Construction and Evaluation of a Rat Model of Abnormal Uterine Bleeding

doi:10.12300/j.issn.1674-5817.2024.132

Abstract

Abstract:

Objective By simulating the etiology of abnormal uterine bleeding-ovulatory dysfunction (AUB-O) and establishing a rat model of abnormal uterine bleeding (AUB), this study aims to provide an experimental platform for investigating pathological mechanisms and developing therapeutic drugs for AUB. Methods After acclimation, 24 adult (10-week-old) female SD rats were randomly divided into a normal control group (6 rats) and a model group (18 rats). The normal control group was housed in a barrier environment, while the model group underwent bilateral ovariectomy via dorsal approach in the same environment and rested for one week before starting to receive modeling drugs. In the model group, from Days 1 to 3 of modeling, each rat received a daily subcutaneous injection of 0.5 mg estradiol into the dorsal region. From Days 4 to 7, a daily subcutaneous injection of 5.0 mg progesterone was administered. On Day 6, rats received bilateral injections of 0.5 mL soybean oil per uterine cavity (total 1.0 mL) via the same dorsal surgical incision. On Day 8, mifepristone (10 mg/kg) was administered via oral gavage. The estrous cycle stage and its dynamic changes were continuously monitored during modeling. Uterine bleeding was recorded during the 48-hour observation period post-modeling. Serum and uterine tissue samples were collected from the model group at 0, 12, 24, 36, and 48 h after mifepristone administration, while the normal control group was sampled at 36 h. The samples were subjected to HE staining, serum sex hormone ELISA, immunohistochemistry, TUNEL apoptosis staining, Western blotting, transcriptome sequencing, and bioinformatics analysis for comprehensive evaluation of the AUB rat model. Results The AUB rats exhibited uterine bleeding, endometrial detachment and injury, incomplete uterine restoration, inflammatory cell infiltration in the endometrium, enhanced tissue apoptosis, and structural damage of the stroma, glands, and vasculature. Compared with the normal control group, the levels of serum follicle-stimulating hormone (FSH), estradiol, and luteinizing hormone (LH) were significantly increased in the AUB rats (P<0.05). The vascular density of the endometrium was significantly reduced (P<0.05). The expression of vascular endothelial growth factor (VEGF) was qualitatively observed to be markedly enhanced at the site of endometrial detachment but significantly decreased around the stromal blood vessels (P<0.01). Matrix metalloproteinase-9 (MMP-9) expression was qualitatively observed to be strongly upregulated at the site of endometrial injury but significantly reduced in the non-detached stroma and glands (P<0.01). Endometrial stromal cell apoptosis was significantly enhanced (P<0.01). The expression levels of fibroblast growth factor 2 (FGF2) and endothelin-1 (ET-1) in uterine tissues were significantly decreased (P<0.05). After comparing the transcriptome sequencing results of uterine tissues between AUB and normal rats, a total of 4 723 differentially expressed genes were identified, including 2 191 up-regulated genes and 2 532 down-regulated genes. KEGG enrichment analysis revealed that these differentially expressed genes were significantly enriched in pathways related to inflammation, immune apoptosis, cell signal transduction, proliferation and differentiation, and muscle contraction, among others. Conclusion An AUB rat model can be successfully established using a sequential administration protocol of estrogen, progesterone, and mifepristone to simulate the etiology of AUB-O. In this model, endometrial injury is associated with inflammation and apoptosis, with pathological manifestations influenced by abnormal vasoconstriction and impaired endometrial regeneration. This rat model closely recapitulates pathological characteristics of non-structural AUB observed in clinical practice, making it a validated experimental platform for exploring the pathological mechanisms and therapeutic interventions of non-structural AUB.

Key words: Abnormal uterine bleeding, Animal models, Rats, Decidualization, Incomplete restoration

CLC Number:

Q95-33

LIAN Hui,JIANG Yanling,LIU Jia,et al. Construction and Evaluation of a Rat Model of Abnormal Uterine Bleeding[J]. Laboratory Animal and Comparative Medicine, 2025, 45(2): 130-146. DOI: 10.12300/j.issn.1674-5817.2024.132.

Figures/Tables 7

Figure 1 Schematic diagram of estrogen and progesterone level fluctuations during the menstrual cycle and flowchart of the preparation process for a rat model of abnormal uterine bleedingNote： Curves in the figure represent the changes in estrogen and progesterone levels during the normal human menstrual cycle. The process in the figure illustrates the protocol for preparing a rat model of abnormal uterine bleeding.

Figure 2 Detection of changes in the estrous cycle in abnormal uterine bleeding model rats using vaginal exfoliated cell smear （methylene blue staining， ×200）Note： A， Morphological changes of vaginal exfoliated cells of a representative rat in the normal control group during one estrous cycle （Days 1， 2， 3， and 4 correspond to proestrus， estrus， metestrus， and diestrus， respectively， showing cyclical changes）； B， Morphological changes of vaginal exfoliated cells of a representative rat in the model group from ovariectomy， hormone induction to progesterone withdrawal （Days 1， 2， 3， and 4 correspond to diestrus， proestrus， estrus， and estrus， respectively， showing a non-cyclic pattern）. In figures， black arrows （?） indicate nucleated epithelial cells， asterisks （*） mark keratinized epithelial cells， and boxes （□） denote white blood cells. In figures， the scale bar is 50 μm.

Figure 3 Uterine morphology， bleeding patterns， and histopathological changes of endometrial tissues in abnormal uterine bleeding model ratsNote： A， Uterine morphology and the HE-stained images of endometrial tissue in normal control rats； B-F， The uterine morphology in AUB model rats at 0 h， 12 h， 24 h， 36 h， and 48 h after progesterone withdrawal， along with vaginal cotton balls collected for uterine bleeding assessment every 12 hours （lower right corner）， and the HE-stained images of endometrial tissue at the corresponding time points. The two asterisks （*） in each low-magnification HE-stained image indicate the two areas observed under high magnification， and the black arrows （?） point to the structures and pathological phenomena such as the endometrial epithelium， glands， necrotic stroma， blood vessels， and bleeding. In the HE-stained images， the scale bars from left to right column are 500 μm， 50 μm and 50 μm， respectively.

Figure 4 Changes in serum sex hormone levels， injury and repair of uterus blood vessels and stroma， and vascular contractile function of uterus in abnormal uterine bleeding model ratsNote： A， ELISA was used to measure serum levels of sex hormones， including FSH， E2， LH， and PROG， in normal control rats and the AUB model rats at 36 h after progesterone withdrawal （n=6）. B， Immunohistochemistry was performed to assess the expression of vascular endothelial marker platelet endothelial cell adhesion molecule-1 （CD31）， angiogenesis-related protein vascular endothelial growth factor （VEGF）， and stromal injury/repair protein matrix metalloproteinase-9 （MMP-9） in uterine tissues of both groups，along with statistical analysis of the results （n=3； scale bars： 100 μm）. Additionally， TUNEL staining of rat endometrium was conducted， and the percentage of positive cells was quantified ［n=3； asterisks （*） in low-magnification images （scale bars： 100 μm） indicate regions selected for high-magnification observation （scale bars： 25 μm）］. C， Western blotting was employed to detect the expression of fibroblast growth factor 2 （FGF2） and endothelin-1 （ET-1） in uterine tissues of both groups， along with relative quantification （n=3； data presented as mean ± standard error （SE）； Compared with normal control group， nsP＞0.05， *P<0.05， **P<0.01）.

Figure 5 Transcriptomic analysis of differentially expressed genes in uterine tissue of abnormal uterine bleedingmodel ratsNote： A， The number of genes in rat samples from the normal control rats and the AUB model rats at 36 h after progesterone withdrawal （Model-36 h）（compared with normal control group， ****P<0.000 1）； B， Results of principal component analysis； C， Venn diagram showing the differences in expressed genes between the two groups ［The blue area represents the gene set with significantly decreased expression （Down） in the Model-36 h compared to normal control group， while the red area represents the gene set with significantly increased expression （Up） in the Model-36 h compared to normal control group］； D， Volcano plot of differentially expressed genes （down-regulated differentially expressed genes meeting fold change criteria in blue， non-meeting fold change criteria in light blue； up-regulated differentially expressed genes meeting criteria in red， non-meeting criteria in light red； non-significantly expressed genes in gray. The plot labels some of the differentially expressed genes with higher fold changes， including both down-regulated and up-regulated ones）； E， Differentially expressed gene clustering heatmap by group， where the intensity of color represents the value of data points （grouping and clustering are based on similarity， and genes with higher fold changes in up-regulation and down-regulation are labeled in the heatmap）.

Table 1 Basic information of the top 10 up-regulated and the top 10 down-regulated differentially expressed genes in uterine tissues between abnormal uterine bleeding model rats and normal control rats

编号 No.	基因符号 Gene symbol	log₂差异倍数 log₂(fold change)	q值 q-value	功能概述 Summary
Up-regulated genes
1	Scin	4.173	4.546×10^-191	Predicted to enable actin filament binding activity and phosphatidylinositol-4,5-bisphosphate binding activity. etc.
2	Btc	5.961	7.347×10^-158	Enables epidermal growth factor receptor binding activity and growth factor activity. etc.
3	Serpinb11	9.021	1.630×10^-142	Predicted to enable serine-type endopeptidase inhibitor activity. etc.
4	Slc16a12	6.266	9.424×10^-126	Enables creatine transmembrane transporter activity. etc.
5	Tprg1	8.356	2.229×10^-118	Predicted to be active in cytoplasm. etc.
6	Olfm4	8.147	2.346×10^-108	Predicted to enable cadherin binding activity and structural molecule activity. etc.
7	Tnfaip6	6.344	3.099×10^-93	Predicted to enable several functions, including carboxylesterase activity. etc.
8	Nck1	2.451	1.127×10^-84	Predicted to enable several functions, including eukaryotic initiation factor eIF2 binding activity. etc.
9	Actn4	1.188	1.391×10^-83	Enables ubiquitin protein ligase binding activity. etc.
10	Tmbim1	3.027	1.062×10^-82	Predicted to enable calcium channel activity and death receptor binding activity. etc.
Down-regulated genes
1	Gstm5	-4.393	3.077×10^-61	Enables glutathione transferase activity and identical protein binding activity. etc.
2	Armh4	-5.671	4.422×10^-54	Predicted to enable TORC2 complex binding activity. etc.
3	LOC102548820	-7.032	2.774×10^-52	-
4	Arg2	-4.338	1.561×10^-50	Enables arginase activity and nitric-oxide synthase binding activity. etc.
5	Frem2	-6.413	6.963×10^-47	Predicted to be involved in anatomical structure morphogenesis and cell adhesion. etc.
6	Mppe1	-1.346	1.575×10^-44	Predicted to enable GPI anchor binding activity. etc.
7	Lef1	-3.875	3.022×10^-43	Predicted to enable several functions, including DNA binding activity. etc.
8	Smad9	-4.048	8.538×10^-42	Predicted to enable DNA-binding transcription factor activity, RNA polymerase Ⅱ-specific. etc.
9	Miga1	-1.944	4.052×10^-41	Predicted to enable protein heterodimerization activity and protein homodimerization activity. etc.
10	Bmpr1b	-2.515	1.387×10^-38	Predicted to enable several functions, including ATP binding activity. etc.

编号 No.	基因符号 Gene symbol	log₂差异倍数 log₂(fold change)	q值 q-value	功能概述 Summary
Up-regulated genes
1	Scin	4.173	4.546×10^-191	Predicted to enable actin filament binding activity and phosphatidylinositol-4,5-bisphosphate binding activity. etc.
2	Btc	5.961	7.347×10^-158	Enables epidermal growth factor receptor binding activity and growth factor activity. etc.
3	Serpinb11	9.021	1.630×10^-142	Predicted to enable serine-type endopeptidase inhibitor activity. etc.
4	Slc16a12	6.266	9.424×10^-126	Enables creatine transmembrane transporter activity. etc.
5	Tprg1	8.356	2.229×10^-118	Predicted to be active in cytoplasm. etc.
6	Olfm4	8.147	2.346×10^-108	Predicted to enable cadherin binding activity and structural molecule activity. etc.
7	Tnfaip6	6.344	3.099×10^-93	Predicted to enable several functions, including carboxylesterase activity. etc.
8	Nck1	2.451	1.127×10^-84	Predicted to enable several functions, including eukaryotic initiation factor eIF2 binding activity. etc.
9	Actn4	1.188	1.391×10^-83	Enables ubiquitin protein ligase binding activity. etc.
10	Tmbim1	3.027	1.062×10^-82	Predicted to enable calcium channel activity and death receptor binding activity. etc.
Down-regulated genes
1	Gstm5	-4.393	3.077×10^-61	Enables glutathione transferase activity and identical protein binding activity. etc.
2	Armh4	-5.671	4.422×10^-54	Predicted to enable TORC2 complex binding activity. etc.
3	LOC102548820	-7.032	2.774×10^-52	-
4	Arg2	-4.338	1.561×10^-50	Enables arginase activity and nitric-oxide synthase binding activity. etc.
5	Frem2	-6.413	6.963×10^-47	Predicted to be involved in anatomical structure morphogenesis and cell adhesion. etc.
6	Mppe1	-1.346	1.575×10^-44	Predicted to enable GPI anchor binding activity. etc.
7	Lef1	-3.875	3.022×10^-43	Predicted to enable several functions, including DNA binding activity. etc.
8	Smad9	-4.048	8.538×10^-42	Predicted to enable DNA-binding transcription factor activity, RNA polymerase Ⅱ-specific. etc.
9	Miga1	-1.944	4.052×10^-41	Predicted to enable protein heterodimerization activity and protein homodimerization activity. etc.
10	Bmpr1b	-2.515	1.387×10^-38	Predicted to enable several functions, including ATP binding activity. etc.

Figure 6 Transcriptomic analysis of differentially expressed genes in uterine tissue of model rats with abnormal uterine bleedingNote： A， GO functional analysis of up-regulated differentially expressed genes； B， GO functional analysis of down-regulated differentially expressed genes； C， KEGG enrichment analysis of up-regulated differentially expressed genes； D， KEGG enrichment analysis of down-regulated differentially expressed genes.

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