursing, Universitas Gadjah Mada -Dr. Sardjito \nGeneral Hospital with number: KE/FK/0581/EC/2021.  \n \n3. Results \nWeight of mice with a volume of fresh chocolate cyst \npulp injected are presented in Table 1. \nTable 2  shows the results of the anatomical, \npathological examination of fresh chocolate cyst pulp \ninduction. Induction using fresh cyst slurry in six mice \nresulted in endometriosis mice models (Figure 1).  \n \nTable 1. Code, mice weight, and volume of fresh chocolate cyst pulp \nNo. Mice code Mice weight (g) the volume of fresh chocolate cyst pulp (mL) \n1 C1 27 0.135 \n2 C5 23 0.115 \n3 D1 24 0.12 \n4 D3 23 0.115 \n5 D4 26 0.13 \n6 D6 27 0.135 \n \n\nIvanna Beru Brahmana, et al. 534 \n      Volume 10, July 2025       Journal of Obstetrics, Gynecology and Cancer Research \nTable 2. Results of anatomical, pathological examination of fresh chocolate cyst pulp induced  \nNo Subject Result \n1 C1 Induction of fresh cyst pulp External endometriosis cyst \n2 C5 Induction of fresh cyst pulp Endometriosis tissue \n3 D1 induction of fresh cyst pulp hemosiderosis \n4 D3 induction of fresh cyst pulp Stromatosis \n5 D4 induction of fresh cyst pulp Stromatosis \n6 D6 induction of fresh cyst pulp Stromatosis \n \n \n \n \n \n \n \n \n \n \n \n \n \nFigure 1. Anatomical, pathological features of induction of fresh chocolate cyst pulp, magnification 40x and 100x \nC1 : External endometriosis cyst wall, part of the cyst wall is found hemosiderophage, magnification 100x \nC5 : Endometrial cyst in fat, magnification 100x \nD1 : Chocolate cyst wall found hemosiderophage, magnification 40x \nD3 : Stromatosis in fat, magnification 100x \nD4 : Stromatosis in fat, magnification 100x \nD6 : Stromatosis in fat, magnification 100x \n \nThe induction of fresh chocolate cyst pulp was \nrepeated in 30 Mus musculus mice in the next study, \nand the 30 mice were also 100% successful in \nbecoming endometriosis mice models. Anatomical \nPathology in the 30 mice showed the presence of \nhemosiderophages or stromatosis as a manifestation of \nthe formation of endometriosis (Figure 2). \n \n \n \n \n \n \n \n \n \n \nFigure 2.  Anatomical pathology description of the \ninduction of fresh cyst pulp in 30 mice in the next study, \nmagnification 100x \n4. Discussion \nPreliminary research was carried out on six mice \nMus musculus strain Balb/c induced with fresh \nchocolate cyst pulp. Each mouse was coded and \nweighed. The volume of fresh chocolate cyst slurry was \ncalculated to be induced as much as 0.1 ml/20 gr of the \nweight of the mice ( Table 1). Furthermore, in the next \nstudy, the other 30 mice were given the same treatment \ninduced into the endometriosis model according to \nthese calculations. The presence of external \nendometriosis cysts, hemosiderophages, and \nstromatosis is in accordance with Bulun's 2019 \ndescription of endometriosis. Endometriosis is said to \nhave three clinical forms, one of which is an \nendometriotic implant located on the surface of \nsubperitoneal fat tissue, as found in this induction of \nfresh chocolate cyst slurry. Two other forms of \nC1 C5 D1 \n \n \n \n  \nD3 D4 D6 \n \n \n \n \n \n \n  \n\n\n535 Induction of Fresh Chocolate Cyst Pulp \n      Volume 10, July 2025       Journal of Obstetrics, Gynecology and Cancer Research \nendometriosis are ovarian cysts (endometriomas) and \nrectovaginal nodules (3).  \nIn this study, endometriosis formed by discovering \nsmall round cell colonies with morphologically similar \nshapes to the epithelium. There are also single cells \nwith typical fibroblast -like morphology (17). Six Mus \nmusculus sp strain Balb/c mice induced with fresh \nchocolate cyst pulp showed the formation of \nendometriosis in the form of external endometriosis \ncysts, hemosiderophages, and stromatosis, at 40x and \n100x magnification, using an Olympus BX51 \nmicroscope. In a subsequent study, the colonies of \nsmall round cells with epithelial -like morphology and \nsingle cells with typical fibroblast -like morphology \nwere also found in 30 mice induced by fresh chocolate \ncyst pulp. The presence of hemosiderophages and \nstromatosis in the 30 induced mice indicated that the  \nexperimental animals had succeeded in becoming a \nmodel of endometriosis with this protocol. This \ninduction protocol of fresh chocolate cyst pulp into \nendometriosis mice model is expected to be one of the \nmodels for involved animal study in the future. \nPrevious endometriosis mouse models used different \nimplants. During surgical procedures, samples were \ntaken from the myometrial and endometrial tissues of \nindividuals diagnosed with adenomyosis (15). \n \n5. Conclusion \nThe endometriosis model could be performed by \nintraperitoneal injection of fresh chocolate cyst pulp on \nday zero followed by intraperitoneal cyclosporine \nevery day for 14 days. On the 1 st and 5th day, the mice \nwere injected with estradiol intramuscularly. \n \n6. Declarations \nAcknowledgments \nWe would like to thank the Research and Innovation \nInstitute of the Universitas Muhammadiyah \nYogyakarta as the funder of this research, and Doddy \nSutanto, Fertility Consultant Obstetrics & Gynecology \nSpecialist from RSIA Gladiool Magelang, Central \nJava, Indonesia, who helped carry out this research. \n \nEthical Considerations \nThis research has received ethical approval from the \nMedical and Health Research Ethics Committee \n(MHREC) Faculty of Medicine, Public Health and \nNursing, Universitas Gadjah Mada -Dr. Sardjito \nGeneral Hospital with number: KE/FK/0581/EC/2021.  \n \nAuthors' Contributions \nAll authors were involved in planning and \nsupervising the work. IBB performed the conceived \nand designed the analysis, collected the data, \ncontributed data or analysis tools, and wrote the paper. \nS performed the analysis and wrote the paper. MP, RC \nand IP performed the analysis and wrote the paper. All \nauthors discussed the results and commented on the \nmanuscript. \n \nConflict of Interest \nThe authors declare no conflict of interest. \n \nFund or Financial Support \nThis study was funded by Research and Innovation \nInstitute (LRI) Universitas Muhammadiyah \nYogyakarta. \n.\n \n \n \n \n \n1. de Ziegler D, Borghese B, Chapron C. \nEndometriosis and infertility: pathophysiology \nand management. Lancet. 2010;376(9742):730–\n8. [DOI:10.1016/S0140-6736(10)60490-4] \n2. Burney RO, Giudice LC. Pathogenesis and \npathophysiology of endometriosis. Fertil Steril. \n2012;98(3):511–9. \n[DOI:10.1016/j.fertnstert.2012.06.029] \n3. Bulun SE. Endometriosis. In: Yen and Jaffe’s \nReproductive Endocrinology. 7th ed. Elsevier; \n2019, p. 609-42.  \n4. Morotti M, Vincent K, Becker CM. Mechanisms \nof pain in endometriosis. Eur J Obstet Gynecol \nReprod Biol. 2017;209:8 –13. \n[DOI:10.1016/j.ejogrb.2016.07.497] \n5. 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J Obstet Gynecol Cancer Res. 2025;10(7):532-6. \nDownload citation:                             RIS | EndNote | Mendeley |BibTeX |","source_license":"CC0","license_restricted":false}