Assessment of expression of stem cell markers in eutopic and ectopic endometrium in women with ovarian endometriotic cysts

Classic Sampson’s theory about the development of endometriosis is based on the fact that endometrial cells gain entrance to the abdominal cavity during the retrograde menstruation [1].

As far as back 1978, V.A. Pryanishnikov postulated that stem cells play the role in the cyclic regeneration of the endometrium [2]. And 30 years later, an assumption about the role of stem cells in the pathogenesis of endometriosis was made in a number of works [3]. Cell populations with the qualities of stem cells were found on the basal membrane of the endometrium [4]. Their detection opened new perspectives in the study of endometrium pathogenesis [5]. Two populations of stem cells, namely mesenchymal stem cells and epithelial progenitor cells, were identified in the endometrium in the research of 2007, and in 2011 stem cells were already identified in the foci of endometriosis [6, 7]. The obtained data led to the conclusion that endometriosis implants were formed due to the spread of stem cells of the endometrium through the fallopian tubes during the menstruation [5].

Stem cells role can be confirmed by the fact of increased invasion in the culture of stromal endometrial cells, received from patients with endometriosis [6]; however, the question about triggering mechanism, which is able to activate stem cells in eutopic and ectopic endometrium, is still open [8].

Genetic and immunological factors, potentiating the survival of cells in ectopic foci, may play an important role. Numerous studies revealed genome differences between eutopic and normal endometrium, confirming the fact that endometrial stem cells/progenitors can differ in women with and without endometriosis [9]. Higher levels of cloning were found in the normal endometrium in comparison with eutopic endometrium and higher levels were in eutopic endometrium in comparison with ectopic one [7]. This fact supports the opinion that stem cells/progenitors require external factors for the activation of proliferation and differentiation in ectopic foci.

Perivascular endometrial mesenchymal stem/stromal cells (eMSC) are localized both in basal and in functional layer of the endometrium. The assumption about their spread to the abdominal cavity during menstruation is confirmed by the data that menstrual blood of women with endometriosis contains more fragments of basal endometrium in comparison with the control group [9]. eMSC can be identified by special markers, including double positivity CD146 and platelet derived growth factor beta (PDGFRβ) and SUSD2+ (recognized with the help of antibodies W5C5) [10]. Probably, the answer to the question, why 90% of women monthly experience spread of endometrial mesenchymal cells, and endometriosis starts only in 6-10% of them, could be given by the studies aimed at examination of differences between expression 146+PDGFRβ+ endometrial stem cells in the functional layer in comparison with the basal one [11].

SSEA (stage-specific embryotic antigen 1, in other words CD15) which is a marker of differentiated pluripotent stem cells of a human (or neutrophils, in which CD15 mediates phagocytosis and chemotaxis) was found in ectopic peritoneal endometrial foci during the studies of 2013 [12]. CD15 is a molecule of carbohydrates adhesion and can be expressed on glycoproteins, glycolipids and proteoglycans. In the year 2016 during the studies of expression of the given marker in the endometrioid heterotopias and autological endometrium, A.M Khachatryan et al. discovered its increased expression in the epithelial component of endometriosis foci [13]. According to the authors, the increased expression confirms relevant immaturity of endometrioid heterotopias.

Taking into consideration the growing interest in the role of stem cells in the endometriosis pathogenesis, we decided to conduct a study of cells, positive to PDGFR, which is protein, regulating proliferation, differentiation and growth of cells, and also SSEA-1, which is a mediator of cellular interaction in eutopic and ectopic endometrium in women with ovarian endometriotic cysts and in normal endometrium.

Materials and Methods

The study included 94 patients of reproductive age, and the patients were divided into 2 groups according to the presence or absence of ovarian endometriotic cysts. Group I included 70 patients with ovarian endometriotic cysts and Group II included 24 patients with unexplained infertility without endometriosis.

Reproductive age, ultrasound signs of ovarian endometriotic cysts, laparoscopically and morpho­logically confirmed diagnosis of ovarian endometriotic cyst became the criteria of inclusion into Group I (main). Women of reproductive age who underwent laparoscopy revealing no endometriosis were enrolled in Group II (the comparative). Patients with extragenital endometriosis, pelvic varicose veins, ovarian tumor or tumors of other localization and also with accompanying decompensated extragenital pathology were excluded from the study.

All the studies were carried out with the permission of local ethics committee (protocol №50/5, dated on 18th November, 2014) and with the voluntary informed consent of patients for the participation in the research.

Patients were offered to fill in a questionnaire for the analysis of anamnestic data before the operation. Then we performed analysis of clinical and anamnestic indices and data of gynecologic examination.

At the next stage of study, the patients of Group I were performed endoscopy to diagnose external genital endometriosis, to confirm the presence of ovarian endometriotic cyst and to estimate the severity of endometriosis (according to the reviewed classification of the American Fertility Society). In the course of operation endometriotic cyst was removed; salpingovariolysis and hysteroscopy with sampling for immunohistochemical study were performed. Patients of Group II underwent laparoscopy, chromohydrotubation and hysteroscopy; endometrial biopsy for the immunohistochemical study was taken. Unexplained infertility served the indication for using laparoscopy. Operative interventions were performed in the patients of both groups within the period from the 8th till the 11th days of the menstrual cycle.

Ultrasound scanning of pelvic organs was performed within the period from the 5th to the 7th days of the menstrual cycle with the help of ultrasound diagnostic equipment «Toshiba (Eccocce) SSA340» using convex electronic endovaginal probe with the frequency of 3.5-6.5 MHz (Japan).

Laparoscopic surgery and hysteroscopy were performed with artificial ventilation of the lungs under general anesthesia. The operations were done using the equipment of the company “Karl Storz” (Germany) at the operating theatre of gynecological department. Therewith, cystectomy in women from Group I was performed using the technique of preserving maximally normal tissue of the ovaries [14, 15].

The material obtained in the course of operation was placed in disposable containers with the fixing solution (10% buffered formalin of neutral reaction), the ratio of the solution to the tissue was 10 and more to 1. Cuts with the width of more than 4 microns were obtained with the help of rotation microtome Accu-Cut SRM, Sakura.

Immunohistochemical test was prepared according to the standard scheme of histological test. Histological cuts were dewaxed before staining. The following antibodies were used during the work:

• antibodies PDGFR Rabbit Polyclonal 0.1 ml (Spring Bioscience Corporation);
• mouse monoclonal antibodies to CD15 0.1 ml (Cell Marque Corporation, 6600 Sierra College Park Boulevard Rocklin, Ca 95677 United States).

Immunohistochemical reaction results were estimated with the help of rating scale: 0, 1+, 2+, 3+; herewith 0 was for the absence of membrane staining or presence of weak staining of less than 10% of cells, 1+ was for weak membrane staining of more than 10% of cells, 2+ was for moderate membrane staining of more than 10% of cells or strong membrane staining of less than 30% of cells, 3+ was for strong full staining of membranes of more than 30% of cells. Since such coding of variables demonstrated their low variability and low informative value, it was decided to use their exact percentage expression.

For obtaining quantitative features in the compared groups we estimated medium and its standard deviation, medians with the explicitation of 25% and 75% of percentile (1-3 quartiles). For the estimation of intergroup differences non-parametrical Mann-Whitney test for the independent samples was used, and fourfold tables were analyzed. In case of fourfold tables analysis with the expected presence of less than 10 in one box, we calculated criteria χ2 with Yates’ correction, which allowed us to reduce the probability of type I error, which means finding differences in those places where there are no differences. In those cases when the number of expected observations in any box of the fourfold table was less than 5, we used Fisher’s exact test for the estimation of the significance level of differences.

Statistical processing of original features was performed using Statistica software package, version 12.5, EXCEL 2010, SPSS 25.002 and package MedCalc 18.9.1.

Results and Discussion

According to the clinical and anamnestic estimation of patients with ovarian endometriotic cysts and patients without endometriosis, there were no differences in age, age at menarche, length of menstrual cycle and algomenorrhea between groups (р = 0.6). Statistically meaningful differences were found concerning hyperpolimenorrhea (p = 0.003) (Table 1, 2).

Analysis of different gynecological diseases revealed high frequency of cervical intraepithelial neoplasia in patients with endometriosis in comparison with those without endometriosis (Table 3).

The degree of endometriosis stage and adhesive process was defined according to the classification of the American Society for Reproductive Medicine (1996). In 100% of cases, patients with ovarian endometriotic cysts had III-IV stage of endometriosis spreading and the severity of adhesive process was much higher than in patients of Group 1 (Table 4).

According to the obtained results, statistically significant differences were discovered in patients of Group I in eutopic and ectopic endometrium with all markers both in stroma and in glands (р < 0.05), herewith CD15 and PDGFR levels were higher in ectopic endometrium in comparison with the eutopic one (Table 5).

Thereafter, test items were analyzed in eutopic and normal endometrium. Statistically significant differences of PDGFR and CD15 were noticed in glands with the increase in eutopic endometrium (Table 6).

Thus, statistically meaningful differences between eutopic and ectopic endometrium for both markers were found both in stroma and in glands.

The number of CD15 (SSEA) positive cells was identical both in eutopic and in normal endometrium in stroma. According to the mathematical analysis, the number of these cells is limited, but it statistically significantly increased in eutopic endometrium. Wherein, their number in ectopic endometrium exceeds manifold their number in eutopic endometrium, which confirms the fact of possessing endometrioid heterotopias of higher expression of stem cells markers [12, 13].

The fact that ectopic endometrium is characterized by the significant increase of PDGFRβ positive cells in comparison with eutopic one is very important. Herewith, we noticed only slight differences between eutopic and normal endometrium for this marker. The increase of level of PDGFRβ positive cells is evident of possible increase of eMSC in eutopic and ectopic endometrium in endometriosis.

Conclusion

Since the year 2007, the attention of doctors has been focused on the importance of the stem cells role in the pathogenesis of endometriosis. This fact does not exclude, and on the contrary, unifies all currently existing theories of this pathology development. The complexity of stem cells identification is the main constraint for the study of this question. In the presented work we studied two stem cells markers not only inside the endometriotic cysts, but also in eutopic endometrium of women with endometrioma. Eutopic endometrium was also compared with the normal one (in women without endometriosis). As a result, we noted a significant increase of PDGFR and CD15 positive cells in ectopic endometrium. Eutopic and normal endometrium have insignificant but statistically meaningful difference in CD15 and PDGFR in gland with the dominance in eutopic endometrium. Impressive increase in number of PDGFR and CD15 positive cells inside the endometriotic cysts confirms the role of stem cells in endometriosis pathogenesis.

The question about what «motivates» stem cells to migrate to the peritoneal cavity and, in particular, to the ovaries, forming sites of endometriosis is the topic for further studies, where the unequivocal role will belong to the study of genetic and epigenetic factors. The fact that eutopic endometrium of patients with ovarian endometriotic cyst is slightly, but still different from the normal endometrium still requires explanation.

In the long term, such studies will make it possible to substantiate etiopathogenetic therapy, aimed at decreasing the “influx” of stem cells to the endometriosis foci.