Decreased expression of Syndecan- 1 (CD138) in the endometrium of adenomyosis patients suggests a potential pathogenetic role

Walid Shaalan: Investigation, data curation, formal analysis, visualization, writing—original draft. Mohamed Gamal Ibrahim: Methodology, data curation, supervision. Ariana Plasger: Funding acquisition, visualization. Nourhan Hassan: Funding acquisition. Ludwig Kiesel: Resources. Andreas N Schüring: Supervision, conceptualization, methodology, project administration. Martin Götte: Conceptualization, project administration, methodology, supervision, funding acquisition; all authors: writing—review & editing.

This study received Institutional Review Board approval according to the principles of the Helsinki Convention and was approved by the local ethics committee (registration no. 1IX Greb) on September 19, 2001, renewed December 6, 2012 (Ethikkommission der Ärztekammer Westfalen‐Lippe und der Medizinischen Fakultät der WWU), and all participants gave written consent. All relevant clinical data relating to the patients were documented. Clinical data were obtained from the patient records.

This research was funded by EU H2020‐RISE project TRENDO, grant # 101008193 (to M.G.), the Clinician–Scientist‐Programme CareerS Starter, grant CS‐Starter‐2023‐27 of the Medical Faculty of Münster University (to A.P.), and the German Academic Exchange Service (DAAD) German–Egyptian Research Long‐Term Scholarship program (GERLS) Grant 91664677 (to N.H.).

The age in both premenopausal groups, aged 30–53 years old, showed no significant difference, with a mean age of 42.5 and 43.2 years in the adenomyosis group and control group, respectively (Table  2 ). Moreover, no significant difference was observed in patients with adenomyosis compared to controls regarding parity, whether nulliparous or multiparous. Comparison between the two studied groups according to age (years). Note : t , Student's t ‐test; p , p ‐value for comparing between the two groups. Figure  1A shows an example of the glandular staining of the endometrium in the control group, while Figure  1B shows the glandular staining of the eutopic endometrium of adenomyosis patients. Figure  1C shows the SDC‐1 expression in the ectopic endometrium of adenomyosis patients. Figure  1D shows the membranous staining of the luminal epithelium of the eutopic endometrium using 100× magnification to display the endometrial junction. Immunohistochemical staining of Syndecan‐1 (Sdc‐1) in different patient cohorts at 200× magnification. Sdc‐1 expression is displayed as membranous staining of the glandular epithelium in the control group (A) and in the adenomyosis patients (B, C, D). Sdc‐1 expression in adenomyosis patients via staining of eutopic endometrium (B) and ectopic endometrium (C). Staining of eutopic endometrium and the endometrial junction for Sdc‐1 at 100× magnification (D). In the first step, we investigated the SDC‐1 expression in the three groups; ectopic endometrium compared to the eutopic endometrium of adenomyosis patients compared to the control group using the Kruskal–Wallis test. Analysis of the samples demonstrated the following differences between the three groups (Figure  2 ). The protein expression was reduced at a significant level in the glandular cells of the ectopic endometrium and the eutopic endometrium in the adenomyosis group compared to the control group. The SDC‐1 expression showed no significant difference between the eutopic endometrium and the ectopic endometria of patients with adenomyosis. Comparison of the three studied groups according to Syndecan‐1 (Sdc‐1) expression. Sdc‐1 expression was determined via “histoscore” (H‐score) considering membrane staining intensity for different intensity levels. Results are shown as means ± SD for ectopic endometrium (left, n  = 21) and eutopic endometrium (center, n  = 21) staining in adenomyosis patients compared to endometrium in a control group (right, n  = 14). Means were compared via Kruskal–Wallis test. p ‐value is shown if statistically significant at p  ≤ 0.05. The analysis of SDC‐1 expression in proliferative and secretory endometrium of adenomyosis patients revealed that the expression of epithelial SDC‐1 does not correlate with the cycle phase in patients with adenomyosis and there were no significant differences in the staining index of glandular immunostaining for SDC‐1 as shown in Figure  3A . Like the findings in the adenomyosis group, Figure  3B showed that the immunostaining of Syndecan‐1 does not correlate with the cycle phase in the glandular epithelium of the control group. Expression of Syndecan‐1 (Sdc‐1) in correlation with menstrual cycle phases (Proliferative vs. Secretory). Sdc‐1 expression was determined via “histo‐score” (H‐score) considering membrane staining intensity for different intensity levels. Results are shown as means ± SD for adenomyosis patients group (A) and control group (B). Means were compared via Mann–Whitney U ‐test and p ‐value was determined as statistically significant at p  ≤ 0.05, ns, non‐significant.

Gynecological diseases such as endometriosis, endometrial cancers, and adenomyosis are believed to arise from abnormalities in endometrial cell proliferation. 27 Our study sheds light on the significant association of SDC‐1 in human adenomyosis uterine tissues, providing novel insights into potential endometrial stem cell markers in the etiology of this disorder. We reported in our study the impact of SDC‐1 on cellular functions in the human endometrium. So far, Syndecan‐1, a heparan sulfate proteoglycan, has been shown to influence invasiveness, inflammatory cytokine secretion of epithelial endometriotic cells, 28 and matrix metalloproteinases (MMPs) expression which are consistently expressed across the entire menstrual cycle, suggesting their potential role in the physiological stabilization of the endometrium. 29 Previous studies have investigated the expression of Syndecans in the endometrium, revealing that all Syndecans were expressed within the human endometrium of normal cycling women; however, the significant increase of Syndecan‐1 and ‐4 expression in the whole endometrium during the secretory phase suggests their role in the regulation of the cycling endometrium. 19 In addition, Chelariu‐Raicu et al. reported a significant increase in the expression of Syndecan‐4 in the glands and stroma of patients with endometriosis compared with the controls, without any observation regarding menstrual‐cycle dependent expression. 30 This is in contrast to our findings for SDC‐1 expression in adenomyosis patients. In our study, we observed the expression patterns of SDC‐1 in both eutopic and ectopic endometrial tissues from women with adenomyosis and compared these patterns with a control group. Previous studies have demonstrated cyclical variation in SDC‐1 expression in healthy endometrium, with higher levels observed in the secretory phase compared to the proliferative phase. 31 , 32 However, our control group did not display this same cyclical pattern. Our control group includes a variety of benign gynecological conditions, not exclusively healthy endometria. This group encompasses cases such as fibroids, which could potentially influence the endometrial environment and, consequently, the expression of SDC‐1. 33 The presence of such conditions within the control group likely contributed to the observed lack of a clear cyclical pattern in SDC‐1 expression. This variation within the control group highlights the complexity of studying endometrial tissue and suggests that the benign pathologies included in the control group may have affected the overall expression of SDC‐1. In our pilot study, we observed a significant decrease of SDC‐1 immunohistochemical staining in the ectopic endometrium compared to the control endometrium, highlighting a regulatory duality in cell proliferation mechanisms that appear to be tissue‐ or tumor‐specific. SDC‐1 overexpression has been linked to hormone‐related cancers like ovarian and endometrial cancer as described by Davies et al. 34 and has been associated with the occurrence and progression of endometrial cancer, which may relate to the promotion of tumor growth and angiogenesis, 35 and is considered a negative prognostic marker regarding the overall survival and the Progression‐free survival. 35 , 36 Conversely, in non‐hormonal dependent tumors such as lung and cervix cancers, the malignant transformation is associated with the downregulation of SDC‐1, aggressive phenotypes of cancers, and poor prognosis. 37 , 38 The role of SDC‐1 in breast tumors remains controversial, with studies reporting conflicting correlations with disease severity and prognosis. Some histopathological studies have demonstrated a correlation of SDC‐1 expression with poorly differentiated grade, high proliferation index, and triggering the hematogenous metastatic spread of breast cancer cells. 39 , 40 In contrast, Loussouarn et al. found a significant correlation between the loss of epithelial SDC‐1 expression and stromal expression with a high grade of malignancy and relapse‐free survival in invasive ductal carcinoma. 41 Furthermore, along with our study, previous work revealed that SDC‐1 decrease may contribute to the pro‐invasive properties of miR‐10b in breast cancer cells and modulate the Notch signaling pathway in triple‐negative inflammatory breast cancers. 24 , 42 Putting alongside our findings and previous research in endometriosis and cancer cells, we speculate on the potential role of SDC‐1 in adenomyosis development. Furthermore, as emerging evidence, decreased apoptosis, increased proliferation, and higher ability for migration and invasion were reported in the eutopic endometrium of endometriosis as described before by Song Y et al. 43 We can conclude that the decrease of SDC‐1 in adenomyotic patients in our study may suggest a role in promoting the invasiveness of endometriotic islands within the myometrium. As previously mentioned, the hormonal‐dependent tissues and tumors are known to be correlated with the up−/and downregulation of Syndecans. In our study, we exposed that there were no significant differences between SDC‐1 expression in both the proliferative and secretory phases, where the low representation of the secretory phase should be considered to affect our ability to detect such differences. Moreover, the adenomyosis cases included in this study were classified as diffuse in pathology reports according to the usual reporting standard for hysterectomy specimens. In contrast, focal adenomyosis is reported in cases involving local resection. 44 This classification was consistent across all cases, providing a uniform basis for our analysis of syndecan‐1 expression. We did not observe stromal staining of syndecan‐1, which is consistent with a previous report by Germeyer et al. 19 In contrast, Lai et al. reported stromal staining of SDC‐1 and its downregulation after ovulation. 45 Also in this study, epithelial/glandular staining of SDC‐1 was substantially more prominent than stromal staining. We can therefore only speculate that technical differences between these studies such as the time of color development affecting the sensitivity of the immunostaining, intensity of the counterstaining potentially masking weak staining, or differences in the scoring systems could be an underlying reason for this discrepancy. Although the correlation between stem cell‐associated markers (such as SDC‐1) and the menstrual cycle remains controversial, 46 , 47 , 48 , 49 our study found no association between the glandular expression of SDC‐1 and the cycle phase in adenomyosis patients. In contrast, few studies found an increased expression of adult stem cell markers, such as SOX‐2 and Musashi‐1, in the proliferative phase for patients with endometriosis. 50 , 51 In contrast to the hormonally‐dependent cells of endometriosis, adenomyotic endometrium appears unresponsive to progestogenic suppression and may benefit from stem cell transplantation into uteri for therapy. 52 , 53 In accordance with the latter findings, we can suggest that there is a broad spectrum of uterine pathologies in different species that are reliant on uterine stem cell functions and their possible future role as therapeutic agents in the treatment strategy of adenomyosis worth more study. Syndecans are subject to ectodomain shedding by MMPs, which are regulated in a menstrual cycle‐dependent manner. 29 , 54 Moreover, SDC‐1 depletion in endometriotic cells resulted in altered expression of MMP2 and MMP9, 20 suggesting that SDC‐1 expression in the control group may vary based on the endometrial subregion and cycle phase. Indeed, Germeyer et al. previously described an upregulation of SDC‐1 in the secretory compared to the proliferative phase. 19 While the mean values for the SDC‐1 score were higher in the secretory vs proliferative phase also in our control group, we did not detect significant differences due to high variability, suggesting that future studies on larger sample collectives may confirm cycle‐dependent changes. Our study underscores SDC‐1 as a potential molecular target in adenomyosis; however, additional research is needed to understand its functions and mechanisms in disease pathogenesis. Our findings support the role of stemness‐related molecular markers in adenomyosis, but a potential drawback is that the sample size was limited, especially secretory endometrium samples (15%) of the included adenomyosis samples, therefore, for some indices, no statistical significance is reached. With a larger sample size and comprehensive studies of adult stem cells in the pathogenesis of adenomyosis, we can explore more therapeutic avenues. Currently, only a little literature studying the role of stem‐related factors in the development of adenomyosis has been published. Therefore, our study provides valuable support for the hypothesis that the dysregulation of some stem cell‐related markers may contribute to the establishment of adenomyosis. However, our study marks SDC‐1 as a potential molecular target in adenomyosis, offering promising prospects for expanding adenomyosis treatment modalities, we have to keep in mind the limitations given by the small sample size, in particular in the secretory phase. Such a pilot study, covering both the proliferative and secretory phases will be warranted in the future for detailed comparisons of the expression of Syndecan‐1 during different stages of the menstrual cycle.

Our findings regarding the significant alterations in SDC‐1 expression support the role of stemness‐related molecular markers contributing to the pathogenesis of adenomyosis. Furthermore, our observation that SDC‐1 expression was dependent on the uterine compartment was found in the limited exclusive expression in the endometrium, underscores the complexity of this disorder. Given the reliance of adenomyosis on estrogen, our study delving into the cyclical variation of these markers suggests a need for further investigation to fully elucidate their role in disease progression. By shedding light on the interplay between stemness‐related markers and adenomyosis, our results pave the way for expanding treatment modalities. However, as this is a pilot study, these findings should be considered preliminary. Further research is necessary to elucidate the exact mechanisms by which SDC‐1 may contribute to adenomyosis, including potential interactions with other molecular pathways.

Adenomyosis is considered a benign uterine disease, affecting about 19.5% of women in the reproductive age period. 1 Histopathologically, it is characterized by the presence of ectopic endometrial tissue (endometrial glands and/or stroma) within the myometrium, surrounded by hyperplastic and hypertrophic smooth muscle. 2 , 3 Clinical manifestations of adenomyosis typically include heavy menstrual bleeding and dysmenorrhea. 4 Despite its prevalence, the precise etiology and pathogenesis of adenomyosis remain unclear. Several proposed mechanisms include the invagination of basal endometrium, local hyperestrogenism, and mechanical forces manifesting as hyper‐ or dysperistalsis. 5 , 6 , 7 , 8 , 9 , 10 , 11 Additionally, studies have suggested a role for estrogen‐induced epithelial‐mesenchymal transition (EMT) in adenomyosis development. 12 , 13 Factors such as multiparity, age, previous uterine abrasion, local hyperestrogenism, elevated s‐prolactin levels, and autoimmune factors are considered potential risk factors for adenomyosis. 14 , 15 , 16 The tissue injury and repair (TIAR) theory is now widely accepted and suggests that uterine hyperperistalsis (i.e., increased peristalsis), during early periods of reproductive life induces repetitive micro‐injures at the endometrial‐myometrial interface. 17 In turn, a process of tissue repair occurs at endometrial‐myometrial interface, accompanied by EMT, with increased local production of estrogen (local hyperestrogenism). The latter induces more hyperperistalsis and subsequently chronic tissue injury. Recently, Ibrahim et al. identified a putative stem cell‐like population termed pale cells in the endometrial‐myometrial interface of the fundocornual raphe in adenomyosis. They were seen migrating from the basal endometrium into the underlying stroma suggesting a possible involvement in the development of adenomyotic foci in the myometrium. 18 Syndecans are stemness‐related proteins that act as coreceptors for ligands relevant in the menstrual cycle, such as fibroblast growth factor (FGF), heparin‐binding growth factor (HBEGF), vascular endothelial growth factor (VEGF), and other adhesion molecules. 19 Syndecan‐1 (SDC‐1) was shown to influence invasiveness, matrix metalloproteinase (MMP) expression, and inflammatory cytokine secretion of endometriotic cells. 20 In cancer research, SDC‐1 has been extensively studied and implicated in cancer progression. Increased Syndecan‐1 expression has been observed in multiple myeloma where it has been linked with cancer progression by mediating the effects of growth factors in cells. 21 Moreover, SDC‐1 expression was shown to be upregulated in ductal breast carcinomas and associated with factors of angiogenesis and lymphangiogenesis. 22 Other studies have shown that patients suffering from endometrial cancer have increased SDC‐1 expression, which regulates the endometrial hyperplasia that can progress to endometrial cancer. 23 In contrast, reduced epithelial expression of SDC‐1 in breast cancer may promote EMT and invasiveness. 24 This study aims to investigate the presence and distribution of the stemness‐related factor Syndecan‐1 in women with and without adenomyosis to find out if Syndecan‐1 may have a possible role in adenomyosis.

The authors declare that they have no conflict of interest regarding this study.

Adenomyosis lesions (ectopic endometrium) and the corresponding eutopic endometrium of adenomyosis patients were acquired during 2016–2017 at the Department of Gynecology and Obstetrics of Münster University hospital from 21 Premenopausal women aged 30–53 years (mean 41) undergoing hysterectomy for benign conditions (Table  1 ). Upon clinical suspicion of adenomyosis, the hysterectomy specimens were thoroughly examined through extra multi‐slicing in different regions to identify adenomyosis lesions macroscopically. Only patients with histopathologically proven adenomyosis were included. Histopathologically, adenomyosis was defined as the presence of endometrial glandular and stromal cells at least 2.5 mm below the endometrial‐myometrial junction with surrounding myometrial hyperplasia and hypertrophy. 2 Participants with adenomyosis with another pathological disease or genital tumors were excluded. All participants included in this study were not undergoing any hormone treatments at the time of or before sample collection. Preoperative indications and the histopathologic diagnosis of the adenomyosis and non‐adenomyosis groups. Endometrial specimens from 14 uteri from reproductive‐age women who had undergone hysterectomies for benign gynecological conditions other than endometrial disease were included in the control group. Endometrial tissues were grouped into proliferative and secretory phases according to the histopathologic criteria of Noyes. 25 Within the adenomyosis group, 17 cases were in the proliferative phase, three cases were in the secretory phase, and only in one case the endometrium was atrophic. In the control group, there were 11 cases in the proliferative phase and three cases were in the secretory phase. Endometrial specimens were fixed in 10% formalin and paraffin‐embedded using standard procedures, which were documented at the Institute of Pathology of Münster University Hospital. Consecutive 3 μm sections were cut from paraffin blocks and placed on poly‐L‐lysine‐coated coverslips. The dried coverslips were deparaffinized, rehydrated, and treated with target antigen retrieval solution (pH 6 0; DAKO) for 35 min in a steamer, followed by three washes in phosphate‐buffered saline (PBS). The sections were blocked with peroxidase (DAKO, Glostrup, Denmark), followed by a second block with 10% Aurion BSA (DAKO) for 30 min. Slides were then washed in PBS and incubated with anti‐Numb antibodies (1:70, rabbit polyclonal IgG, Santa Biotechnology) as a negative control or with anti‐Syndecan‐1 antibodies (1:100, mouse‐anti‐human SDC‐1, Serotec) diluted with DAKO Real Antibody diluent, for 1 h at room temperature. In negative controls, the primary antibodies were omitted. After three more washes with PBS (5 min), the slides were incubated with anti‐rabbit or/and anti‐mouse EnVision systems, corresponding to the source of the primary antibody, for 30 min. The slides were then washed with PBS three more times (5 min each). The immunohistochemical signal was detected using AEC‐substrate chromogen (3‐amino‐9‐ethylcarbazole substrate) incubated for 6 min at room temperature according to the manufacturer (DAKO). Sections were shortly washed with PBS, counterstained with Mayers haemalum (Merck, Darmstadt, Germany), and embedded in GelTol Aqueous Mountain Medium (Immunotech). The light microscopic evaluation was performed by the first observer; WS and confirmed by the second observer; MGI, and both were blinded to the patient's data. Microscopy was performed using a Zeiss Axiophot100 microscope equipped with a CCD camera and Axiovision Software (Zeiss, Göttingen, Germany). Staining intensity was evaluated using a magnification of 200× per stained section. Expression of SDC‐1 was strongly detected in the glandular epithelium and the luminal epithelium but did not reveal any significant detectable immunoreactivity in the stroma and underlying smooth muscles. Therefore, the evaluation system and scoring have considered only glandular and luminal staining. This approach was chosen to capture the overall expression pattern of SDC‐1 across the entire endometrial tissue. The membranous staining of the glandular and the luminal epithelium was evaluated by a semi‐quantitative approach used to assign an H‐score (or “histo” score). 26 First, membrane staining intensity (0, negative staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining) was determined for each cell in a fixed field. The percentage of cells at each staining intensity level was calculated, and finally, an H‐Score for each intensity level was seen. The percentage of cells at each staining intensity level was calculated, and finally, an H‐score was assigned using the following formula: 1 × ( % cells 1 + ) + 2 × ( % cells 2 + ) + 3 × ( % cells 3 + ). The final score, ranging from 0 to 300, gives a more emphasized relative value to higher‐intensity membrane staining in the given sample. Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Qualitative data were described using numbers and percentages. The Kolmogorov–Smirnov test was used to verify the normality of the distribution. Quantitative data were described using range (minimum and maximum), mean, standard deviation, and median. The significance of the obtained results was judged at the 5% level. Comparing Syndecan‐1 expression between groups, the Kruskal–Wallis test and Mann–Whitney U ‐test were applied. A p  < 0.05 was considered statistically significant.