Expressions of IGF-1, pAKT, and pS6 in Uterosacral Ligaments of Patients with Pelvic Organ Prolapse

Abstract

Introduction and Hypothesis

The objective was to investigate the expressions and significance of IGF-1, pAKT, pS6, and type I and type III collagens in uterosacral ligaments of patients with pelvic organ prolapse (POP).

Methods

A total of 38 patients with POP who underwent hysterectomy or pelvic floor reconstruction were selected as the POP group. Another 38 patients who underwent hysterectomy owing to nonpelvic floor dysfunction were selected as the control group. The POP quantitative staging scores of the two groups were III–IV and 0–I respectively. The histological manifestations of the abandoned uterosacral ligament tissues near the cervix were evaluated using hematoxylin–eosin staining and Masson staining. The expressions of IGF-1, plGF-1R, pAKT, pS6, and type I and type III collagen fibers were detected using immunohistochemistry and Western blotting.

Results

Hematoxylin–eosin and Masson staining showed that the number of POP constituent fibrocytes decreased, the arrangement was disordered, and the distribution of collagen fibers was sparse compared with the control group. Immunohistochemical analysis showed that the expressions of IGF-1, pIGF-1R, pAKT , pS6, and type I and type III collagen in uterosacral ligament tissues of the POP group significantly decreased. Western blotting demonstrated that the expressions of IGF-1, pIGF-1R, pAKT, pS6, and type I and type III collagen in the POP group significantly decreased.

Conclusion

The expressions of IGF-1, pAKT, pS6, and type I and type III collagen decreased in the uterosacral ligament tissues of POP patients, and the IGF-1/AKT/mTORC1 signaling pathway regulated by IGF-1 may be related to the occurrence of POP.

Introduction

Pelvic organ prolapse (POP) in or outside the vagina is mainly caused by the dysfunction or structural damage of the female pelvic support structure, including the pelvic floor muscle group and fibrous connective tissue. In recent years, the incidence of POP has been increasing year by year [1]. The prevalence of symptomatic POP falls within the range 3% to 6%, whereas anatomical prolapse is present in as many as 50% of women [2]. Collagens, as critical components of the extracellular matrix (ECM) structural proteins, the main types present in pelvic connective tissues include type I and type III collagens, which are considered the main determinants of soft-tissue strength. Type I collagen fibers are widely present, flexible, and can resist tension well. Type III collagen is dominant in tissues that require greater flexibility, expandability, and are subject to periodic stress [3]. Studies have demonstrated that abnormal collagen metabolism, characterized by altered collagen I/III ratios, is associated with weakened pelvic floor tissues in patients with POP [4–6]. The uterosacral ligaments near the cervix mainly consist of smooth muscle cells, fibroblasts and their ECM, nerves, blood vessels, and lymphoid tissues [7].

Insulin—like growth factor-1 (IGF-1) is an important endocrine growth factor in the human body. It has a wide range of biological functions and can promote cell growth, differentiation, wound repair, tissue fibrosis, and proliferation [8]. After IGF-1 activates IGF-1R on the cell membrane, it can further activate the downstream mTORC1 signaling pathway, promote the synthesis of intracellular proteins, and increase the number of collagen fibers [9]. In recent years, the role of IGF-1 in promoting cellular protein synthesis has attracted growing attention from scholars. IGF-1 is reportedly pivotal in promoting biological processes such as muscle fibrosis and bone healing [10–12]. The IGF-1/AKT/mTORC1 signaling pathway plays an important role in the function of fibroblasts, and the inhibition of this pathway affects the proliferation and migration of fibroblasts [13, 14]. IGF-1 activates the AKT/mTORC1 pathway, which in turn promotes collagen synthesis in fibroblasts. This mechanism is essential for tissue fibrosis and the repair process [15]. IGF-1, pAKT, and pS6 are important components of this pathway.

However, whether this pathway regulated by IGF-1 is related to the collagen metabolism of uterosacral ligament fibroblasts in POP has not been reported. Herein, we aimed to examine the expressions of IGF-1, pAKT, pS6, ColI, and ColIII in the uterosacral ligament tissues of POP patients, and preliminarily explore the association between the IGF-1/AKT/mTORC1 pathway and the occurrence and development of POP.

Materials and Methods

Patient Selection

A total of 38 patients undergoing hysterectomy or pelvic floor reconstruction owing to POP in Anqing Municipal Hospital between July 2023 and February 2024 were selected (POP group). The POP quantitation (POP—Q) scores were III—IV. Another 38 patients who underwent hysterectomy owing to non—POP gynecological benign tumors were selected as the control group. Their POP—Q scores were 0—I.

Sample Size Section Basis

A pilot study (N = 10 per group) indicated a large effect size (Cohen's d = 1.3) for IGF-1 expression. Using G*Power 3.1 (α = 0.05, power = 80%) a minimum of 34 subjects per group were required. To account for potential technical variability, we enrolled 38 subjects per group, aligning with methodologies from prior POP biomarker studies [16].

Clinical data of age, body mass index (BMI), gestational number, number of vaginal deliveries, and POP—Q score were collected in both groups.

Exclusion Criteria

1. Endometriosis

2. A history of hormone replacement therapy in the past 3 months

3. Comorbid with immune system disease or connective tissue disease

4. Comorbid with a malignant tumor

5. Comorbid with urinary and bowel incontinence

Determination Method of Endometriosis

Endometriosis was comprehensively judged through patient history inquiry, gynecological examination, ultrasound examination, and MRI examination when necessary. For suspected cases, relevant indicators such as serum CA125 were also used for auxiliary diagnosis.

Reasons for Excluding Incontinence

Urinary incontinence may affect the mechanical environment and metabolic state of the pelvic floor tissues, interfering with the study of factors related to POP. To more accurately explore the relationship between the expression of IGF-1, pAkT, pS6, and type I and type III collagens in the uterosacral ligament tissues patients with POP and POP itself, we excluded patients with urinary incontinence to reduce the interference of confounding factors.

Specimen Selection and Treatment

Both the patients with POP and the control patients were operated on by the same senior gynecological expert. During each operation, the tissue was held with toothless forceps, and a part of the tissue was cut using scissors or blades while keeping the uterosacral ligaments tension free. The sampling site was selected from the uterosacral ligament 0.5–1.0 cm from the cervix, and about 0.5 cm × 0.5 cm × 0.5 cm of the isolated uterosacral ligament was collected. Two samples were taken from each patient. One sample was stored in a neutral fixed solution with 10% volume fraction. The other sample was washed with normal saline on the tissue surface to coagulate the blood, put into a frozen tube, and stored in the refrigerator at −80 °C as soon as possible. During the sampling process, attention should be paid to protecting the specimen and reducing the pulling and squeezing of the tissue, so as not to affect the subsequent experimental operation.

Hematoxylin–Eosin and Masson Staining

The uterosacral ligament tissues stored in the fixed solution were dewatered and embedded. Then, tissue slices 4 μm thick were prepared using a microtome, and stained with HE and Masson to observe histology and collagen fiber deposition respectively.

Immunohistochemical

Immunohistochemical (IHC) sections of IGF-1, plGF-1R, pAKT, pS6, ColI, and ColIII expressions in uterosacral ligament tissues were treated with HE staining, and a sodium citrate antigen repair solution (0.01 mol/l, pH 6) was applied after the patch. For antigen repair, a goat serum working fluid was used to block the nonspecific antibody, and primary antibodies were added in the corresponding proportion: anti-IGF-1 (1:50, sc-74116; Santa Cruz), pS6 antibody (1:50, ab225676; Abcam,) and pAKT (1:100, 4060 T; CST), pIGF-1R (1:100, PA5-104774; Invitrogen), ColI (1:100, bs-10423R; Bioss), and ColIII antibody (1:100, bs-0948R; Bioss). Then the secondary antibody was added for DAB color development. The nucleus is blue under microscopy, and the target protein is brown. The average optical density of each protein staining region was measured on IPP 6.0.

Western Blotting Assay

Western blot of the expressions of IGF-1, IGF-1R, plGF-1R, AKT, pAKT, S6, pS6, ColI, and ColIII were detected in the uterosacral ligament tissues stored in the refrigerator at −80 °C, and proteins were extracted. After electrophoresis with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyvinylidene difluoride (PVDF) membranes were added to primary antibodies: anti-IGF-1 antibody (1:2000, SC-74116; Santacruz), anti-IGF-1R antibody (1:1000, AB182408; Abcam), anti-PIGF-1R antibody (1:1000, PA5-104774; Invitrogen), anti-S6 antibody (1:1000, AB225676; Abcam), anti-PS6 (1:500, AB215214; Abcam), anti-Akt antibody (1:5000, A18675; ABclonal), anti-PAKt (1:2000, 4060 T; CST), anti-ColI, anti-ColIII antibodies (1:1000, bs-10423r and bs-0948r; Bioss), and anti-GAPDH antibody (1:30 000, 60,004–1 1-IG; Three Eagle). Then, the second antibody was added for incubation and exposure development. The gray value of each strip was measured on Image J 1.52. The results were expressed as the gray value ratio of the target protein to the gray value of GAPDH (internal reference), and the gray value of the film was analyzed with Image-Pro Plus.

Statistical Processing

All data were expressed as mean ± standard deviation, and statistical analysis was performed on GraphPad Prism 6 (La Jolla, CA, USA). Age, body mass index (BMI), number of pregnancies, and number of vaginal deliveries were compared between the two groups using two-independent-sample t test. The IHC data were evaluated using the Mann–Whitney U test. Western blot data were compared using unpaired t test, Welch t test, and Pearson test. The test level was α = 0.05.

Results

Baseline Characteristics

The comparison of general clinical data showed no significant differences in age, BMI, pregnancy times, or vaginal delivery times between the two groups (p > 0.05) (Table 1). To ensure the comparability of the two groups, the control group was matched with the POP group according to key characteristics such as age and BMI.

Table 1.

Comparison of general clinical data between the two groups

	Control group (n = 38)	Pelvic organ prolapse group (n = 38)	t	p
Age (years)	58.95 ± 11.32	60.53 ± 11.38	0.5920	0.5558
BMI (kg/m2)	23.78 ± 2.11	23.08 ± 4.06	0.9105	0.3667
Gravidity	3.33 ± 0.79	3.31 ± 0.52	0.1753	0.8614
Parity	2.5 ± 0.81	2.69 ± 0.71	1.083	0.2827

Mean ± SD of patient characteristics

No significant differences between the two groups were observed

Hematoxylin–Eosin and Masson Staining

Hematoxylin–eosin staining results are shown in Fig. 1. In the control group, fibroblasts were arranged regularly under microscopy, and ECMs (collagen fibers, elastin, etc.) were evenly distributed among the cells. The fibroblasts of the uterosacral ligament in the POP group were disordered under microscopy, the cell number was significantly lower than in the control group, and the ECMs were sparsely distributed and unevenly colored. The results of Masson staining are shown in Fig. 2. The collagen fibers are blue, and the muscle fibers are red. The tissue staining of the uterosacral ligament in the control group was dense and dark blue with a regular arrangement of cells. However, the tissue staining of uterosacral ligaments in the POP group was obviously lighter than that in the control group, and there were more blank spaces interlaced with a few cells, which showed a disordered structure.

Fig. 1.

Hematoxylin–eosin (HE) staining results of the uterosacral ligaments in the two groups. A For the control group (× 100), B for the POP group (× 100), C for the Control group (× 200), and D for the pelvic organ prolapse (POP) group (× 200). Collagen fibers after HE staining are pink. In the control group, fibroblasts were arranged regularly, and the extracellular matrix (ECM; collagen fibers, elastin, etc.) was evenly distributed among the cells. In the POP group, the fibroblasts were disordered, the cell number was significantly lower than in the control group, and the ECM (collagen fibers, elastin, etc.) was sparsely distributed and unevenly colored

Fig. 2.

Masson trichrome staining results of uterosacral ligaments in the two groups. A For the Control group (× 100), B for the POP group (× 100), C for the Control group (× 200), and D for the POP group (× 200). Collagen fibers are blue and muscle fibers are red. In the control group, the uterine and sacral ligaments after staining were dense and dark blue with a regular arrangement of cells. In the POP group, the staining intensity of the uterosacral ligaments tissue was significantly lighter than that of the Control group, there were more blank spaces with a few cells interlaced, and the structure was disordered

Immunohistochemical Analysis

Immunohistochemical assay revealed the expression of IGF-1, plGF-1R, pAKT, pS6, ColI fibers, and ColIII fibers in the uterosacral ligament tissue of subjects in the POP and control groups. Expression levels of IGF-1 (median = 0.0021 vs 0.0137, p < 0.01), pIGF-1R (median = 0.0080 vs 0.0265, p < 0.01), pAKT (median = 0.0018 vs 0.0170, p < 0.01; Fig. 3), pS6 (median = 0.0007 vs 0.0062, p < 0.01), ColI fibers (median = 0.0236 vs 0.0468, p < 0.01), and ColIII fibers (median = 0.0088 vs 0.0325, p < 0.01) were significantly lower in the POP group than in the control group (Fig. 4).

Fig. 3.

Immunohistochemistry analysis of IGF-1, pIGF-1R,and pAKT expressions in the uterosacral ligament tissues of women with or without pelvic organ prolapse (POP; × 400). Sections of the uterosacral ligament tissues were stained with antibodies against IGF-1, pIGF-1R, and pAKT. The percentages of positively stained areas were quantified using Image J, and integrated optical density (IOD)/Area for quantitative analysis (Mann–Whitney U test). ****p < 0.0001

Fig. 4.

Immunohistochemistry analysis of pS6, ColI, and ColIII expression in the uterosacral ligament tissues of women with or without POP (× 400). Sections of the uterosacral ligament tissues were stained with antibodies against pS6, ColI, and Col III. The percentages of positively stained areas were quantified using Image J, and integrated optical density (IOD)/Area for quantitative analysis (Mann–Whitney U test). ****p < 0.0001, ***p < 0.001

Western Blot Analysis

Western blot results verified that the levels of ColI fibers (0.484 ± 0.082 vs 0.826 ± 0.095, p < 0.01), ColIII fibers (0.406 ± 0.144 vs 0.669 ± 0.076, p < 0.05) in the POP group were substantially decreased compared with those in the control group. Compared with the control group, the expression levels of IGF-1, plGF-1R, pAKT, and pS6 were significantly down-regulated in the POP group (0.265 ± 0.129 vs 0.679 ± 0.039, p < 0.01, 0.073 ± 0.027 vs 0.192 ± 0.048, p < 0.05, 0.108 ± 0.036 vs 0.338 ± 0.020, p < 0.01, 0.084 ± 0.013 vs 0.148 ± 0.026, p < 0.05) (Fig. 5). These Western blot data agreed with IHC analysis performed on samples for the same markers (Figs. 3, 4).

Fig. 5.

a Western blot of IGF-1, IGF-1R, plGF-1R, AKT, pAKT, S6, pS6, ColI, and ColIII in control and POP groups. b Relative intensity (unpaired t test or Welch t test). *p < 0.05, **p < 0.01

Discussion

Our main results demonstrated that the expression levels of IGF-1, pAKT, pS6, ColI, and ColIII in the uterosacral ligament tissues of patients with POP were significantly lower than those in patients without POP. Also, we observed evident structural changes in fibrous connective tissues and a reduction in collagen fiber distribution. These findings strongly support our initial hypothesis that IGF-1 might be involved in the pathological process of POP by activating collagen fiber generation through the mTORC1 signaling pathway.

The significant decrease in the expression of IGF-1 in patients with POP is likely to disrupt the normal activation of the IGF—1/AKT/mTORC1 signaling pathway. As IGF-1 is an important growth factor, the activated IGF-1 receptor (IGF-1R) on the cell membrane further activates the corresponding signaling pathway in the downstream cytoplasm, and ultimately stimulates the signal transmission to the nucleus, regulating the biological processes such as gene transcription and protein translation. It plays a crucial role in the fibrosis process of many tissues [15, 17, 18]. In our study, the reduced expression of IGF-1 may lead to a weakened activation of this pathway, resulting in a decrease in the synthesis of ColI and ColIII. As collagen fibers are essential for maintaining the integrity and toughness of pelvic floor connective tissues, The decreased collagen content in patients with POP may be associated with the weakening of the pelvic floor support structure, which could play a role in the occurrence and progression of POP (Fig. 6).

Fig. 6.

The IGF-1—mediated IGF-1/AKT/mTORC1 signaling pathway may be related to the pathological process of pelvic organ prolapse

Our results are consistent with those of previous studies that reported lower expression levels of IGF-1, ColI, and ColIII in the vaginal wall fibroblasts of patients with POP. For example, Yin et al. found that the expression levels of these biomarkers in vaginal wall fibroblasts of patients with POP were significantly lower than those in the normal group, and low-level IGF-1 reduced ColI expression by regulating the AKT/mTOR/p70S6 K pathway [19, 20]. However, compared with other studies, our research has some differences. For instance, Kerkhof et al. [21] reported that there was no significant difference in the expression of ColI and ColIII in the anterior vaginal wall tissues of patients with POP (POP—Q II—III). However, Sun et al. [22] reported that within the uterosacral ligament tissues of patients with POP in the advanced stages (POP-Q II–III), the expression level of COL1a1 remained unchanged, whereas that of COL3a1 was elevated. Additionally, Yucel et al. [23] found that compared with subjects without POP, patients with POP (POP—Q II—III) had reduced expression of type I collagen and increased expression of type III collagen in the uterosacral ligaments. These discrepancies may be attributed to differences in tissue sampling (such as sampling from the vaginal wall versus the uterosacral ligaments) [24] and patient inclusion criteria (such as different POP—Q stages or parity) [25]. Future research should standardize the sampling methods and patient selection criteria to improve the comparability of research results.

Our findings have potential clinical implications for the diagnosis and treatment of POP. In terms of diagnosis, the expression levels of IGF-1, pAKT, pS6, ColI, and ColIII in the uterosacral ligament tissues could potentially serve as novel biomarkers for POP. By identifying the abnormal expression of these factors before the appearance of obvious clinical symptoms, early intervention can be carried out to prevent the progression of the disease.

Regarding treatment, the IGF-1/AKT/mTORC1 signaling pathway may be a promising therapeutic target for POP. Developing drugs or therapies that can upregulate the expression of IGF-1 or activate this signaling pathway may promote the synthesis of collagen fibers and strengthen the pelvic floor support structure. For example, gene therapy or targeted drug delivery systems could be explored to specifically enhance the activity of this pathway in the uterosacral ligament tissues. However, further pre-clinical and clinical studies are needed to evaluate the safety and effectiveness of these potential treatment strategies.

Despite the valuable insights provided by our study, several limitations need to be acknowledged. First, we did not consider the severity and duration of POP in our analysis. The severity of POP maybe associated with the degree of decrease in the expression of IGF-1, pAKT, pS6, and collagen fibers. Additionally, the duration of the disease may also affect the expression of these biomarkers over time. Future studies should conduct a more in-depth analysis of the relationship between POP severity, duration, and the expression of these factors to better understand the time–effect and dose–effect relationships. Second, owing to the complexity of the disease and the limitations of specimen acquisition, the exact molecular mechanism by which the IGF-1-regulated PI3 K/AKT/mTOR signaling pathway is involved in the occurrence of POP remains unclear. Although we observed a decrease in the expression of related factors in this pathway, the specific upstream and downstream regulatory mechanisms need to be further investigated. More in-depth basic research, such as in vitro cell experiments and animal models, is required to clarify the detailed molecular mechanisms.

To address the limitations of our study, future research could take several directions. First, longitudinal studies with a larger sample size should be conducted to comprehensively analyze the relationship between POP severity, duration, and the expression of IGF-1, pAKT, pS6, and collagen fibers. This will help in establishing a more accurate model for predicting the progression of POP. Second, in-depth in vitro and in vivo studies are needed to elucidate the detailed molecular mechanisms of the IGF—1/AKT/mTORC1 signaling pathway in POP. By using gene knockout or overexpression techniques in cell lines and animal models, we can better understand how this pathway regulates the synthesis and degradation of collagen fibers in the uterosacral ligament tissues. Finally, translational research should be carried out to develop and evaluate potential therapeutic strategies based on our findings. This includes the pre-clinical evaluation of drugs or therapies targeting the IGF-1/AKT/mTORC1 signaling pathway and subsequent clinical trials to assess their safety and effectiveness in treating POP patients.

In conclusion, our study provides important evidence for the potential role of the IGF-1/AKT/mTORC1 signaling pathway in the occurrence of POP. However, further research is required to fully understand the underlying mechanisms and develop effective diagnostic and therapeutic approaches.

Authors’ Participation

H.X.: project development, conducted the experiments, and wrote the manuscript; H.X., X.W., and D.X.: conducted the experiments, and collected and analyzed data; Q.L.: collected and analyzed data; Gensheng Wang: was responsible for the protocol and reviewed the manuscript.

Funding

This study was funded by 2022 Annual Natural Science Research Projects of Anhui Universities (2022 AH050735).

Data Availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics Approval and Consent to Participate

This study protocol was approved by the Ethical Committee of Anqing Municipal Hospital. Informed consent was obtained from all participants before sample collection.

Conflicts of interest

None.