GRN, NOTCH3, FN1, and PINK1 expression in eutopic endometrium – potential biomarkers in the detection of endometriosis – a pilot study

Endometriosis (EM) is defined as the presence of functional endometrium in any location outside the uterine cavity. The ectopic endometrial cells can be present in the ovaries, peritoneum, and fallopian tubes and even in distant organs such as the lung and brain [15]. EM typically causes dysmenorrhea, dyspareunia, and pelvic pain and may contribute to infertility [9]. It affects approximately 10–15% of women of reproductive age and can even be found in 35–50% of women suffering from pelvic pain and/or infertility [14]. However, the prevalence might be underestimated due to non-specific symptomatology and late diagnosis caused by a lack of non-invasive methods for detecting. According to recent estimates, the diagnosis of EM is delayed by an average of 7–11 years [18]. So far, there is no reliable clinical marker and the gold standard diagnostic method is invasive laparoscopy [19]. EM can be graded according to the revised American Society for Reproductive Medicine (rASRM) staging system in minimal, mild, moderate, and severe stages of the disease (ASRM I–IV), depending on the character, expansion, and localization of the lesions as well as the presence of adhesions [16]. It is the most common international classification of EM; however, the infiltration depth and severity of symptoms are not included in this classification. None of the patients included as ASRM I and II had a deep infiltration.

Although various theories about the pathogenesis of EM have been submitted, none of them is proven so far and the exact causes and pathogenetic pathways of this heterogenetic disease are still not fully understood [42]. Furthermore, it has been suggested that within the disease, there are distinct entities and pathogenesis [24]. Clinical studies have shown different implantation rates in women with mild versus severe EM, assuming a difference in function and biochemistry of these two stages of EM [20].

Although EM is considered a benign disease, it shares several characteristics with malignancy including excessive proliferation, cellular invasion, peripheral metastasis, inflammation, and estrogen dependency [21]. Patients with EM even suffer a slightly increased risk of developing ovarian carcinomas, especially of the endometrioid and clear cell subtypes [1, 7, 28].

Considering these circumstances, EM is one of the most common female health disorders and has a great impact on the patient’s quality of life [36]. Thus, it is necessary to identify potential molecular biomarkers for diagnosis and regulatory factors, underlying the progress of EM.

Herein, the objective of our study was to investigate molecular differences in the expression of specific genes and proteins in eutopic endometrium from women with and without EM. We analyzed changes of these proteins and genes in different stages of the disease in an attempt to determine the degree of the disease without the necessity of an abdominal surgery. We wanted to validate the assumption of a potential instinct pathogenesis and entity of mild and severe endometriosis by showing a difference in gene and protein expression. Furthermore, we tried to investigate proteins that have the potential for use as clinical biomarkers to enable an earlier diagnosis without the need for laparoscopy. We focused on proteins that are already known to be involved in inflammatory diseases, cell adhesion, and migration as they are assumed to play an important role in the pathogenesis of endometriosis. Furthermore, we wanted to investigate proteins that have already been described in connection with implantation, cell development, or blastocyst maturation. Therefore, we chose to examine the gene and protein expression of progranulin (GRN), neurogenic locus notch homolog protein (NOTCH3), fibronectin (FN1), and phosphatase and tensin homolog–induced kinase 1 (PINK1).

In this study, we show differences in protein and gene expression of selected genes among the different degrees of EM, according to the ASRM classification, as well as compared with the endometrium of healthy controls. For the first time, we demonstrate an association of GRN, NOTCH3, FN1, and PINK1 with the disease of EM. Their role in other tissues and physiological or pathological mechanisms has been described in many other studies [3, 10, 30, 39]; the regulation of the selected proteins in human EM however remains to be examined.

GRN is a pleiotropic glycoprotein and growth factor with proliferative, invasive, and anti-inflammatory properties [17]. Despite the fact that a couple of studies have attributed GRN a role within blastocyst development and implantation of mice and mink [12, 13, 31], very little is known of its function in human endometrium. In our study, GRN mRNA expression is considerably upregulated in women with EM compared with that in healthy controls, which may contribute to its role in inflammation and cell proliferation process. Moreover, our findings of GRN protein expression show a significant continuous reduction in expression level the higher the degree of the disease. While Qin et al. demonstrated that the addition of recombinant GRN in blastocyst culture media promoted blastocyst hatching, adhesion, and outgrowth, rabbit anti-mouse GRN IgG reduced that effect [31]. The reduction of GRN in the endometrium of women with EM may have a negative effect on blastocyst development, potentially contributing to a reduction of implantation rates in affected women, as seen in women with EM according to the severity of the disease [37].

There are few studies revealing Notch expression in the endometrium [2, 25, 26]. It is known that the Notch signaling pathway plays a fundamental role in the development of diverse organisms including mammals [3]. Currently, the Notch system is believed to impact differentiation, proliferation, and apoptosis and last but not the least influence organ formation and morphogenesis [3]. In addition, the Notch genes seem to influence the regulation and remodeling of the vascular system [34], including angiogenesis in endometrial cancer [27]. As little is known about the function of Notch signaling in the reproductive tract, this is the first study showing the effects of NOTCH3 in EM. Similar to the findings of Mikhailik et al. (25) describing NOTCH 1–3 expression in both, endometrial stromal and epithelial cells, we also observed a positive staining in these two cell types. Furthermore, while Shawber et al. described NOTCH3 as the only Notch ligand not expressed in capillary endothelial cells but in pericytes in the preimplantation uterus of mice [35], we found a moderate immunopositivity of NOTCH3 in the endothelium of endometrial blood vessels. Similar to the findings of Cobellis et al. showing a nuclear expression of NOTCH1 and NOTCH4 in the endometrium [11], we also detected positive immunostaining of NOTCH3 exclusively in the nuclear cell compartment. Therefore, our results indicate that not only NOTCH1 and 4 but also NOTCH3 plays an important role in angiogenesis of the endometrium. Our investigations on mRNA level exhibited a particular elevated level of NOTCH3 gene expression in women with minimal EM compared with women without EM, as well as an overall elevated expression. The fact that Notch signaling protein expression is increased in human endometrial carcinoma cells compared with that in healthy endometrial cells [27] is in line with the assumed effect of NOTCH3 in proliferation and differentiation [3]. Furthermore, this observation poses again the question of the parallel operations between EM and malignancy. Caused by its overexpression in several types of cancers, NOTCH3 has already been investigated as a target for anticancer drugs [6]. In addition, recent studies showed an activation of the Notch domain and its abnormal signaling in connection with chronic inflammatory diseases leading to pathological fibrotic processes [22], which underlines the possible relevance of NOTCH3 in the development of EM. Therefore, our findings of overexpression of NOTCH3 in patients with EM may open a new perspective of NOTCH3 as a target for EM therapy, particularly in early disease.

FN1 is a glycoprotein of the extracellular matrix, which is involved in important cellular mechanisms including cell adhesion, migration, wound healing, blood coagulation, and even metastasis [30]. Some studies already analyzed FN1 and its role in the endometrium and implantation [8, 40, 41], as well as in pathogenesis of EM [29, 32]. Sapkota et al. identified in a meta-analysis of SNPs the association of the FN1 locus with EM, mainly with moderate-to-severe EM [32]. Based on these genomic findings, we wanted to investigate the role of FN1 directly in eutopic endometrial tissue of women with EM. Others, like Beliard et al., suggested FN1 could have a function in persistence of endometriotic lesions and found elevated FN1 receptor expression in the endometriotic glands in peritoneal lesion compared with the eutopic endometrium of healthy controls [5]. While they had a small number of eutopic and ectopic tissues during different phases of the menstrual cycle, they did not find a difference in FN1 expression between the tissues [5]. In contrast to their study, we used only mid-to-late proliferative samples with clearly determined stages of EM disease, where we were able to detect changes in protein expression showing a stage-dependent reduction of FN1 expression, suggesting that not only the receptors are regulated in endometrium of women with EM, but also the FN1 protein itself plays a role in endometriosis. Furthermore, while Beliard et al. demonstrated an upregulation of FN receptor within epithelium of ectopic lesions [5], FN1 as seen in our IHC is exclusively expressed in the capillary endothelium and endometrial stromal cells and not in the epithelium. The localization of FN1 in the endothelium could potentially contribute to its role in angiogenesis that is depicted in tumor pathogenesis [23]. As FN1 and its receptors are believed to be important in mammalian reproduction and placentation [8], with a particular impact on blastocyst adhesion and implantation [40], the dysregulation of FN1 in the eutopic endometrium of women with EM may potentially affect the implantation potential of patients with EM. However, this remains to be further analyzed.

Mutations in PINK1 seem to play an important role in the pathogenesis of Parkinson’s disease [38]. Besides that, little is known about PINK1’s function in other human tissues. Herein, we investigated for the first time the role of PINK1 in the eutopic human endometrium of women with and without EM. Our data show a mild expression of PINK1 in stromal cells and a moderate expression in the glandular epithelium of the human endometrium of healthy women as well as in the endometrium of all different types of EM. We even could find a mild increase, however not significant, in women with endometriosis ASRM I and even further with ASRM II, which gets lost in ASRM III. Interesting enough, Barodia et al. describe that a deficit of PINK1 results in increased mitochondrial autophagy leading to an accumulation of reactive oxygen radicals [4]. Recent studies defined oxidative stress as an important factor in the pathophysiology of endometriosis due to an imbalance between reactive oxygen species and antioxidants, resulting in an inflammatory response within the peritoneal cavity [33]. While we do not observe a reduction in PINK1 expression in women with EM, we experience a dysregulation of PINK1 with a particular high gene expression in minimal disease that vanishes over the progression of the disease. The importance of this finding within the proliferative phase of the cycle remains to be elucidated further.

To the best of our knowledge, this is the first study assessing GRN, NOTCH3, FN1, and PINK1 among the different degrees of EM. We could show differential expression of these genes and proteins within the different stages of EM as well as to healthy endometrium. These findings may support the suggestion of a potentially distinct entity and physiopathology between minimal, mild, and moderate EM already notable within the eutopic endometrium. Furthermore, our results open a new field of interest regarding potential biomarkers of EM as well as a better understanding of differences in gene and protein expression in women with EM. However, a prospective validation is required for the actual function of the investigated proteins and genes in EM and their use as clinical biomarkers.