Introduction

As a major gynecological cause of hospital admissions [1] which affects 6–10% of the female population at reproductive age [2], endometriosis etiopathogenesis is not fully understood. Because of its impact on physical and psychological health of patients [3, 4] and of its socioeconomic impact [5], endometriosis can be considered a public health problem and deserves further investigation [1, 6,7,8,9]. It is characterized by implantation and growth of endometrial tissue outside the uterine cavity [10], and it is often associated with dysmenorrhea, deep dyspareunia, acyclic pelvic pain, infertility and alterations of the intestinal and urinary habits [11, 12].

There seems to be a similarity between endometriosis and cancer [13,14,15,16], particularly deep infiltrating endometriotic lesions, shares features with neoplastic processes, such as self-sufficiency in growth signals, insensitivity to anti-proliferative signals, resistance to apoptosis, sustained angiogenesis, tissue invasion, and genomic instability [17, 18]. Additionally, endometriosis and some types of cancer have alterations in the expression of oncogenes and tumor suppressor genes [19, 20] that are related to features that are commonly observed in both diseases, such as proliferation, apoptosis and cell cycle arrest [21, 22].

There is a correlation of a few oncogenes involved in cell cycle regulation, cell repair mechanism, replication and DNA repair and programmed cell death [23,24,25,26] such as cyclins p16INK4a (p16) and p21WAF1 (p21), with the progression of tumors [27]. Some studies suggest the involvement of increased p16 in tumor proliferation, invasiveness and angiogenesis by inhibiting cellular proliferation thus protecting cells from malignant transformation [28, 29]. Increased p21 and p16 expression have been described in endometrioma and endometrial polyps, respectively, suggesting a role of cyclins in some endometrial disorders [30,31,32]. Even though the involvement of p21 and of p16 in the more invasive types of endometriosis should be expected, none study has yet described an altered expression of such cell cycle biomarkers in deep infiltrating endometriosis.

Other proteins involved with cell cycle regulation in tumorigenesis are lamins. Lamins (A, B and C) are a group of proteins found inside the inner layer of the nuclear membrane of all cells [33] and are associated with the nuclear membrane structure maintenance. Lamin B1 is particularly involved with proliferation [34]. Moreover, higher lamin B1 seems to be associated with prostate and liver cancer, whereas lower lamin B1 was seen in breast, pancreas, and others types of cancer [35,36,37,38], commonly connected to an increased proliferative capacity and apoptosis instability of tumors, as that protein is able to prevent or delay apoptosis depending on the amount of lamin present [39].

Although there is a robust amount of evidence showing the role of p16, p21 and lamins in cancer and other diseases, there are hitherto no reports of their involvement on the development and maintenance of endometriosis, particularly in the deep infiltrating phenotype. Due to the clear involvement of cell cycle regulation in proliferative, invasiveness and apoptotic processes, the present study aims to test the hypothesis that p16, and p21 are higher and lamin B1 is lower in endometriotic lesions as compared to the eutopic endometrium, suggesting the presence of a higher number of senescence cell in endometriotic tissue.

Materials and methods

Setting

This study is part of the Women’s Health Program of the Hospital Israelita Albert Einstein, carried out in the Experimental Laboratory Center of the Education and Research Institute of the Hospital. The patients were screened by a single gynecologist and divided into two groups: with and without endometriosis.

This study was approved by the Committee on Human Research of the Hospital Albert Einstein (Number 56229916.9.0000.0071; São Paulo, Brazil), which is part of the Ethics Committee of the Brazilian Ministry of Health (CONEP).

Patients

The endometriosis group (n = 8) consisted of patients with unsatisfactory pelvic pain improvements after clinical treatment, who maintained complaints of dysmenorrhea, deep dyspareunia, chronic pelvic pain and/or cyclic intestinal alterations with proven histological diagnosis of the disease. The control group (n = 8), consisted of women submitted to laparoscopy for benign gynecologic diseases (uterine fibroids or ovarian benign cysts) and had confirmation of absence of any endometriosis focus at the time of the surgical procedure. All patients were between 18 and 45 years old, had eumenorrheic cycles, with intervals of 26–34 days between the cycles and non-use of hormonal therapy 3 months prior to surgery, including GnRH analogues, progestins and combined hormonal contraceptives. In the endometriosis group, 4 patients were in the secretory phase, 3 in the proliferative phase and 1 in the menstrual phase of the cycle. In the control groups, 5 women were in the menstrual phase, 2 in the proliferative phase and 1 in the secretory phase of the cycle. For division criteria, proliferative phase was considered from day 5 after menstruation until day 15, the secretory phase, from day 16 until day 34 of menstrual cycle.

Collection and processing of biopsy samples

Endometriosis group had resection of endometriotic lesions, including deep retrocervical lesions and rectum wall nodules [39]. Endometrium was collected using a Pipelle curette from both groups. The biopsies obtained from both endometrium and endometriotic lesions were transported in sterile cryotube immersed on ice to the experimental laboratory of the Hospital Israelita Albert Einstein. The material was frozen and kept in a freezer at − 80 °C until the experiments were carried out.

The tissue was immersed on Tissue-Tek OTC Compound before cryosectioning. Cryosections of the frozen material were made and fixed on a correctly identified slide and stored at − 80 °C until used for immunofluorescence or immunohistochemistry procedures. For immunofluorescence, and immunohistochemistry techniques, all selected tissue fragments were sectioned in 5 μm thickness.

Immunofluorescence for p16 [25]

The cross-sectional samples were thawed; fixed in 4% paraformaldehyde; washed in PBS with 0.5% Tween; immersed in Triton X-100 solution. Subsequently, the samples were incubated overnight with monoclonal antibody to p16INK4A (1:100, Rabbit monoclonal (EPR1473) to CDKN2A/p16INK4a tag: Abcam) mixed in 0.5% Tween and 5% BSA. The slides were then washed in PBS with 0.5% Tween and the secondary antibody (1:600, Goat Anti-Rabbit IgG H & L (Alexa Fluor®) (ab150116)) was added. The evaluation was performed through photographs using a confocal microscope.

Immunohistochemistry for lamin B1, p16 and p21

Immunohistochemistry was accomplished using the marking platform BenchMarck ULTRA IHC/ISH from Ventana Medical System Inc. (Tucson, Arizona, USA]. Basically, it followed a standard protocol for frozen materials. Briefly, slides were blocked with hydrogen peroxide; incubated with primary antibody Lamin B1 (H-90), Rabbit IgG—200 μg/mL—(1:200, Santa Cruz, SC-20682); monoclonal antibody to p16INK4A (1:150, Rabbit monoclonal (EPR1473) to CDKN2A/p16INK4a tag: Abcam); monoclonal antibody p21 [1:200, Rabbit monoclonal (EPR3993), Abcam]. The samples were incubated with polymer, chromogen and stained with hematoxylin. To finalize the procedure, the slides were dehydrated in alcohol for assembly with coverslip and mounting medium.

Histometry [40, 41]

The samples were cryosectioned 4 times each with 4 cuts in between each section and 4 different fields of photographs were taken for histometry procedure, resulting in 16 digitalized images per samples to be analyzed. The captured images were used to quantify the findings through a computer-based image analysis system (Olympus cellSens Dimension 1.16). Each corresponding protein detected field was histometrically evaluated by area percentage; a blinded experiment for histometric assessment was carried out.

Statistical analysis

Primarily, data analysis was done qualitatively. From the results produced by histometry (quantification of cells or amount of stained areas per field) percentage values were obtained and compared with the groups of the binary questions (lesion vs. eutopic endometrium of patients with endometriosis and endometrium of patient with endometriosis vs. patient free disease endometrium). All data analyses were performed using SAS software, version 6.11 (Statistical Analysis System Institute Inc., Cary, NC, USA). A multilevel linear mixed effect regression model was used to analyze the histometric results of p16 and lamin B1 in the groups. Results are presented as least square mean ± standard error. Statistical significance was defined as p < 0.05.

Results

No significant differences between control and endometriosis groups concerning mean age (38.25 ± 6.54 and 38.38 ± 6.80 years, respectively) or mean body mass index (21.99 ± 2.79 and 22.48 ± 4.21 kg/m2, respectively) were found out.

Validation studies were conducted using standard immunohistochemistry and immunofluorescence protocols and adjusted according to the obtained results [25]. For lamin B1, p16 and p21 antibodies, uterine tubal, ovarian carcinoma, and rectum tissue were used as positive control, respectively.

Histometric measurement revealed detection of lamin B1 with a reduced presence in endometriotic lesions when compared with eutopic endometrium (13.06% ± 5.60 and 20.94% ± 5.60, respectively). Endometrium from control patients (36.28% ± 5.60) had no lamin B1 detection significantly different when compared to endometriotic lesions nor endometrium from patients with endometriosis (Fig. 1). Data are presented as mean ± standard error mean (SEM).

Fig. 1
figure 1

Lamin B1 protein detection on ectopic and eutopic endometrium. Representative immunohistochemistry images of lamin B1 staining [brown] of uterine tubal as positive control (a); normal endometrium (b); eutopic endometrium (c) and ectopic endometrium (d). Graphical representation of the percentage of lamin B1 stained cells analyzed by histometry (e). Significance is indicated with an *p < 0.001

Full size image

Results of p16 immunofluorescence and immunohistochemistry analysis (Fig. 2a–h) generated no significant differences among those three groups (Fig. 2i). After all validations, the p21 was not identified in lesions and eutopic endometrium from both groups (Supplementary Fig. 1), thus this enzyme was not used for any comparisons in this study.

Fig. 2
figure 2

P16 protein detection on ectopic and eutopic endometrium. Representative immunohistochemistry images of p16 staining [brown]: ovarian carcinoma as positive control (a); normal endometrium [33,29 ± 3,72 (b); eutopic endometrium [22,52 ± 3,72 (c); ectopic endometrium [21,31 ± 3,72] (d). Representative immunofluorescence images of p16 staining [red]: ovarian carcinoma as positive control (e); normal endometrium (f) eutopic endometrium (g); ectopic endometrium (h). Graphical representation of the percentage of p16 stained cells analyzed by histometry (i). Data are presented as mean ± standard error mean [SEM]

Full size image

For the P16 analysis, the sample size (n = 8) was suitable with a 80% statistical power, and 5% of significance, considering a difference of at least 5 ± 3.52% (absolute value ± standard deviation) between groups (women with endometriosis and control without endometriosis) [42].

Discussion

In the present study, no differences in cyclins commonly associated with cancer were observed; however, lamin B1, an important protein responsible for structural integrity of the nucleus, cellular apoptosis and senescence [43, 44], was reduced in deep infiltrating endometriosis lesions (Fig. 1). Reduced lamin B1 has also been described in gastric and breast cancer [38, 45]. These changes can cause cell cycle arrest and proliferation inhibition, leading to apoptosis or senescence, and senescence could help the maintenance of some types of cancers [46]. Proliferation inhibition associated to lamin B1 loss and was found explicitly in senescent fibroblasts, keratinocytes and human diploid cells [43, 47,48,49,50,51]. In addition, the direct knockdown of lamin B1 triggers cells to exhibit premature senescence phenotypes [52]. Dutta et al. evaluating oxidative stress and preterm premature rupture of the membranes, using lamin B1 as one of the markers, suggest that lower lamin B1 expression in preterm premature rupture of the patient membranes was associated with loss of nuclear envelope integrity and with cellular senescence, pointing the latter as a potential mechanism involved in premature rupture [53]. In the endometriosis scenario, a reduced lamin B1 can be related to apoptosis resistance observed in endometriosis [22, 54, 55], even though the possibility of the involvement of senescent cells in the endometriosis pathology should not be ruled out.

Alterations in lamin B1 expression itself may not lead to cellular senescence, but additional stress on lamin B1 depleted cells may enhance and accelerate cellular senescence [44, 46, 50, 51]. It is known that endometriosis patients have higher pro inflammatory cytokines, macrophages and metabolites in the peritoneal cavity [12, 54]. Oxidative stress and increased estrogen concentration are also endometriosis features [56,57,58,59] which are considered pro-senescence factors. Thus, we speculate that the lower lamin B1 detection in endometriotic lesions described in the present study, coupled with additional peritoneal stress inherent of endometriosis patients, potentially indicate the presence and involvement of senescent cells in ectopic endometrium, therefore, helping the lesion maintenance and progression of the disease. This situation would aid in the maintenance of the characteristic disease inflammatory microenvironment through the senescence secretory phenotype, inducing the establishment of more senescent cells in the endometriotic lesions. However, it is unclear if the reduced lamin B1 detection indicates cellular senescence in endometriosis and whether this finding is a cause or consequence of the disease.

Besides commonly associated with malignant diseases [28], p16 is a robust marker of senescence cells and aging [27]. Here, we demonstrated p16 detection in deep endometriosis lesions, but no difference was seen among the studied groups, possibly because of the small and heterogeneous group of patients, since no distinction was made regarding the menstrual cycle phase. p16 could be a more sensitive marker and estrogen menstrual cycle variation could be regulating p16 expression [60]. Few studies correlate endometriosis with p16 expression. p16 expression was measured in two groups of polypoid endometriosis. One group had p16 equally expressed in comparison with non-polypoid endometriosis patients, in the other group, p16 was similarly expressed as in normal endometrium polyps, a disease that usually affects older individuals, with age-associated senescent cells [31]. The authors suggest that these results reflect the activation of cellular senescence pathways in polypoid endometriosis, corroborating with our hypotesis. Similarly, Moritani et al. assessed the p16 expression in endometrial polyps and endometrial hyperplasia, describing increased p16 expression in stromal cells of endometrial polyps that can be associated with the senescence state of these cells [61].

Conclusion

In conclusion, although using a limited but well characterized group of samples, this report was able to describe for the first time a reduced presence of lamin B1 in endometriosis lesions compared with eutopic endometrium from patients with deep infiltrating endometriosis, associated with no differences in p16 and with the absence of p21. Even though endometriosis has common features with cancer, as proliferation and invasion, the oncogenes [p21 and p16] that regulate these features in tumorigenesis were not dysregulated in our groups. The present results indicate that the role of lamin B1 and p16 should be further elucidated in the context of endometriosis pathogenesis and in the involvement of senescent cells in endometriosis.