Induction of Fresh Chocolate Cyst Pulp into Endometriosis Mice Model

& Objective: A chocolate cyst is a type of endometriosis in addition to the appearance of endometrial lesions. The limitations of obtaining these samples led to trying to induce fresh chocolate cyst pulp to become a model of endometriosis mice. Chocolate cyst pulpy can be obtained through cyst puncture with no need to perform laparoscopy or laparotomy. This study was conducted with the aim of proving that induced mice with fresh chocolate cyst pulp samples succeeded in becoming endometriosis models of mice.

& Methods: Fresh chocolate cyst slurry was injected as much as 5.10-3 mL/g of mice weight intraperitoneally on day zero. Mice of Mus musculus strain Balb/c were injected with cyclosporine 1.4.10-2 mg/g of the weight of mice intraperitoneally daily for 14 days. On the 1st and 5th day, the mice were injected with estradiol 2.6.10-4 mL/g of the weight of the mice, intramuscularly. Induction research on the endometriosis mice model was conducted at the Pharmacology Laboratory of the Universitas Gadjah Mada, Yogyakarta, Indonesia. On the 15 th day, the mice were necropsied for histopathological examination at the Anatomical Pathology Laboratory, Faculty of Medicine, Public Health & Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, with Hematoxylin-Eosin staining.

A total of 6 mice induced with fresh chocolate cyst pulp histopathologically showed signs of endometriosis. Histopathologically readings showed external endometriosis cyst (1 mouse), endometriosis tissue (1 mouse), hemosiderosis (1 mouse), and stromatosis (3 mice).

The six mice were induced with fresh chocolate cyst slurry were 100% successful in becoming endometriosis mice models, as a novelty in endometriosis studies using experimental animals.

Anatomical pathology examination, Endometriosis mice model, Fresh Chocolate cyst pulp Received: 2025/01/18 Accepted: 2025/04/26 Published Online: 30 Jun. 2025 Corresponding Information: Ivanna Beru Brahmana, Department of Obstetrics and Gynecology, Medical Study Program, Faculty of Medicine and Health Sciences, Universitas Muhammadiyah Yogyakarta, Indonesia Email: [email protected] Copyright © 2025, This is an original open -access article distributed under the terms of the Creative Commons Attribution-noncommercial 4.0 International License which permits copy and redistribution of the material just in noncommercial usages with proper citation . 1. Introduction Endometriosis is defined as a chronic gynecological disease in which endometrial tissue is found outside the uterine cavity (1), especially in the pelvic and ovarian tissue (2,3). About 10% of women complain of endometriosis worldwide (4,5). The two main complaints of endometriosis are pain and infertility (5– 9). Pain complaints are found in around 10-15%, while infertility complaints are found in 50% of cases (10), or both are found together in 35-50% of cases (11). Endometriosis therapy takes a relatively long time, averaging about 3 -6 months. It creates a social and economic burden due to a significant reduction in productivity and health costs during treatment (5,12,13). It is concerning that endometriosis therapy shows a high recurrence rate, reaching 21.5% after two years of therapy and 50% after five years of therapy (14). On the other hand, the effectiveness of endometriosis treatment is still limited (5). An effective endometriosis treatment continues to be found. Therefore, more research continues to be carried out. Research begins by using experimental animals. Experimental animal models of endometriosis in the form of mice have been carried out by implan ting myometrial and endometrial tissues of patients with adenomyosis through surgery (15). 533 Induction of Fresh Chocolate Cyst Pulp Volume 10, July 2025 Journal of Obstetrics, Gynecology and Cancer Research Endometriosis has three clinical forms: Endometriotic implants on the peritoneal surface of the pelvis and ovaries or in the subperitoneal fat tissue, Ovarian cysts (endometriomas), and Rectovaginal nodules (3). Chocolate cyst as a form of endometriosis is possible with a simpler operation. This action is in the form of a cyst puncture, namely by puncturing the contents of the chocolate cyst so that a chocolate cyst pulp is obtained. The chocolate cyst slurry was tested to be induced in experimental animals by selecting Mus musculus mice to become endometriosis mice models. Induction of chocolate cyst pulp in experimental animals was published in 1992 but has not shown the

of endometriosis formation (16). The large number of chocolate cyst cases has given rise to the idea of trying to induce fresh chocolate cyst pulp obtained from chocolate cyst puncture surgery. The fresh chocolate cyst gruel was induced in Mus musculus mice due to the need to conduct rese arch using endometriosis mice models. The present study was conducted with aim to prove that mice induced with fresh chocolate cyst pulp samples succeeded in becoming endometriosis models of mice. 2. Materials and Methods This experimental study was conducted by inducing female mice Mus musculus sp strain Balb/c with fresh chocolate cyst pulp. Fresh chocolate cyst pulp Chocolate cyst pulp was obtained from chocolate cyst puncture surgery on chocolate cyst sufferers. The chocolate cyst slurry from the puncture results was taken to the experimental animal laboratory to be prepared and induced in the experimental animal. Mice models of endometriosis Female Mus musculus sp. strain Balb/c mice as research subjects aged 1.5 -2 months, weighing about 20-40 grams, were placed in cages with a minimum height of 12 cm. The cage was well -ventilated and quiet and had lighting (12 hours of dark and light), room temperature (20-24oC), and relative humidity 45- 65%. They were given food and drank standard BR1, and were in good health, had no physical disability, and had normal activities. The six induced mice were obtained from livestock facilities and infrastructure at the Pharmacology and Toxicology Laboratory, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia, which was also the place for animal testing. Fresh chocolate cyst pulp was washed with Phosphate-Buffered Saline (BPS) twice at 3000 rpm at 4oC. The supernatant was removed, and then PBS was added again. On Day Zero (D0), induction was started by injecting fresh chocolate cyst pulp intraperitoneally at a dose of 0.1 mL/20 g or 5.10-3 mL/g weight of mice. For 14 days, mice have been conditioned to experience immunodeficiency by giving intraperitoneal injections of cyclosporine at a dose of 0.28 mg/20 g or 1.4.10 -2 mg/g weight of mice. Endometriosis is an estrogen -dependent disease, and this condition was made by injecting estradiol on the First Day (D1) and the Fifth Day (D5) intramuscularly at a dose of 0.0052 ml/20 g or 2.6.10 -4 mL/g weight of mice. On Day 15 (D15), the mice were sacrificed by cervical dislocation, which was previously administered intraperitoneally with 0.1 mL of ketamine anesthesia. After the mice were immobile, the surgery was performed to remove the uterine organs, ovaries, and fatty tissue around the uterus and ovaries. Samples were fixed with 10% buffered formalin, sent to the Anatomical Pathology Laboratory for Hematoxillin - eosin (HE) examination, and read microscopically with an Olympus BX51 microscope with 40x and 100x magnification. This research has received ethical approval from the Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada -Dr. Sardjito General Hospital with number: KE/FK/0581/EC/2021. 3. Results Weight of mice with a volume of fresh chocolate cyst pulp injected are presented in Table 1. Table 2 shows the results of the anatomical, pathological examination of fresh chocolate cyst pulp induction. Induction using fresh cyst slurry in six mice resulted in endometriosis mice models (Figure 1). Table 1. Code, mice weight, and volume of fresh chocolate cyst pulp No. Mice code Mice weight (g) the volume of fresh chocolate cyst pulp (mL) 1 C1 27 0.135 2 C5 23 0.115 3 D1 24 0.12 4 D3 23 0.115 5 D4 26 0.13 6 D6 27 0.135 Ivanna Beru Brahmana, et al. 534 Volume 10, July 2025 Journal of Obstetrics, Gynecology and Cancer Research Table 2. Results of anatomical, pathological examination of fresh chocolate cyst pulp induced No Subject Result 1 C1 Induction of fresh cyst pulp External endometriosis cyst 2 C5 Induction of fresh cyst pulp Endometriosis tissue 3 D1 induction of fresh cyst pulp hemosiderosis 4 D3 induction of fresh cyst pulp Stromatosis 5 D4 induction of fresh cyst pulp Stromatosis 6 D6 induction of fresh cyst pulp Stromatosis Figure 1. Anatomical, pathological features of induction of fresh chocolate cyst pulp, magnification 40x and 100x C1 : External endometriosis cyst wall, part of the cyst wall is found hemosiderophage, magnification 100x C5 : Endometrial cyst in fat, magnification 100x D1 : Chocolate cyst wall found hemosiderophage, magnification 40x D3 : Stromatosis in fat, magnification 100x D4 : Stromatosis in fat, magnification 100x D6 : Stromatosis in fat, magnification 100x The induction of fresh chocolate cyst pulp was repeated in 30 Mus musculus mice in the next study, and the 30 mice were also 100% successful in becoming endometriosis mice models. Anatomical Pathology in the 30 mice showed the presence of hemosiderophages or stromatosis as a manifestation of the formation of endometriosis (Figure 2). Figure 2. Anatomical pathology description of the induction of fresh cyst pulp in 30 mice in the next study, magnification 100x 4. Discussion Preliminary research was carried out on six mice Mus musculus strain Balb/c induced with fresh chocolate cyst pulp. Each mouse was coded and weighed. The volume of fresh chocolate cyst slurry was calculated to be induced as much as 0.1 ml/20 gr of the weight of the mice ( Table 1). Furthermore, in the next study, the other 30 mice were given the same treatment induced into the endometriosis model according to these calculations. The presence of external endometriosis cysts, hemosiderophages, and stromatosis is in accordance with Bulun's 2019 description of endometriosis. Endometriosis is said to have three clinical forms, one of which is an endometriotic implant located on the surface of subperitoneal fat tissue, as found in this induction of fresh chocolate cyst slurry. Two other forms of C1 C5 D1 D3 D4 D6 535 Induction of Fresh Chocolate Cyst Pulp Volume 10, July 2025 Journal of Obstetrics, Gynecology and Cancer Research endometriosis are ovarian cysts (endometriomas) and rectovaginal nodules (3). In this study, endometriosis formed by discovering small round cell colonies with morphologically similar shapes to the epithelium. There are also single cells with typical fibroblast -like morphology (17). Six Mus musculus sp strain Balb/c mice induced with fresh chocolate cyst pulp showed the formation of endometriosis in the form of external endometriosis cysts, hemosiderophages, and stromatosis, at 40x and 100x magnification, using an Olympus BX51 microscope. In a subsequent study, the colonies of small round cells with epithelial -like morphology and single cells with typical fibroblast -like morphology were also found in 30 mice induced by fresh chocolate cyst pulp. The presence of hemosiderophages and stromatosis in the 30 induced mice indicated that the experimental animals had succeeded in becoming a model of endometriosis with this protocol. This induction protocol of fresh chocolate cyst pulp into endometriosis mice model is expected to be one of the models for involved animal study in the future. Previous endometriosis mouse models used different implants. During surgical procedures, samples were taken from the myometrial and endometrial tissues of individuals diagnosed with adenomyosis (15). 5. Conclusion The endometriosis model could be performed by intraperitoneal injection of fresh chocolate cyst pulp on day zero followed by intraperitoneal cyclosporine every day for 14 days. On the 1 st and 5th day, the mice were injected with estradiol intramuscularly. 6. Declarations Acknowledgments We would like to thank the Research and Innovation Institute of the Universitas Muhammadiyah Yogyakarta as the funder of this research, and Doddy Sutanto, Fertility Consultant Obstetrics & Gynecology Specialist from RSIA Gladiool Magelang, Central Java, Indonesia, who helped carry out this research. Ethical Considerations This research has received ethical approval from the Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada -Dr. Sardjito General Hospital with number: KE/FK/0581/EC/2021. Authors' Contributions All authors were involved in planning and supervising the work. IBB performed the conceived and designed the analysis, collected the data, contributed data or analysis tools, and wrote the paper. S performed the analysis and wrote the paper. MP, RC and IP performed the analysis and wrote the paper. All authors discussed the results and commented on the manuscript. 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