A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
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 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.
Thin endometrium (TE) is a serious condition in obstetrics and gynecology that often affects women of childbearing age. TE is diagnosed when the endometrium thickness measures less than 7 mm on an ultrasound scan, accompanied by a normal uterine cavity, and is closely associated with pregnancy failure1,2. It is estimated that approximately 1.5%-9.1% of women undergoing in vitro fertilization (IVF) treatment will experience TE, making it a growing challenge in reproductive medicine3,4,5. The most common causes of TE include improper endometrium repair following surgical separation of intrauterine adhesions (IUA) and curettage, which are often accompanied by disrupted blood vessel distribution and sparse glands6,7,8. To date, the cellular and molecular mechanisms underlying TE remain unclear. Recovery of the endometrium in TE patients is time-consuming, although estrogen treatment and low-dose aspirin therapy have been explored as potential interventions9. Therefore, an in-depth study of the pathogenesis of TE is the most direct approach to addressing the challenges of this condition. However, studies on the pathogenesis of TE rely on animal models, making the selection of an appropriate model crucial.
Most histopathological studies of thin endometrium (TE) have shown that impaired proliferation of epithelial cells and macrophages, decreased expression of ovarian steroid hormone receptors, excessive deposition of extracellular matrix, and cellular senescence are the most significant pathological features of TE6,9,10. Currently, various rat models have been developed to mimic TE, including models induced by scratching11,12,13, ischemia14, thermal injury15, and chemical injury16,17,18,19. A rat model is induced by scratching the endometrium with a needle or catheter, which often results in intrauterine adhesions (IUA) rather than TE11,12,20,21,22. An ischemia-induced TE model in rats has also been reported, where endometrial ischemia is achieved by performing bilateral uterine artery ligation, leading to reduced endometrial thickness. However, this method is time-consuming, requiring three months, and is not widely used for research14. In the thermal injury-induced model, an artificial insemination tube is used to infuse 85 °C preheated water into one side of the uterine horn through the confluence of the two sides, making it more complex than other methods15. The chemical-induced model involves injecting 95% ethanol into the exposed uterine horn to damage the entire endometrium. This model offers the advantages of low cost and a short experimental period for studying the pathological mechanisms and treatments in TE, but it is primarily used in rats rather than mice16,17,23. Although various animal models exist, especially rat models, each has its limitations. They can only simulate certain aspects of TE, and few mouse models closely replicate the disease characteristics of TE while also being convenient and versatile for research.
In this context, we developed a novel thin endometrium (TE) mouse model by infusing 95% ethanol into the uterine cavity using a time-gradient approach with a modified method24 (Figure 1). The results showed that all the mice successfully developed TE when the infusion time ranged from 1-3 min, displaying typical characteristics such as reduced endometrial thickness, gland reduction, and increased endometrial fibrosis. These findings suggest that our mouse model is a suitable tool for studying TE and can serve as a platform for developing future TE treatments.
This study was approved by the Institutional Animal Care and Use Committee of Shenzhen Zhongshan Obstetrics & Gynecology Hospital (formerly Shenzhen Zhongshan Urology Hospital), where all animal experiments were conducted. Female C57BL/6 mice (age 6-8 weeks, weight 18-20 g) were used in this study. All animals were housed in a specific pathogen-free (SPF) environment in the same room and acclimatized for one week before the experiments in a room without specific pathogens at (22 ± 1) °C under a 12-h light/dark cycle. They were provided with free access to food and water. The details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Identification of estrus
NOTE: The estrus phase in mice is comparable to the late luteal phase in humans, with a relatively thick endometrial lining at this time. Selecting mice in estrus for model induction ensures that they are in a relatively consistent physiological state. Additionally, the results of endometrial thinning are more pronounced when the endometrial thickness is relatively thick. For details on this procedure, refer to previously published reports25,26.
2. Group designing
NOTE: To assess the impact of various durations of 95% ethanol infusion on the stability of the TE model, six treatment groups were established. One group underwent sham surgery without ethanol administration to serve as a control for the procedural effects. Mice with ethanol-induced TE were randomly divided into five treatment groups based on the infusion time (n = 5 per group) as follows:
3. Induction of TE model in mice
4. Sample collection
5. Tissue dehydration
NOTE: Tissue dehydration is achieved using an automatic tissue processor (see Table of Materials).
6. Paraffin embedding
NOTE: Perform these steps using a modular tissue embedding center (see Table of Materials).
7. Paraffin sectioning
8. Hematoxylin and eosin staining
NOTE: Hematoxylin and Eosin staining is performed using an automated slide stainer machine (see Table of Materials).
9. Masson staining
10. Imaging and analysis
11. Statistical analyses
The key features of thin endometrium (TE) are decreased endometrial thickness and glandular density, along with increased endometrial fibrosis. This method successfully replicated these characteristics in the model mice. Data analysis revealed a significant decrease in endometrial thickness in the 1-min group (222.3 µm ± 13.96 µm vs. 359.2 µm ± 12.41 µm, P < 0.05), the 2-min group (168.7 µm ± 17.57 µm vs. 359.2 µm ± 12.41 µm, P...
TE is characterized by insufficient cell proliferation and dysfunctional cells, closely linked to infertility, recurrent miscarriage, and placental abnormalities2,3. Unfortunately, there is currently no effective therapy for TE. Animal models play a crucial role in studying this condition. Between 2014 and 2024, rats were used as model organisms in 16.4% of 208,000 studies (34,200 studies) and mice in 22.7% (47,300 studies). The increasing use of mice in experime...
The authors have nothing to disclose.
We gratefully thank the anonymous referees for their important and helpful comments. This work was supported by the Shenzhen Science and Technology Project (No. JCYJ20220818103207016) and the Guangdong Basic and Applied Basic Research Foundation (No. 2024A1515010478).
Name | Company | Catalog Number | Comments |
Anesthesia Machine | RWD Life Science | R530 | Mobile inhalation anesthesia machine for small animals |
0.9% saline | Hubei Kelun Pharmaceutical | C230817A2 | 10 mL, medical injection |
75% ethanol | LIRCON | 6303060031 | 500 mL, disinfectant reagent |
95% ethanol | Guangzhou Chemical Reagent Factory | 64-17-5 | 500 mL, chemical reagent |
Absorbable sutures | Jinhuan Medical | CR537 | Thickness: 5-0; Length: 90 cm |
Aqueous ammonia | MilliporeSigma | 1336-21-6 | 1000 mL, chemical reagent |
Automatic Tissue Processor | Leica | TP1020 | 100 embedding boxes can be processed at one time |
C57BL/6 mice | Experimental Animal Center of Southern Medical University | ||
Eosin-Y | BASO | BA4024 | 1000 mL, used for the staining of paraffin sections, frozen sections, etc |
Haematoxylin | BASO | BA4041 | 1000 mL, used for the staining of paraffin sections, frozen sections, etc |
HALO Image Analysis Platform | Indica labs | The instrument features ease-of-use and scalability, powerful analytical capabilities, and the fastest processing speed | |
Hemostatic clamps | HUAYON | 18-5021 | 1.8 cm in total length with 0.7 cm jaw |
Hemostatic forceps | HUAYON | 18-5020 | 10 cm in total length |
HistoCore Rotary Microtome | Leica | 149BIO000C1 | Slice thickness ranges from 1 to 60 μm |
Indorphor | ADF | 1005 | 500 mL, disinfectant reagent |
Isoflurane | RWD Life Science | R510-22-10 | 100 mL, active ingredient 100% isoflurane |
Masson's trichrome staining | SOLARBIO | G1340 | 7 × 100 mL for 100 tests |
Microscope slide | Gene Tech | GT100511 | Length: 75 cm; Width: 25 cm |
Modular Tissue Embedding Center | Leica | EG1150 C | The instrument contains a cold stage and a heated paraffin distribution module, providing flexibility for the embedding work |
Natural resin | SAKURA | 4770 | Resin-coated film, Suitable for histology staining |
Olympus SLIDEVIEW VS200 | PANOVUE | VS200 | The instrument captures high-quality virtual slide images and enables advanced quantitative image analysis |
Paraformaldehyde fix solution | Servicebio | G1011 | 500 mL, universal tissue fixative (neutral) |
Surgical forceps | HUAYON | 18-1300 | 2.2 mm straight, 12.5 cm wide |
Surgical scissors | HUAYON | 18-0110 | 10 cm, stainless steel surgical scissors |
Syringe | Kindly | 60017031 | 1 mL, disposable sterile syringe with needle |
Tissue cassettes | CITOTEST | 80106-1100-16 | White; flow-through slots; 0ne-piece integral lid; labeling areas are located on three sides |
Tissue-Tek Prisma Plus | SAKURA | DRS-Prisma-P-JCS | The processing capacity is 60 slides at one time |
Xylene | Guangdong Guanghua Sci-Tech | 1330-20-7 | 1000 mL, organic solvent |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved