In recent years, the incidence of endometriosis is about 10–15% and has a trend of increasing year by year [10]. More and more evidence has confirmed the multifactorial nature of endometriosis, which is the result of the combined effects of anatomical, immune, reactive, hormonal, genetic, epigenetic, and environmental factors in affected women ADDIN NE.Ref.[11]. Guideline for the diagnosis and treatment of endometriosis recommend that medical treatment be preferred in patients without infertility and those with adnexal masses < 4 cm in diameter. Surgery may be considered in patients with infertility or adnexal mass ≥ 4 cm in diameter, and in patients who do not respond to medical therapy ADDIN NE.Ref.[12]. Currently, laparoscopic surgery is still the standard treatment for ovarian endometrioid cysts ADDIN NE.Ref.[13, 14]. The recurrence rate of ovarian endometrioid cysts in patients without appropriate postoperative treatment can be as high as 40% within 2 years ADDIN NE.Ref.[15]. Medical treatment is recommended for patients who do not desire fertility after surgery. First-line drugs include non-steroid anti-inflammatory drug (NSAID), oral contraceptives and high-potency progesterone, and second-line drugs include GnRH-a and levonorgestrel intrauterine system (LNG-IUS) ADDIN NE.Ref.[12]. At present, GnRH-a is still the "gold standard" for drug treatment of endoheterosis ADDIN NE.Ref.[12]. Pharmacologic doses of GnRH agonists administered for six months have been found to produce amenorrhea and anovulation in numerous controlled, randomized clinical studies ADDIN NE.Ref.[16]. Therefore, the patients included in this study were treated with GnRH-a for only 3 times after surgery, and the patients were followed up closely after treatment. Even for patients receiving appropriate postoperative treatment, the recurrence rate is still about 10% ADDIN NE.Ref.[17]. For such patients, if they have no fertility requirements, surgery or ultrasound-guided puncture can be performed. GnRH-a therapy is recommended to be given after surgery, and then other drugs may be used for long-term maintenance therapy ADDIN NE.Ref.[12]. Due to such a high recurrence rate and the lack of effective biological markers, many scholars have started to study the related cell, protein and gene biomarkers in urine, blood, cervical mucus and other body fluids from a non-invasive perspective, but the results are still not ideal.

The NF -κB pathway is one of the major markers of inflammation, which can regulate cell proliferation, apoptosis and inflammatory processes [18]. NF-κB regulates various proinflammatory such as Tumor necrosis factor(TNF-α), Interleukin(IL-1, IL-6, IL-12), chemokines(CXCL1, CXCL2), recombinant human chemokine-5, adhesion molecules(vascular cell adhesion factor-1) and so on, involved in the activation and recruitment of inflammatory regulatory cells. The NF-κB pathway is down-regulated in normal endometrium [19], but its expression is increased in all stages of endometriosis [20]. Previous studies have demonstrated that women with endometriosis have increased NF-κB expression that regulates the expression of aberrant cytokines through autocrine self-amplifying cycles of cytokine release and NF-κB activation. These lead to amplification and maintenance of the proinflammatory local environment, promoting the survival and growth of endometrial cells in endometriosis patients and reducing the clearance of retrogradely transported endometrial fragments, which promotes the development of endometriosis [21, 22]. Therefore, given the high recurrence rate of endometriosis, we hypothesized that high expression of NF-κB may lead to the formation and maintenance of proinflammatory local environment that allows the growth and invasion of ectopic endometrial cells, which promotes the recurrence of endometriosis.

In our study, the expression intensity of NF-κB in the first recurrence group was higher than that in the non-recurrence group. As mentioned above, NF-κB plays an important role in the occurrence and development of ovarian endometrioid cysts. Patients with recurrent endometrioid cysts may be more prone to recurrent ovarian endometrioid cysts due to stronger expression of NF-κB and more severe inflammatory reactions in vivo. Nf-κB can regulate the expression of genes and play a key role in the development and progression of cancer, especially in inflammatory tumors, such as the proliferation, migration and apoptosis of cancer cells, and is related to the occurrence of lung cancer [23], nasopharyngeal carcinoma [23], breast cancer [5], liver cancer [24] and other tumors. In recent years, NF-κB has also been studied as a new target for the diagnosis or treatment of tumors, such as endocrine therapy of breast cancer [5]. In the study of the tumor and inflammatory diseases, scholars have found that EMs seems to fit another disease – endometriosis very well. EMs has many characteristics similar to malignant cells, such as invasion and proliferation of ectopic cells. In this study, the high expression of NF-κB in the recurrence group suggests that the recurrence group is more prone to adhesion and invasion. Nf-κB may also be used as a target for the treatment of ovarian endometrioid cysts, reducing the inflammatory response of patients with ovarian endometrioid cysts by reducing the expression of NF-κB, to achieve the purpose of controlling the recurrence.

Gonzalez-ramos et al. [22] found that the NF-κB pathway is related to the occurrence of endometriosis in the early development stage. In this study, the expression intensity of NF-κB in the first recurrence group and the recurrence group was significantly higher than that in the non-recurrence group, and NF-κB promoted the occurrence of ectopic lesions. One of the NF-κB subunits of Rel family proteins is p65, which assembles as a dimer in the cytoplasm. In the inactive state, NF-κB binds to its inhibitor IκB, forming the NF-κb-IκB complex. After activation, NF-κB translocates to the nucleus, where IκB is phosphorylated in response to different stimuli. IL-6 and IL-8 in endometriosis can thus be activated. Inhibition of the action of the complex can reduce the maintenance and development of Ems [25], so patients with higher NF-κB expression are more likely to develop endometriosis. The expression of NF-κB was stronger in the patients with postoperative recurrence in this study, which can be speculated that the higher the expression of NF-κB, the stronger the inflammation, and the easier the recurrence of the ovarian endometrioid cyst.

IER3 plays a key role in the regulation of cell proliferation and apoptosis, and its overexpression can inhibit or enhance cell apoptosis according to the nature of stress [26]. Positive expression of IER3 in ovarian cancer [7] and pancreatic cancer [27] is associated with a good prognosis, while increased expression of IER3 in other diseases has a poor survival rate, such as acute myeloid leukemia, bladder cancer, liver cancer, breast cancer, Sezary syndrome and colorectal cancer [6], this may be related to the excess IER3 mediating cancer cell survival. IER3 has been widely reported in cancer, but rarely in EMs with oxidative stress and inflammatory diseases. Given the great advances in diagnostic imaging (such as transvaginal ultrasound and nuclear magnetic resonance), the diagnosis of endometrioid cysts tends to be simplified and become a structured process based on a combination of patient interviews, clinical examination and imaging. The diagnosis of endometriosis is not difficult, but recurrence is very troublesome [28]. Endometriosis is deeply affected by oxidative stress. Samimi et al. [29] found that the concentrations of glutathione peroxidase and catalase were higher in EMs ectopic cells, and in EMs follicular fluid, Higher concentrations of malondialdehyde (MDA), NO and reactive oxygen species (ROS) were observed. As a stress-inducer gene, IER3 was involved in oxidative stress and inflammatory reactions. Reactive oxygen species (ROS) can also activate the NF-κB pathway, promote oxidative stress-mediated proinflammatory signals, and promote the development of endometriosis [30]. The IER3 promoter has been shown to contain consistent sequences of several transcription factors, including NF-κB, p53, cMyc, VDRE, Sp1, p300, and Sox [31]. Studies have shown that IER3 plays a regulatory role in apoptosis, inflammation, immune system regulation, tumorigenesis and other cellular functions mainly through NF-κB, PI3K/Akt and MAPK/ERK pathways [6]. Therefore, we hypothesized that NF-κB promotes the development of endometriosis by targeting IER3 expression induced by inflammation and oxidative stress. The results of this study were consistent with that of IER3 expression in almost all ovarian endometrioid cysts, and the expression intensity of IER3 in the recurrence group was higher than that in the non-recurrence group, and the difference was statistically significant (P < 0.05), indicating that the patients with recurrent ovarian endometrioid cysts had strong oxidative stress and inflammatory response in the body. The high expression of IER3 provides a good basis for the recurrence of the ovarian endometrioid cyst.

In this study, the expression of NF-κB and IER3 was highly expressed in patients with recurrent ovarian endometrioid cysts. The area under the ROC curve of NF-κB and IER3 expression score in the tissues of patients with ovarian endometrioid cysts in the primary operation was 0.867. NF-κB and IER3 have a high accuracy in predicting the recurrence of ovarian endometrioid cysts, which may be related to the disease recurrence by enhancing the inflammatory and oxidative stress response in vivo. Multivariate Cox survival regression showed that the expression of IER3 > 4.5 (HR = 3.418,95%CI: 1.227 ~ 9.523, P = 0.019), NF-κB > 4.5 (HR = 5.491,95%CI: 1.600 ~ 18.838, P = 0.007) was an independent risk factor for postoperative recurrence, and EFI score (HR = 0.791,95%CI: 0.637 ~ 0.983, P = 0.035) was a protective factor for postoperative recurrence. The expression levels of NF-κB and IER3 in the two recurrence group was significantly higher than those in the non-recurrence group, which also suggested that the level of oxidative stress in patients with recurrent ovarian endometrioid cyst was higher. Therefore, drugs targeting NF-κB and IER3 may inhibit the recurrence of ovarian endometrioid cysts.

The high expression of NF-κB and IER3 suggests that the risk of ovarian endometrioid cyst recurrence may be higher. For patients who are more prone to recurrence, more systematic treatment should be taken to avoid recurrence after surgery. Studies have shown that an NF-κB inhibitor, DHMEQ, inhibited the migration and invasion of human endometriosis stromal cells, and DHMEQ is particularly effective in suppressing disease models by intraperitoneal administration [32]. Therefore, we can further study the effects of NFKB inhibitors and IER3 inhibitors on endometriosis stromal cells, so as to further verify the inhibitory effect of these inhibitors on endometriosis through animal models, with a view to developing drugs that can more effectively inhibit the occurrence and development of endometriosis. Moreover, patients who plan to become pregnant should be encouraged to become pregnant as early as possible, and individualized precise medication should be realized to reduce patients' pain. Regrettably, the sample size of this study is relatively small, the data results are not representative, and other types of EMs cases (such as deep infiltrating type and superficial peritoneal EMs, etc.) are not included. Therefore, the sample size should be expanded and the types of cases should be enriched in subsequent research to get more convincing conclusions.