Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients

Abstract

Background: Current evidence suggests that endometrial-derived stem cells, spilled in the peritoneal cavity via retrograde menstruation, are key players in the establishment of endometriotic lesions. The aim of this study was to determine the presence and distribution of the stemness-related factors OCT4, SOX15, TWIST1 and DCAMLK1 in women with and without endometriosis.

Methods: Immunohistochemical analysis was used to determine stromal and epithelial expression of OCT4, SOX15, TWIST1 and DCAMLK1 in endometriosis patient (EP) endometrium (n = 69) and endometriotic tissue (n = 90) and in control endometrium (n = 50). Quantitative Real-Time PCR of OCT4, SOX15 TWIST1 and DCAMLK1 was performed in paired samples of EP endometrium and endometriotic tissue. Co-immunofluorescence staining was performed for OCT4 and SOX15. For statistical analyses we used unpaired t-test, Fisher combination test and Spearman test. For paired analyses, paired t-test and McNemar test were used.

Results: We detected a significant correlation between the expression of the established stem cell marker OCT4 and the stemness-related markers SOX15 (p < 0.001) and TWIST1 (p = 0.002) but not DCAMLK1. We showed a colocalization of SOX15 and OCT4 in epithelial and stromal cells of endometriotic tissue by coimmunofluorescence. A concordant expression of OCT4 and SOX15 in the same sample was observed in epithelial cells of the endometriotic tissue (71.7%). The expression of stemness-related factors was not associated with proliferative or secretory phase of the menstrual cycle in endometriosis patients but was found to be differentially expressed during the menstrual cycle in the control group. Increased expression of epithelial OCT4, SOX15 and TWIST1 was detected in endometriotic tissue compared to EP endometrium in paired (p = 0.021, p < 0.001 and p < 0.001) and unpaired analysis (p = 0.040, p < 0.001 and p = 0.001).

Conclusion: Our findings support the hypothesis that upregulation of stem cell-related factors contribute to the establishment of endometriotic lesions.

Trial registration: The study was approved by the institutional review board (545/2010 on 6th of May 2014) of the Medical University of Vienna ( http://ethikkommission.meduniwien.ac.at/fileadmin/ethik/media/dokumente/register/alle_2010.pdf ).

Keywords: Endometriosis; OCT4; SOX15; Stem cells; TWIST1.

Fig. 1

Immunohistochemical analyses of OCT4, SOX15, TWIST1 and DCAMLK1 in eutopic and ectopic endometrium. Anti-OCT4 and anti-SOX15 antibodies were applied at a dilution of 1:500 and 1:300, respectively, and yielded nuclear staining in eutopic (a, c) or ectopic tissue (b, d). Anti-TWIST1 antibody was applied at a dilution of 1:1200 and yielded cytoplasmatic and nuclear staining in eutopic (e) and ectopic (f) lesions. For evaluation, only nuclear staining was analyzed. Anti-DCAMLK1 antibody was applied at a dilution of 1:500 and yielded cytoplasmatic staining in eutopic (g) or ectopic tissue (h). Magnification = 200x

Fig. 2

Expression analyses in 49 paired cases with endometriosis. IHC was used to analyze the protein expression of OCT4 (a), SOX15 (b), TWIST1 (c) and DCAMLK1 (d). Results are expressed as mean score of the immunohistochemical staining ± SD. Epithelial and stromal expression was analyzed separately. All p-values of paired comparisons were analysed by paired t-test

Fig. 3

Expression analyses in 50 control patients, and 110 patients with endometriosis (eutopic and ectopic). IHC was used to analyze the protein expression of OCT4 (a), SOX15 (b), TWIST1 (c) and DCAMLK1 (d). Results are expressed as mean score of the immunohistochemical staining ± SD. Epithelial and stromal expression was analyzed separately. Comparisons with the control group were analysed by t-test, comparisons between EP endometrium and endometriotic tissue were performed using Fisher combination test

Fig. 4

Relative mRNA expression levels in 49 paired cases with endometriosis. Quantitative Real Time PCR was used to analyze the mRNA expression levels of OCT4 (a), SOX15 (b), and TWIST1 (c). Expression levels were normalized to ß-actin and GAPDH. Expression levels were shown as mean ± SD. All p-values were analysed by paired t-test

Fig. 5

Colocalization of OCT4 and SOX15 in human endometriotic tissue. Paraffin sections of human endometrium were processed for immunofluorescence microscopy. OCT4 (a), SOX15 (b), and DAPI (c) stainings are shown. OCT4/SOX15 merged picture (d) denotes colocalization in all epithelial cells whereas few stromal cells were only positive for OCT4 (white arrows)

Fig. 6

Expression levels of epithelial OCT4, SOX15, TWIST1 and DCAMLK1 in proliferative and secretory phase of the menstrual cycle in control patients. Results are expressed as mean scores of the immunohistochemical stainings ± SD. All p-values of subgroup comparisons were analysed by t-test