Endometrial polyps (EPs) are composed of endometrial glands and fibrotic endometrial stroma with thick-walled blood vessels [1]. They can be single or multiple, with diameters ranging from several millimeters to several centimeters [2]. EPs often occur in 40–50-year-old and postmenopausal women [3]. Up to 82% of EP patients have no clinical symptoms, and a few may show abnormal uterine bleeding [4].

The formation of EPs may be related to the imbalance of estrogen and progesterone receptors, inflammation, oxidative stress, abnormal expression of cytokines, and an imbalance between cell proliferation and apoptosis [5, 6, 7, 8]. Evidence suggests that EPs are closely related to estrogen. However, after menopause, despite the decline in ovarian function and the decrease in systemic circulating estrogen levels, the incidence of EPs does not decrease. A study has found that postmenopausal EP is mainly due to the absolute lack of progesterone, which results in the improper functioning of progesterone receptors, thereby promoting the development of EPs [9].

From the perspective of immunology, the change in cytokines may cause the entire immune response to shift in a certain direction, resulting in a completely different outcome. Helper T cells (Th) and regulatory T cells (Treg), both subsets of CD4⁺ T lymphocytes, play pivotal roles in immune modulation and stimulation [10]. The cytokines secreted by these cells are intimately associated with the processes of inflammation and cellular proliferation. In recent years, with the discovery of Th17 cells, the traditional Th1/Th2 balance model has been further developed into the T helper 1 (Th1)/T helper 2 (Th2) and T helper 17 (Th17)/Regulatory T (Treg) balance model [11]. Under normal circumstances, the number of these subsets of cells maintains a dynamic balance through the mutual regulation of cytokines secreted by them, and participates in the regulation of body immunity. It has been found that the subsets of Th1, Th2, Th17 and Treg cytokines, representative of CD4⁺ T cells, are related to the development of EPs. However, the relationship between the equilibrium state of Th1/Th2, Th17/Treg and EPs in EPs has not been reported. In this experiment, Th1 representative cytokines such as interleukin-2 (IL-2), tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interferon-gamma (IFN-$\gamma$), Th2 representative cytokines interleukin-4 (IL-4), interleukin-6 (IL-6), Th17 representative cytokines interleukin-17 (IL-17), and Treg representative cytokine transforming growth factor-$\beta$ (TGF-$\beta$) were used to detect the cytokines in the tissues of patients with EPs. It is expected to explain the pathogenesis of EPs from the perspective of immunology and provide some theoretical basis for the pathogenesis of EPs.

In this study, we found that: (1) In the tissue of patients with single and multiple Eps, the balance of Th1/Th2 and Th17/Treg subsets of CD4⁺ T cells shifted to Th1 and Th17, which may be an important reason for the occurrence of EPs in the single polyp group. (2) Compared with the multiple polyps group, the cellular immune balance Th17/Treg of EPs in the single polyp group shifted more significantly to Th17, indicating that the shift of Th17/Treg balance to Th17 was not more pronounced with the increase in the number of polyps. (3) Different from premenopausal single and multiple EPs, in the postmenopausal EP patients, the balance of Th1/Th2 and Th17/Treg subsets of CD4⁺ T cells shifts to Th1 and Treg, which may be an important cause of postmenopausal EP.

Th1 cells primarily secrete IL-2, TNF-$\alpha$, and IFN-$\gamma$, guiding both the innate and acquired immune systems to produce cytokines and antibodies, activate macrophages, and promote cellular immunity, which is linked to inflammation and tissue damage [12]. In this study, we found that there was a high expression of Th1 cytokines in EPs tissues of single, multiple and postmenopausal single polyps, which was consistent with most scholars’ studies. TNF-$\alpha$ is highly expressed in EPs, and the expression of TNF-$\alpha$ and IL-6 is negatively correlated with the expression of progesterone receptor. It is analyzed that the decrease in progesterone receptors weakens the inhibitory effect of progesterone on estrogen receptors, resulting in a sustained high level of estrogen receptors, resulting in polyps [13]. In EP patients with infertility, the value of TNF-$\alpha$ in uterine lavage fluid and serum before surgery were significantly higher than in the infertility patients without EPs in the middle stage of endometrial proliferation. The concentrations of TNF-$\alpha$ in uterine lavage fluid and serum in patients with EPs after surgery were significantly lower than those before surgery (p 0.05) [14]. The levels of IFN-$\gamma$ in serum and endometrium were significantly higher than those in infertile patients without EPs [15]. Based on this, we can speculate that the high expression of Th1 cytokines mediates inflammation and immune injury, leading to the occurrence of EPs.

Th2 cells stimulate B cells to produce antibodies, promote eosinophil and mast cell differentiation, inhibit immune inflammation and reduce tissue damage. In this study, we found that there was a low expression of Th2 cytokines in both single and postmenopausal EP patients, which was consistent with another study: during the proliferative phase of the endometrium, the contents of IL-6 and IL-10 in uterine lavage fluid of EPs patients increased significantly [16]. Based on this, we can speculate that the low expression of Th2 cytokines and insufficient immune protection lead to the occurrence of EPs.

Under normal circumstances, Th1 and Th2 cells are in dynamic balance and interact with negative feedback regulation. In normal reproductive women, the balance of Th1/Th2 cells moves to Th2 [17]. The results showed that the expression levels of Th1 cytokines IL-2, IFN-$\gamma$ and TNF-$\alpha$ in the peripheral blood of patients with EPs were significantly increased, while the expression levels of Th2 cytokines IL-4 and IL-6 were decreased (p 0.05). The expression of IL-10 was slightly lower, but there was no significant difference (p $>$ 0.05). The above results confirmed that the occurrence of EPs is related to the balance/shift of Th1/Th2 cells to Th1. Reversing the imbalance of Th1/Th2 can prevent the postoperative recurrence of EPs. Changing the expression of cytokines in the uterine cavity of patients with EPs can shift the balance of Th1/Th2 cells towards Th2, reducing the local inflammatory reaction. The content of INF-$\gamma$ significantly decreases, while the content of IL-6 significantly increases. This can prevent the recurrence of EPs.

Th17 cells are a new class of CD4⁺ T cell subsets that can secrete IL-17 at high levels and participate in inflammatory responses and autoimmune diseases [18]. In this study, we found that IL-17 was highly expressed in the epithelium, stroma and glands of Eps in both the single and multiple polyps group. Some studies have found that the Th17 response is up-regulated in the peripheral blood of patients with EPs, leading to increased secretion of many pro-inflammatory factors, such as IFN-$\gamma$, TNF-$\alpha$, IL-17, and IL-6 [19, 20]. This supports our conclusion that the high expression of Th17-type cytokines in the polyps of premenopausal EP patients promotes inflammatory responses, thereby contributing to the occurrence of premenopausal EPs.

Treg cells secrete TGF-$\beta$, which participates in the regulation of immunosuppression. TGF-$\beta$ may play an important regulatory role in the proliferation and fibrosis of intimal stroma and the formation and development of thick-walled vessels in EPs [5, 21]. In this study, we found that there was a high expression of TGF-$\beta$ in the epithelium, stroma and glands of EPs in both the single and multiple polyps group. This is consistent with other findings that the expression of TGF-$\beta$1 in stroma cells of EPs is significantly higher than in the normal endometrium, whether in the proliferative phase or secretory phase. The increased expression of TGF-$\beta$1 in EP tissue can increase the risk of postoperative uterine adhesion due to the overexpression of TGF-$\beta$1, which increases the production of extracellular matrix and inhibits the growth of epithelial cells [22]. Therefore, we conclude that the high expression of TGF-$\beta$1 in premenopausal polyps promotes interstitial hyperplasia, fibrosis and the formation and development of thick-walled blood vessels. However, the immunosuppressive effect is relatively insufficient, thus contributing to the occurrence of premenopausal EPs.

This study had several limitations. First, the sample size collected for the study is limited; except for 19 cases of postmenopausal single polyps, there were only 20 cases in each of the other groups. However, it meets the minimum sample size required for statistical analysis. In addition, this study only involved experimental analysis of specimens collected from surgeries of EP patients. As the research is still ongoing, we did not choose to include peripheral blood for further validation analysis. Furthermore, the expression of cytokines in this study was assessed using immunohistochemistry, rather than the more commonly used and more precise flow cytometry. Finally, this study was limited to finding the differential expression of inflammatory factors in EPs and cannot directly prove a causal relationship.

To sum up, the regulation of the uterine environment depends not only on the balance of Th1/Th2 cells but also on the balance of Th17/Treg cells. In the tissues of patients with single and multiple EPs, the balance of CD4+ T cell subsets Th1/Th2 and Th17/Treg shifts towards Th1 and Th17. Compared with the multiple group, the cellular immune Th17/Treg balance of EPs in the single group shifted more significantly to Th17, indicating that the shift of Th17/Treg balance to Th17 was not more obvious with the increase of the number of polyps. In the tissues of postmenopausal EPs patients, the balance of CD4⁺ T cell subsets Th1/Th2 and Th17/Treg shifts towards Th1 and Treg expression, and the specific mechanism needs to be further studied.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

HY and SL designed the research study. HY and LB performed the research. SL and LB provided help and advice on the immunohistochemistry. HY analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Changzhou No. 2 People’s Hospital (approval number: 202123).

This project was supported by the research project (Grant No.: ZD202343).

The authors declare no conflict of interest.