The authors wish to acknowledge support from the National Nature Science Foundation of China (82001629, XQS), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116, XQS), and Jiangsu Province Postdoctoral Research Funding (2021K277B, XQS).

All of the animal experiments described were approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School&#39;s Committee on the Use and Care of Animals (No. 20171202). All operations follow appropriate animal care and use agency and national guidelines.
NOTE: Mice were raised in a specific pathogen-free (SPF) environment, with a temperature of 22 &#176;C &#177; 1 &#176;C, relative humidity of 50% &#177; 1%, a light/dark cycle of 12 h/12 h, and free access to food and water.
1. Mouse ovariectomy
<ol>
	<li>Sterilize the surgical instruments with a high-temperature and high-pressure sterilizer 1 day before the operation.</li>
	<li>Weigh 8-week-old C57BL/6 mice and intraperitoneally (i.p)&#160;inject 1% sodium pentobarbital accordingly to anesthetize the mice. The dose used is 40 mg/kg17. This dose provides 40-60 minutes of sedation, which meets the requirements of an ovariectomy. For pre-operative analgesia, inject mice with 5 mg/kg meloxicam (s.c). 
	NOTE: Successful anesthesia in mice can be indicated by the disappearance of deep muscle reflexes and steady breathing. Signs of successful anesthesia include no blinking when touching the inner canthus, no swallowing when pulling the tongue, no leg bending when pinching the skin between the toes, and no bouncing when needling the tail.</li>
	<li>Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia.</li>
	<li>Start the operation when mice are completely anesthetized. Place and fix the mice in the prone position on the operating surface, and shave the hair on the back after wetting them with soapy water. Disinfect the skin with 70% ethanol after shaving.</li>
	<li>Find the operation incision site at 0.5 cm (or 1.0 cm below the rib) on the upper edge of the thigh root of both hind limbs parallel to the spine (Figure 1A). 
	NOTE: A correct incision location is essential for quickly localizing the ovary.</li>
	<li>Take the incision site as the center and disinfect the incision area with iodophor 3x, then place a surgical drape around the incision area.</li>
	<li>Make a longitudinal incision of about 0.5-1.0 cm using scissors. Cut the fascia and passively separate the muscles with tweezers.</li>
	<li>Find a piece of the white fat pad in the incision field close to the lower pole of the kidney through the thin muscle layer (Figure 1B). 
	NOTE: The white fat pad is the most obvious indicator of the location of the ovary.</li>
	<li>Clamp the fat mass with hemostatic clips, and pull the ovary wrapped by the fat pad out of the incision.</li>
	<li>Clamp the junction of the ovary and fallopian tube with curved tweezers, and remove the ovary along the ovarian pedicle with scissors. Stop the bleeding using an electrocoagulation pen or a needle of a 1 mL syringe heated on the alcohol lamp. 
	NOTE: Be sure to preserve the fallopian tube, which is the anatomical landmark for the subsequent injection of sesame oil into the uterine horns.</li>
	<li>Rinse the surgical incision with normal saline to prevent tissue adhesion, use a 4-0 suture to sew the muscle and skin respectively, and disinfect the surgical incision with iodophor again.</li>
	<li>Place the mice in the prone position in the cage and resuscitate them in a 25 &#176;C incubator equipped with a light source and a ventilation system for about 2 h.</li>
	<li>Pay attention to the mice after the operation, put them back in the original feeding place alone until they recover completely, and give them enough water and food.</li>
</ol>
2. Postoperative rest and estrogen and progesterone formulation
<ol>
	<li>Make sure the mice rest for 2 weeks after ovariectomy.</li>
	<li>In an ultra-clean cabinet, add estrogen (2 ng/&#181;L) and progesterone (0.2 ng/&#181;L) into sesame oil in RNA enzyme-free centrifuge tubes.</li>
	<li>Place the centrifuge tubes in a thermostatic water bath at 37 &#176;C and shake the tubes continuously to accelerate dissolution.</li>
	<li>After complete dissolution, divide the sesame oil solution into 100 &#181;L aliqouts for convenient use. Store the prepared estrogen and progesterone solutions in a refrigerator at 4 &#176;C.</li>
</ol>
3. Induced artificial decidualization model
<ol>
	<li>Subcutaneously (s.c) inject 100 ng of estrogen&#160;in 50 &#181;L of sesame oil into each mouse for 3 days, followed by 2 days of rest15. 
	NOTE: Ensure that the estrogen and progesterone have been formulated and placed in different places. Any contamination of estrogen by progesterone&#160;may lead to failure.</li>
	<li>Inject (s.c.) 1 mg of progesterone and 10 ng of estrogen&#160;in 50 &#181;L of sesame oil into each mouse for another 3 days15.</li>
	<li>Operate the mice to induce the artificial decidualization model15 6 h after the third estrogen-progesterone combined injection.</li>
	<li>Find the uterus horn at the lower end of the fallopian tubes and inject 20 &#181;L of sesame oil slowly along with the uterine horn, push the tissue back, suture the incision, and allow the mouse to recover. The approach to operation is the same as the mouse ovariectomy15.&#160; 
	NOTE: Only proceed with the second surgery in the event of a successful ovariectomy (skin incision is well healed, the ovary is completely excised, and the residual end of the fallopian tube is not adhered to the surrounding tissues). 
	NOTE: For inducing the AD model, 20 &#181;L of sesame oil is appropriate; more than 20 &#181;L of sesame oil will easily penetrate the other side.</li>
	<li>Inject (s.c.) 50 &#181;L of sesame oil containing 1 mg of progesterone and 10 ng of estrogen (H.) for another 4 days. See details in Figure 215,18.</li>
</ol>
4. Sample collection
<ol>
	<li>Euthanize the mice (n = 6) by carbon dioxide asphyxiation and collect the uteri. Observe the shape of the bilateral uteri, take photos, and weigh the uterine horns (Figure 3A, B).</li>
	<li>Fix 3-5 mm of the uterus tissue with 10% formalin, embed in paraffin, and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes19 (Figure 4A).</li>
	<li>Embed another 3-5 mm of the uterus tissue in optimal cutting temperature compound (OCT), freeze in liquid nitrogen, and store in a &#8722;80 &#176;C refrigerator. Frozen section the tissue to 10 &#181;m and detect alkaline phosphatase activity in the uterine tissue by the azo coupling method20 (Figure 4B). 
	NOTE: Satisfactory results can be obtained using frozen sections to detect ALP content, not paraffin sections.</li>
	<li>Extract the total RNAs of the uterus with Trizol reagent and perform quantitative real-time PCR (qPCR) on a real-time PCR system (Table of Materials) with qPCR master mix (Table of Materials) to detect the relative mRNA expression of prolactin family 8 subfamily member 2 (Prl8a2), alkaline phosphatase liver/bone/kidney (Alpl), and insulin-like growth factor-binding protein 1 (Igfbp1)15 by normalization to 18S mRNA levels (Figure 4C-E). Follow the PCR conditions: Stage 1, 95 &#176;C for 30 s; Stage 2, 95 &#176;C for 10 s and 60 &#176;C for 30 s; repeat the cycle 40x. A complete list of primer sequences is provided in Table 1.</li>
	<li>Analyze qPCR data using the 2-&#916;&#916;CT method and a t-test method for statistical analysis in the appropriate software. In this study, p &#60; 0.05 was considered statistically significant.</li>
</ol>

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The authors have nothing to disclose.

Decidualization in mice is a spontaneous process depending on the presence of embryos, which is different from humans. However, it has been found that artificial stimulation such as uterine injection of glass beads and uterine laceration can induce decidualization of the endometrium instead of embryos. In addition, researchers found that many factors could induce decidual or participate in decidualization, such as the injection of steroid hormones, prostaglandins, and growth inhibitory factors into the uterine cavity<sup...

With the development of human-assisted reproductive technology, the current clinical pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) has reached or even exceeded that of natural pregnancy. Despite this, many patients in assisted reproduction clinical practice still undergo multiple embryo transfers but fail to achieve pregnancy as desired. However, its specific molecular mechanism is still unclear, so clinical intervention is ineffective, which is one of the significant challenges facing reproductive medicine1,2.
Endometrial factors account for about two-thirds of the causes of IVF failure3. Human embryo implantation is divided into three stages: positioning, adhesion, and invasion4,5,6. The maternal endometrium undergoes a series of changes to meet the arrival of the embryo. Forming an implantation &#34;window period&#34; provides favorable conditions for embryo implantation7,8.
In most mammals, after the blastocyst adheres to the luminal epithelium of the uterus, the stromal cells surrounding the blastocyst rapidly begin to proliferate and differentiate, and the rapid remodeling of the mesenchyme changes its shape and function, leading to embryo implantation5,9,10. The rapid increase in site volume and weight allows the blastocyst to become embedded in the uterine stroma, a process known as decidualization11. The endometrial stroma differentiates and remodels in preparation for pregnancy, while the transition of stromal cells provides space and new signaling connections for decidual cells to perform their functions12,13. Stromal cells transform into decidual cells and secrete many iconic factors such as prolactin (PRL), insulin-like growth factor-binding protein 1 (Igfbp1), and so on. Studies have shown that abnormal decidualization is one of the key reasons for embryo implantation failure, but the cause of abnormal decidualization is still unclear and needs to be further elucidated1,14.
The mouse artificial decidualization model is essential for studying the physiological process and molecular mechanisms underlying decidualization. Artificial decidualization (AD) mainly refers to the process of endometrial decidualization established by artificial methods to simulate pregnancy or the menstrual cycle. In terms of morphology, there is little overall difference between pregnancy decidualization and artificial decidualization15,16. The uterine glands exist in the endometrium before decidua form and disappear after decidualization. Regarding the gene expression, only a slight difference is identified between natural pregnancy decidualization (NPD) and AD15. Consequently, the artificial decidualization model in mice can simulate pregnancy decidualization to explore the unknown pathogenesis and new treatment of human reproductive diseases.
NPD, AD, and in vitro decidualization (IVD) are three methods to achieve mouse decidualization. The NPD model depends on natural pregnancy and is closest to the maternal physiological state, including the effects from embryos. Comparing the differences between implantation and non-implantation sites is a more physiological and convenient approach for studying decidualization. The AD model was developed by using an intrauterine injection of sesame oil as a stimulant to induce decidualization in a pseudopregnant female mouse mated with vasectomized males to avoid the impact from embryos. Both NPD and AD models play essential roles in different research purposes, but they cannot avoid mating failure and within-group differences caused by the different activities of maternal hormone metabolism. IVD is a method depending on the treatment of combined estrogen and progesterone at the cellular level, which requires more stringent experimental conditions and operating ability. However, the in vitro model cannot fully simulate the decidual response under physiological conditions15. Therefore, we propose a simple and improved induction method modified from traditional AD to reduce the effect of endogenous hormones on decidualization. Based on ensuring the success of decidualization induction, it is closer to the physiological state and more suitable for experiments that need to exclude embryo factors.

<table><tbody><tr><td>Estrogen</td><td>Sigma</td><td>E2758</td><td>Hormone supplement</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P0130</td><td>Hormone supplement</td></tr><tr><td>Sesame oil&amp;nbsp;</td><td>Sigma</td><td>S3547</td><td>Hormone supplement</td></tr><tr><td>Sodium pentobarbital&amp;nbsp;</td><td>Dainippon Sumitomo Pharma Co.,Ltd.</td><td></td><td>Anaesthesia</td></tr><tr><td>Meloxicam injection</td><td>Qilu Animal Health Products Co., Ltd</td><td></td><td>Analgesia</td></tr><tr><td>Alkaline phophatase stain kit(kaplow&#039;s/azo coupling method)</td><td>Solarbio</td><td>G1480</td><td>Alkaline phophatase stain</td></tr><tr><td>Eosin</td><td>Servicebio</td><td>G1005-2</td><td>HE stain</td></tr><tr><td>Hematoxylin</td><td>Servicebio</td><td>G1005-1</td><td>HE stain</td></tr><tr><td>ChamQ Universal SYBR qPCR Master Mix</td><td>Vazyme</td><td>Q711-02</td><td>qPCR</td></tr><tr><td>70% ethanol</td><td>Lircon</td><td>ZH1120090</td><td>Disinfect</td></tr><tr><td>Iodophor</td><td>Runzekang</td><td>RZK-DF</td><td>Disinfect</td></tr><tr><td>Erythromycin Eye Ointment</td><td>Guangzhou Baiyunshan</td><td></td><td>Mice eyeball protect</td></tr><tr><td>4-0 suture</td><td>Ethicon</td><td>W329</td><td>Incision suture</td></tr><tr><td>10% formalin</td><td>Yulu</td><td>L25010118</td><td>Tissue fix</td></tr><tr><td>Optimal cutting temperature compound</td><td>Sakura</td><td>4583</td><td>Ssection</td></tr><tr><td>Trizol reagent</td><td>Ambion</td><td>15596018</td><td>qPCR</td></tr></tbody></table>

generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

The mouse decidualization model indexes include the uterus&#39;s general morphology, the mass ratio of the decidualized and non-decidualized uterus, the histological morphology of the endometrium, and the expression level of decidualization marker molecules. The general morphology of the artificial decidualized uterus of mice induced by oil is closer to that of the uterus in pregnancy. The uterine body becomes thick, and the uterine cavity becomes smaller than the non-induced side. The volume and weight of the induced ut...

Watch this Scientific Journal Video about Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research at JoVE.com

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

generation-mouse-artificial-decidualization-model-with-ovariectomy

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

4.2K Views.  The Affiliated Drum Tower Hospital of Nanjing University Medical School. Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Video: Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Medicine

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Biology

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

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Chapters in this video

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Establishing 3D Endometrial Organoids from the Mouse Uterus

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

Establishing a Mouse Model of Thin Endometrium