Developing a mouse endometriosis model using stored chocolate cyst slurry: an experimental study

This laboratory experimental study included 12 female Mus musculus mice. Immunodeficient mice were intraperitoneally injected with a previously prepared chocolate cyst slurry. On the 15th day, the mice were euthanized, and anatomical pathological examination was performed using hematoxylin and eosin (HE) staining.

Anatomical pathology examination revealed stromatosis in the fat tissue of all 12 mice treated with stored chocolate cyst slurry, confirming the presence of endometriosis. The protocol demonstrated a 100% success rate in developing a mouse model of endometriosis.

The successful development of a mouse endometriosis model from stored chocolate cyst slurry using this protocol is expected to contribute to experimental animal studies. Plain Language SummaryResearchers developed a new mouse model for studying endometriosis, a chronic gynecological disease, using stored chocolate cyst pulp. Due to COVID-19, laparoscopic surgeries were paused, prompting the need for alternative research methods. In this study, 12 female mice were injected with a chocolate cyst slurry, and after 15 days, all mice showed signs of endometriosis in their fat tissue. This method achieved a 100% success rate in creating the model, which could help advance understanding of endometriosis and improve treatment strategies. The model offers a promising tool for future experimental studies on the disease. Text is machine generated and may contain inaccuracies. FAQ

Endometriosis is a chronic gynecological disease characterized by the presence of endometrial tissue in the form of glands and stroma outside the uterus[1]. One in 10 women experiences endometriosis between the ages of 12 and 52 years, and it affects nearly 200 million women worldwide[2]. The prevalence of endometriosis in the general population is 0.8%–28.6%[3]; in another study, it ranged from 10.8 per 1000 individuals, and 37% of them experienced infertility[4]. Endometriosis therapy requires a relatively long therapy of at least 3–6 months and a high recurrence rate of between 6% and 67%[5]. Further research on endometriosis necessitates the use of animal models. Traditionally, mouse models have been developed by implanting excised endometrial lesions of patients into the uterine tissue of mice, with the endometrial tissue being obtained through laparoscopic surgery. However, during the COVID-19 pandemic, laparoscopic procedures were halted to reduce the risk of viral transmission. Chocolate cysts, a specific type of endometriosis, can be obtained laparoscopically or through cyst aspiration. This operation was allowed during the pandemic if the lesions were symptomatic. Based on this factor, a research idea emerged to create a mouse model of endometriosis from the induction of stored chocolate cyst slurry. This study presents an experimental mouse model of endometriosis to contribute to endometriosis research.

This research design is a laboratory experiment in the form of induction of endometriosis in female Mus musculus mice with stored chocolate cyst slurry to create a mouse endometriosis model. Stored chocolate cyst slurry The stored chocolate cyst slurry was obtained by cyst aspiration from patients diagnosed with chocolate cysts. The chocolate cyst slurry from the aspiration was stored in a 10 mL syringe in a −20°C freezer for 1 month. Animal testing The study subjects were Mus musculus female mice of a similar strain, 1.5–2 months old, weighing approximately 20–30 g. The experimental animals were placed in cages under the following conditions: a minimum height of 12 cm, good ventilation, quiet, lighting (12-hour dark and light), room temperature (20°C –24°C), relative humidity 45%–65%. The experimental animals were not physically disabled and their activities were normal. All mice that met the inclusion criteria were included in the study, and none were excluded based on the exclusion criteria. The number of experimental animals used was sufficient for the study, but not excessive. Usually, 5–15 experimental animals are necessary, 12 mice were included in the study. Each female Mus musculus mouse that met the inclusion criteria was weighed to calculate the amounts of chocolate cyst slurry, cyclosporine, and estradiol used for each mouse. Each mouse was coded to facilitate the observation and examination of the results. Viability test of the stored chocolate cyst slurry As the chocolate cyst slurry was stored in a freezer for almost a month, it was necessary to test the viability of the cells in the cyst slurry. The viability tests were performed at the Clinical Pathology Laboratory, Fakultas Kedokteran, Kesehatan Masyarakat, and Keperawatan (FKKMK) Universitas Gadjah Mada, Yogyakarta, Indonesia. Induction of endometriosis in the female mouse model The induction of endometriosis was conducted at the Pharmacology and Toxicology Laboratory of the Faculty of Pharmacy, FKKMK UGM, Yogyakarta, Indonesia, using chocolate cyst slurry stored for 14 days. At the beginning of the study (D0), 0.2 mL/20 g of the stored chocolate cyst slurry was mixed with phosphate-buffered saline (PBS) in a ratio of 1:1. The slurry was washed with PBS twice at 3000 rpm at 4°C, after which the supernatant was discarded, and additional PBS was added. The slurry was then injected intraperitoneally at a dose of 0.1 mL per 20 g of body weight. Cyclosporine was administered intraperitoneally at a dose of 0.28 mg per 20 g body weight every day from the first day (D0) to the 14th day (D14) to induce immunodeficiency in the experimental mice. Cyclosporine was dissolved in a 0.5% sodium carboxymethyl cellulose solution. On the first (D1) and fifth (D5) day, estradiol at a concentration of 0.26 mg/kg body weight was injected intraperitoneally at a dose of 0.0052 mL per 20 g of body weight. Owing to endometriosis being estrogen dependent, estradiol was administered to create an estrogen-dependent condition. On D15, the mice were euthanized by cervical dislocation, and the uterus, ovaries, and fat tissue around the uterus and ovaries were collected. The samples were sent to the Anatomical Pathology Laboratory. Ethical consideration This study was approved by the Medical and Health Research Ethics Committee, Faculty of Medicine, Public Health, and Nursing Universitas Gadjah Mada, Dr. Sardjito General Hospital (KE/FK/0581/EC/2021). Statistical analysis The samples were sent to the Anatomical Pathology Laboratory for hematoxylin-eosin (HE) examination and read using an Olympus BX51 microscope at 40×, 100×, and 200× magnification. Twelve slides were prepared for each mouse (a total of 144 slides), with all slides showing the presence of endometriotic cells.

Viability test of apoptotic cells of chocolate cyst slurry The chocolate cyst slurry used in this protocol was stored for 1 month before induction in the experimental animals. Because of this storage time, it is imperative to test the viability of cells and determine whether there were enough (approximately 75%) viable cells.

of anatomical pathology examination with HE The results of anatomical and pathological examinations in the 12 induced mice showed stromatosis in the fat tissue, indicating that the induction of the stored chocolate cyst slurry in all samples was successful (Fig. 2). This resulted in a 100% success rate in creating the mouse endometriosis model.

of viability test for unstained apoptotic cells (A); unextracted sample of apoptotic cells (B); extracted samples of apoptotic cells (C). FITC: fluorescein isothiocyanate; FSC: Forward Scatter; SSC: side scatter. Anatomical pathology of the 12 mice models of endometriosis.

Endometriosis is an estrogen-dependent disease that occurs in 10% of women of reproductive age[6]. The frequency of complaints of pain is approximately 10%–15%, and infertility is 50%[7]. Approximately 25%–50% of infertile women suffer from endometriosis, and approximately 30%–50% of women with endometriosis infertility complaints[8]. Hypothalamic-pituitary-ovarian axis dysfunction, in which the follicular phase is prolonged and the luteal phase is defective due to disturbed peak luteinizing hormone (LH) concentrations, causes infertility problems[9]. The fecundity rate, which is the probability of a woman achieving a live birth per month, is reduced by 2%–10% in women with endometriosis, leading to infertility. In comparison, women without endometriosis have a fecundity rate of approximately 15%–20%[7]. The fecundity rate is the probability that a woman will have a live birth per month. The fecundity rate of untreated patients with endometriosis is approximately 2%–10%, which is lower than that of women without endometriosis, whose fecundity rate is approximately 15%–20%[10]. There are three forms of endometriosis: peritoneal, ovarian, and deep infiltration. All three contain glands and stroma, along with nerves, blood vessels, and inflammatory cells, which cause the complaints of pain and infertility in endometriosis[11]. The histopathological diagnosis of endometriosis was strengthened by the identification of ectopic endometrial epithelium and stroma, as well as fibrosis and infiltration of hemosiderin-laden macrophages. Laparoscopic grades of endometriosis include grades I, II, III, and IV according to the Revised American Society for Reproductive Medicine classification system (r-ASRM) criteria[2]. The primary result of this study is the successful establishment of mice as a model for endometriosis. The procedure for obtaining donor tissue was minimally invasive, achieved through cyst puncture from patients diagnosed with chocolate cysts. The brown cyst slurry used as donor tissue for inducing endometriosis in mice was obtained through this puncture procedure. Cyst puncture is a less invasive procedure compared to laparotomy surgery, which involves the removal of endometriosis lesions from the uterine wall of patients diagnosed with the disease. Cyst puncture involves no major surgery but instead uses a needle-like instrument to puncture through the vagina directly to the cyst, allowing the cyst fluid to be aspirated and collected. The advantage of this model lies in the less invasive method of obtaining donor tissue (brown cyst slurry), compared to surgical procedures. This approach was particularly valuable during the COVID-19 pandemic, when major surgical interventions were limited to emergency cases due to the risks to patient safety. Moreover, cyst puncture is simpler, easier, less costly, and quicker to perform than laparotomy. The high success rate of inducing brown cyst slurry into a mouse model of endometriosis contributes significantly to endometriosis research, which remains an active area of study due to the prevalence of endometriosis affecting approximately 10% of women worldwide. Intraperitoneal placement is also easier to perform than intrauterine placement, given that mice are small animals. Achieving accurate injection into the intraperitoneal cavity is simpler and more reliable than targeting the uterine cavity. Table 1 lists the codes of the mice, weight of each mouse, and volume of the stored chocolate cyst slurry injected into each mouse. The volume of chocolate cyst slurry injected intraperitoneally was 0.1 mL per 20 g body weight. PBS was required in a ratio of 1:1 with the chocolate cyst slurry so that as much as 0.2 mL per 20 g body weight of total solution was injected intraperitoneally. Table 1 - Code, the weight of mice, and volume of stored chocolate cyst slurry. Mouse code Weight (g) The volume of stored chocolate cyst slurry A 1 35.5 0.18 2 31.5 0.16 3 30 0.15 4 34 0.17 5 27 0.14 6 29 0.15 B 1 34.5 0.17 2 29 0.15 3 38 0.19 4 39.5 0.20 5 32 0.16 6 41 0.21 Fig. 1A shows the results of the unstained apoptotic cell viability test. The first step was to use unstained annexin V. Furthermore, fluorescence analysis was performed, which showed the extent of cell viability, apoptosis, and necrosis[12]. Apoptosis is well known as the programmed death of eukaryotic cells[13]. The unstained apoptotic cell viability test was used as a control to identify the positive and negative areas. The side scatter vertical line indicates granularity, whereas the forward scatter horizontal line indicates size. Size is shown by light intensity, where cells with higher intensity show larger cell sizes and cells with low intensity indicate smaller cells. This apoptotic cell viability test uses annexin, which is conjugated with fluorescein isothiocyanate as a phosphatidylserine binder on the surface of cells undergoing apoptosis[14,15]. Flow cytometry can be used to differentiate between living cells, early apoptotic cells, late apoptotic, and necrosis[16]. Cells at Q3 represent 98.1% of the total, indicating viable cells, whereas apoptosis was observed in Q4. Cells that occupied Q2 started to die, indicated by a propidium iodide expression of 1.3%. Dead cells (0.5%) occupied Q1, representing 0.5% of the total cell population. Propidium iodide has been used to differentiate between living, apoptotic, and necrotic cells[14]. Fig. 1B shows the viability of cells in the original, unextracted samples. The viability test of apoptotic cells in the original samples that had not been extracted by flow cytometry showed the percentage of cell death by apoptosis or necrosis, and the number of cells still alive[14]. Flow cytometry revealed that the number of living cells in Q3 was 76.7%. The incidence of apoptosis in the Q4 group was 17.1%. Cells that started to die were observed at the Q2 position were as much as 6.0%, and dead cells (0.2%) were observed at the Q1 position. Fig. 1C shows the viability of cells after extraction. Flow cytometry indicated that the number of surviving cells in Q3 was 72.6%. The incidence of apoptosis in the Q4 cells was 24.1%. Cells that started to die occupied Q2 as much as 3.3%, and no completely dead cells occupied Q1 (0.0). The viability test results showed that the cells in the Q3 position remained alive at 76.7% before extraction and 72.6% after extraction. The results also showed >75% viability before extraction and approximately 75% viability after extraction, which indicates that most of the cells were still alive and fit to be induced in experimental animals. Fig. 2 presents the results of anatomical and pathological examinations of the successful induction of stored chocolate cyst slurry in the 12 induced mice. The success of induction can be seen from the reading of the slides, which showed stromatosis in the fat tissue. This aligns with Bulun’s 2019 description of endometriosis[2]. Endometriosis is said to have three clinical forms, namely, 1. endometriotic implants are located on the peritoneal surface of the pelvis and ovaries, or in the subperitoneal fatty tissue, 2. ovarian cysts (endometriomas), and 3. rectovaginal nodules or deep infiltrating endometriosis[2,17]. Primary endometriosis cell cultures can be isolated directly from fresh tissue of donor patients. In addition, many cases involve ovarian cysts[18] and deep infiltrating endometriosis[19]. The gold standard for diagnosing endometriosis is by performing laparoscopy, with direct visualization of endometrial lesions or histological confirmation[20].

All Mus musculus mice treated with stored chocolate cyst slurry showed endometriosis in the form of stromatosis in fat tissue at 40×, 100×, and 200× magnification using an Olympus BX51 microscope. Thus, the induction of stored chocolate cyst slurry succeeded in becoming a model of endometriosis. Although this protocol showed 100% success in creating a mouse endometriosis model, very few experimental animals were used. Therefore, it is necessary to repeat the protocol in subsequent studies so that the success of this protocol for creating a mouse endometriosis model is validated. Acknowledgments None. Author contributions Each author contributed to the research and drafting of the publication manuscript. I.B.B. designed and conducted the research, collected and analyzed the data, and wrote the publication manuscript. S.S. wrote the publication manuscript. R.C. conducted the research, analyzed the data, and wrote the publication manuscript. Funding(s) This research was supported by the LRI UMY, Indonesia (01/RIS-LRI/I/2022, January 18, 2022). Conflicts of interest All authors declare no conflicts of interest. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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