Expression of SMAD-dependent pathway components in the pathogenesis of hyperplastic processes of the endometrium in benign uterine diseases

Endometrial hyperplastic processes, including endometrial polyps (EP) and endometrial hyperplasia (EH), are among the most common pathologies of the female reproductive system and are often detected in women of reproductive age [1]. These conditions not only diminish the quality of life of patients, causing heavy uterine bleeding, intermenstrual spotting, and anemiabut also increase the risk of malignant transformation, particularly in cases of complex hyperplasia. The presence of concomitant benign uterine diseases, such as leiomyoma and adenomyosis, further complicates this situation by disrupting the homeostasis of endometrial tissues and exacerbating pathological processes [2–5]. Recent studies on the pathogenesis of hyperplastic changes have focused on the molecular mechanisms that regulate cell proliferation and apoptosis. Among the key signaling pathways, the transforming growth factor β (TGF-β) signaling pathway plays a crucial role in regulating cell growth, differentiation, and apoptosis through SMAD-dependent and SMAD-independent cascades [6]. Under normal conditions, this pathway exhibits antiproliferative and pro-apoptotic functions; however, pathological conditions can alter its regulatory role, leading to disturbances in cellular homeostasis and proliferative processes within the endometrium [7].

A particularly important aspect of this research is the examination of the expression of components of the SMAD-dependent signaling pathway, including SMAD2, SMAD3, and SMAD4, in the hyperplastic processes of the endometrium. Several studies have indicated that the expression levels of these proteins increase with hyperplastic changes, suggesting a compensatory mechanism in response to pathological proliferation [8, 9]. However, it remains unclear whether this increase in expression is the primary cause or merely a consequence of the activation of other signaling pathways, such as the AKT-dependent pathway or SMAD-independent TGF-β cascades. An additional complicating factor in pathogenesis is the interaction of TGF-β with the estrogen signaling pathway [10]. It is known that the activation of the estrogen receptor through TGF-β can enhance estrogen signaling, creating a feedback loop that normally promotes self-regulation. However, in instances where endometrial hyperplastic processes coexist with leiomyoma or adenomyosis, this mechanism may be disrupted, leading to further destabilization of proliferative processes and an increase in pathological changes.

Thus, this study aimed to investigate the expression of components of the SMAD-dependent TGF-β signaling pathway in endometrial hyperplastic processes, both in isolation and in conjunction with benign uterine diseases. This study encompasses a comprehensive analysis of molecular markers and their correlation with the clinical and morphological features of the disease, thereby facilitating the identification of the underlying mechanisms of pathogenesis. The study findings will enhance the understanding of the role of the TGF-β signaling pathway in regulating hyperplastic processes and aid in developing targeted therapeutic strategies to improve women's reproductive health.

Materials and methods

A comprehensive examination of 90 patients with endometrial hyperplastic processes was conducted at the Department of Gynecology at V.I. Kulakov NMRC for OG&P of the Ministry of Health of the Russian Federation. The patients were divided into the following groups: EP group (group 1, n=15), EH group (group 2, n=15), EP with adenomyosis (group 3, n=15), EP with myoma (group 4, n=15), EH with adenomyosis (group 5, n=15), and EH with myoma (group 6, n=15). Preoperative counseling included collection of medical history, complaints, and assessment of clinical, laboratory, and instrumental data, followed by intraoperative evaluation of the histological material. This evaluation was conducted in the 1st Department of Anatomical Pathology at V.I. Kulakov NMRC for OG&P using standard methods combined with immunohistochemical studies. A postoperative assessment of the endometrium was performed using instrumental methods and histological and immunohistochemical studies to evaluate the expression of TGF-β, SMAD2, SMAD3, and SMAD4.

The inclusion criteria were as follows: reproductive age between 18 and 45 years; the presence of endometrial hyperplastic processes (EP or EH) identified during ultrasound, confirmed intraoperatively, and verified through pathological examination; the presence of uterine fibroids or adenomyosis detected via gynecological and ultrasound examination confirmed intraoperatively and verified during pathological examination; and informed consent to participate in the study.

The exclusion criteria included malignant neoplasms of any localization, hormonal therapy within six months before surgery, inflammatory diseases of the pelvic organs in the acute stage, severe somatic pathology, and the use of antibacterial drugs one month prior to the study.

Immunohistochemical markers were assessed using a semi-quantitative Immunoreactive index (the (immunoreactivity index IRS) score (Table 1).

Statistical analysis

Statistical processing of the data was performed using Microsoft Office Excel 2015 and MedCalc version 12. The normality of the distribution was assessed using the Kolmogorov–Smirnov test. Continuous variables with a normal distribution were described using arithmetic mean (M) and standard deviation (SD), expressed as M (SD). For continuous variables lacking a normal distribution, the median (Me) and interquartile range (Q1, Q3) were used and expressed as Me (Q1, Q3). Categorical variables were described using counts and percentages. When comparing more than two groups, the Bonferroni correction for multiple pairwise comparisons was applied. The traditional level of type I error (p<0.05) was divided by the number of pairwise comparisons according to the formula m=n(n-1)/2, where n is the number of groups. For the six study groups, the number of comparisons was 15; therefore, the new critical significance level was set at p<0.0033. Kruskal–Wallis test and Spearman correlation analysis were employed to assess the relationship between the expression of TGF-β signaling pathway components. The values of the correlation coefficients were interpreted according to the Chaddock scale: 0–0.3 – weak, 0.3–0.5 – moderate, 0.5–0.7 – noticeable; 0.7–0.9 – high, 0.9–1.0 – very high.

Results

Patients in the study groups did not differ significantly in age, which averaged 35.1 (5.5) years. Obesity was found in 18/90 (20%) of the women. We found no statistically significant differences in the onset of menarche, duration of the menstrual cycle, or duration of menstruation (Table 2).

The main complaint of the patients was heavy menstrual bleeding: 8/15 (53.3%) in groups 1, 3, and 6, 4/15 (26.7%) in group 2, 12/15 (80%) in group 4, and 6/15 (40.0%) in group 5. Dysmenorrhea was noted in 5/15 (33.3%) in groups 2 and 4, 3/15 (20.0%) in groups 1, 3, and 6, and 4/15 (26.7%) in group 5. In addition, patients more frequently complained of intermenstrual spotting and anemia (Table 2).

According to the immunohistochemical study, TGF-β expression was 5.00 (3.5; 7.0) in group 1, 7.00 (6.0; 8.0) in group 2, 8.0 (7.0; 8.0) in group 3, 6.0 (6.0; 7.5) in group 4, 8.0 (8.0; 8.0) in group 5, 7.0 (6.0; 8.0) in group 6 (the variables are expressed as the Me (Q1; Q3)). Statistically significant differences were found between groups 1 and 3 (p=0.001), groups 5 and 1 (p=0.001), and groups 4 and 5 (p=0.034) (Kruskal–Wallis test) (Fig. 1).

According to the immunohistochemical study, SMAD2 expression was 3.0 (2.0; 3.0) points in group 1, 4.0 (4.0; 5.0) points in group 2, 6.0 (5.0; 6.0) points in group 3, 5.0 (4.0; 6.0) points in group 4, 6.0 (5.0; 6.0) points in group 5, 6.0 (5.5; 6.0) points in group 6 (the variables are presented as Me (Q1; Q3)). Statistically significant differences were found between groups 1 and 3 (p=0.001), groups 5 and 1 (p=0.001), groups 4 and 1 (p=0.001), groups 6 and 1 (p=0.001), groups 3 and 2 (p=0.025), groups 5 and 2 (p=0.011), and between groups 6 and 2 (p=0.001) (Kruskal–Wallis test) (Fig. 2).

According to immunohistochemical study, SMAD3 expression was 2.0 (2.0; 3.0) points in group 1, 4.0 (3.0; 4.0) points in group 2, 4.0 (4.0; 5.0) points in group 3, 5.0 (4.0; 5.0) points in group 4, 5.0 (5.0; 5.0) points in group 5, 5.0 (5.0; 6.0) points in group 6 (where the variables are expressed as Me (Q1; Q3)). Statistically significant differences were found between groups 1 and 3 (p=0.001), groups 5 and 1 (p=0.001), groups 4 and 1 (p=0.001), groups 6 and 1 (p=0.001), groups 5 and 2 (p=0.001), groups 4 and 2 (p=0.006), groups 6 and 2 (p=0.001), and groups 6 and 3 (p=0.029) (Kruskal–Wallis test) (Fig. 3).

According to the immunohistochemical study, SMAD4 expression was 3.0 (2.0; 3.0) points in group 1, 4.0 (4.0; 5.0) points in group 2, 4.0 (4.0; 4.0) points in group 3, 4.0 (4.0; 5.0) points in group 4, 5.0 (4.0; 5.0) points in group 5, 5.0 (3.0; 4.0) points in group 6 (the indicators are presented as Me (Q1; Q3)). A statistically significant difference was found between groups 1 and 2 (p=0.001), groups 3 and 1 (p=0.002), groups 5 and 1 (p=0.001), groups 4 and 1 (p=0.001), groups 6 and 1 (p=0.011) (Kruskal–Wallis test) (Fig. 4).

A significant correlation was found between the expression of TGF-β and SMAD2, and a moderate correlation was found between the expression of TGF-β and SMAD3, as well as between the expression of TGF-β and SMAD4 (Table 3). A high correlation was found between the expression of SMAD2 and SMAD3, and a moderate correlation was found between the expression of SMAD2 and SMAD4 and between SMAD3 and SMAD4 (the strength of the correlation was assessed using the Chaddok scale).

To illustrate the obtained results, micrographs of the expression of the studied markers in EH alone and in combination with adenomyosis are shown (Fig. 5).                 

Discussion

The role of the regulation of signaling pathway components in the development of endometrial hyperplastic processes has been extensively studied over the past 20 years. Notably, the AKT-dependent signaling pathway has garnered significant attention, as it has been identified as a central pathway in the pathogenesis of EH, the immediate precursor of endometrioid adenocarcinoma of the uterine mucosa [11–13]. However, subsequent research has revealed that other signaling pathways also contribute to the development of endometrial hyperplastic processes, with the TGF-β-dependent signaling pathway being particularly important (Fig. 6).

TGF-β-dependent signaling pathway can be divided into two main branches: SMAD-dependent and SMAD-independent signaling pathways. The latter group includes key proteins from the JNK, ERK, and MAPK families. Although the role of SMAD-independent pathways has been well studied [14–16], there is limited understanding of the regulation of the SMAD-dependent pathway in the pathogenesis of endometrial hyperplastic processes. This knowledge gap motivated our study to examine the expression of components of the SMAD-dependent signaling pathway. This study not only aimed to clarify their role in the formation of EP and EH, but also to compare the degree of activation of the SMAD-dependent signaling pathway in combined benign uterine diseases, such as adenomyosis and leiomyoma.

Considering the pathogenesis of endometrial hyperplastic processes, it is crucial to recognize that the uterine mucosa is subjected to cyclic influences from steroid hormones, estrogen, and progesterone, and can undergo numerous regeneration cycles throughout a woman's reproductive life. This regenerative potential is facilitated by the presence of unique stem and progenitor cells dispersed throughout the uterine mucosa, which aids in rapid recovery following menstruation [17, 18]. The inhibition of the TGF-β signaling pathway plays a vital role in sustaining the proliferation and regeneration of endometrial mesenchymal stem cells and endometrial epithelial organoids, highlighting the critical function of the TGF-β signaling pathway in endometrial regeneration and restoration [19, 20]. However, the mechanisms regulated by the TGF-β signaling pathway in the endometrium remain poorly understood.

The TGF-β signaling pathway encompasses various secreted ligands, inhibitors, cell surface receptor kinases, and the transcription factors SMAD2/SMAD3 and SMAD4, which are activated through phosphorylation and subsequently translocated to the nucleus to regulate gene expression (Fig. 6). This signaling pathway governs essential developmental processes, including cell migration, differentiation, and proliferation, and is often dysregulated in various cancer subtypes. Mutations in TGF-β pathway components have been identified across different tumor types and are correlated with genes associated with metastasis and reduced patient survival [21]. In the female reproductive system, TGF-β plays a crucial role in maintaining the integrity and development of the uterus and ovaries, controlling processes such as endometrial receptivity, decidualization, and placentation during early pregnancy and throughout gestation [22, 23]. Additionally, components of the TGF-β signaling pathway function as tumor suppressors. Mouse models have demonstrated that conditional deletion of the TGF-β receptor ALK5 or the transcription factors SMAD2 and SMAD3 in the endometrial epithelium and stroma results in an aggressive metastatic form of endometrial cancer [24, 25]. Furthermore, mouse models of endometrial cancer with conditional inactivation of the PTEN gene, as well as the ARID1A gene, exhibit abnormal TGF-β signaling, further confirming that TGF-β pathway inactivation contributes to the metastatic potential of endometrial tumors.

Our study demonstrated that the expression of TGF-β was significantly different in the endometrium of patients with isolated EP, in combination with adenomyosis, and in the endometrium of patients with EH and adenomyosis. From these results, it can be concluded that adenomyosis affects the level of TGF-β expression in both EH and EP. It can also be concluded that TGF-β expression was higher in EH than in EP; however, a statistically significant difference was found only in the groups of patients with EP and EH in combination with adenomyosis. As already noted, among the published studies, there are few observations of TGF-β expression in hyperplastic changes of the endometrium. However, in a study by Gold L.I. et al. A statistically significant progressive increase in immunoreactivity was found for all three TGF-β isoforms in endometrial glands from the normal proliferative state to simple hyperplasia, and from simple hyperplasia to complex hyperplasia. The most noticeable increase was observed when comparing normal proliferative endometrium with complex hyperplasia; the increases were 5.1-, 3.4-, and 2.6-fold for TGF-β1, TGF-β2, and TGF-β3, respectively. However, no further increase was observed during the progression from preneoplastic complex hyperplasia to endometrial carcinoma [8]. In a study by Faraji A. et al., TGF-β expression in the polyp area was significantly lower than that in the unchanged endometrium, but the authors did not report data on the expression of the factor in the adjacent endometrium, which does not allow a comparative analysis of the results of this study with our results [9]. In addition, TGF-β expression in the endometrium of patients with uterine leiomyoma and adenomyosis was studied. In a study by Inagaki N. et al., TGF-β expression was higher in both leiomyomas and adenomyoses than in the control group [26]. In studies by Kishi Y. et al. and Jacobo A. et al., no difference was found in TGF-β expression in ectopic adenomyosis foci and eutopic endometrium of the control group, but the eutopic endometrium of patients with adenomyosis has not been studied [27, 28].

Regarding the study of combined benign lesions of the uterine body and TGF-β expression, we did not find such data in the available literature.

We also studied the expression of components of the SMAD-dependent pathway (SMAD2, SMAD3, and SMAD4). These interconnected components work synergistically to implement proapoptotic and antiproliferative potential. We found an increase in the expression of these proteins in the group of patients with isolated EH compared with the isolated EP group, as well as when comparing the expression in the EP group combined with adenomyosis, compared with isolated polyps, and when comparing the EH group combined with adenomyosis, compared with isolated EH. These proteins have been studied in EH patients. Liu et al. studied the expression of SMAD4 in EH and the development of endometrial carcinoma. A positive expression was detected in all EH cases [29]. Although SMAD family proteins have been actively studied in adenomyosis, no emphasis has been placed on the eutopic endometrium, although increased expression has been reported in adenomyosis foci compared with the control group [27, 30].

In patients with uterine leiomyoma, the expression of SMAD2/3/4 proteins has been studied mainly in ​​the tumor tissue itself. It was shown to be higher than in the normal myometrium, and it was noted that the SMAD-independent pathway of activation of the TGF-β signaling pathway also plays a role in the development of smooth muscle tumors of the uterine body [31, 32]. We also did not find any studies devoted to the combination of benign lesions of the uterine body and expression of proteins of the SMAD family. Thus, in our study, an increase in the components of the TGF-β signaling pathway (namely, its SMAD-dependent cascade, since we studied the expression of the TGF-β protein itself, as well as the SMAD2, SMAD3, and SMAD4 proteins) was observed in various pathological processes of the endometrium. Normally, this pathway performs important functions such as suppression of cell division, induction of apoptosis, and regulation of immune processes. In our study, we have shown that the development of endometrial hyperplastic processes enhances the expression of the components of this pathway, and concomitant benign lesions of the uterine body aggravate this regulatory mechanism, further increasing the expression of both TGF-β and the components of the signaling pathway located lower in the hierarchy of proteins of the SMAD family. Since the function of the TGF-β signaling pathway is antiproliferative and proapoptotic, one would expect a greater decrease in the expression of one or more components of this signaling pathway if its implementation was the main factor in the pathogenesis of endometrial hyperplastic processes and/or benign lesions of the uterine body, since all these conditions are somehow associated with increased proliferation and decreased apoptosis of cellular elements. Nevertheless, in the endometrium of patients with EP and EH, both isolated and in combination with adenomyosis and leiomyoma, we observed an increase in the expression of TGF-β and SMAD2/3/4, although in some cases this was only a trend, and in others, statistically significant differences between the groups. In our opinion, this is due to the fact that the main initiating mechanism of the pathogenesis of endometrial hyperplastic processes is associated with the implementation of SMAD-independent signaling pathways, as well as with the AKT-dependent signaling pathway, the implementation of which generally occurs in parallel with TGF-β regulated signaling pathways. Therefore, the increase in the expression of components of the TGF-β signaling pathway revealed in our study is most likely compensatory, responding to increased proliferation and decreased apoptosis under the influence of parallel signaling pathways. This assumption is supported by studies focusing on precancerous and malignant lesions of the uterine mucosa, which did not show a decrease in the expression of TGF-β signaling pathway components [33, 34]. Numerous studies on the disruption of SMAD-independent signaling pathways have also indirectly confirmed this, as they lead to an imbalance between proliferation and apoptosis.

Another possible mechanism regulating the TGF-β signaling pathway is its connection with estrogen. It has been shown that activation of the estrogen receptor (ER) through the TGF-β signaling pathway can create a feedback mechanism, where TGF-β enhances the estrogen signaling pathway, leading to its faster and more effective inhibition. When the TGF-β signaling pathway is suppressed, ER activity returns to normal levels [35]. However, we did not observe such an inhibition in our study. It is likely that the combination of hyperplastic processes in the endometrium and benign lesions of the uterine body disrupts the feedback mechanism, which leads to the suppression of TGF-β expression owing to the activation of other signaling pathways and the presence of external estrogen-dependent tissues (such as smooth muscle tumors and foci of adenomyosis). This situation prevents the achievement of a balance in regulating proliferative processes.

Conclusion

The results of this study demonstrate the key role of the TGF-β signaling pathway, particularly its SMAD-dependent cascade, in the pathogenesis of hyperplastic processes in the endometrium, such as polyps and hyperplasia, including their association with benign diseases of the uterine body (adenomyosis and leiomyoma). The increased expression of TGF-β and SMAD components (SMAD2, SMAD3, and SMAD4) in these conditions indicates compensatory activation of the signaling pathway in response to increased cell proliferation and impaired apoptosis. These findings underscore the complexity of regulatory mechanisms in the endometrium and the significance of the interaction between the TGF-β pathway and other molecular processes, including estrogen-dependent signaling pathway. These results support the feasibility of further investigation of TGF-β pathway components as potential diagnostic markers and therapeutic targets for the targeted treatment of endometrial hyperplastic processes. An integrated approach, including the immunohistochemical study of key markers, not only enhances diagnostics but also reduces the risk of relapse, representing an important step towards improving the quality of life for women and maintaining their reproductive health.