Accumulating evidence showed that inflammation may be considered as a crucial mechanism in endometriosis. In addition, it was identified that stemness genes may be involved as key mediators in establish endometriosis [24-27].
It has been observed that growth factors, extracellular vesicles, and bioactive agents in secretion released by stem cell into the culture medium in vitro, which is referred to as the conditioned medium, can cause many cell processes and changes in gene expression and ultimately tissue repair and regeneration through paracrine mechanisms [28-30]. In addition, more recent evidence proposed that CM from menstrual blood‐derived MSCs could support against MPP+-induced cytotoxicity in vitro [31].
In the present research study, we have indicated that CM derived from male factor-derived MenSCs (NE-MenSCs) could have a major impact on the phenotype and expression level of inflammatory and stemness genes of menstrual blood-derived stem cells of endometriosis patients (E-MenSCs).
According to our findings, the interleukin-1 (IL-1) and cyclooxygenase-2 (COX-2) inflammatory genes in E-MenSCs had a high level of expression, which after treatment with the CM derived from NE-MenSCs, their expression level decreased and did not show a significant difference compared to NE-MenSCs as control group.
Besides, the tumor necrosis factor- alpha (TNF-α) expression did not change significantly after treatment with CM derived from NE-MenSCs and was still significantly different from control. High expression of TNF-α was observed in E-MenSCs compared to control. In addition, the nuclear factor-kappa B (NF-κB) expression was not significantly different before and after treatment with CM derived from NE-MenSCs. Consistent with previous results, it is possible that NF‐κB activates epithelial cells proliferation and migration most likely via Notch signaling activation resulting in enhancement of re‐epithelialization [32]. Given that there is no change in epithelial cell proliferation and epithelialization in endometriosis, it seems that NF-κB expression could not be different significantly before and after treatment with CM derived from NE-MenSCs.
Unlike TNF-α expression which was not affected, the expression of the IL-1 and COX-2 changed significantly in E-MenSCs in the presence of CM derived from NE-MenSCs compared to control.
While NE-MenSCs illustrated a more elongated, spindle-shaped morphology similar to fibroblast-like cells, E-MenSCs did not. However, the morphology of E-MenSCs changed in the presence of CM derived from NE-MenSCs into a more elongated, spindle-shaped morphology.
Whereas the E-MenSCs before treatment with CM derived from NE-MenSCs demonstrated OCT-4 and NANOG expression level lower than the control, treatment of the E-MenSCs with CM derived from NE-MenSCs resulted in the OCT-4 and NANOG expression level similar to control.
Furthermore, SOX2 and SALL4 stemness genes had a high level of expression in E-MenSCs, which after treatment with the CM derived from NE-MenSCs, their expression level decreased, however, did not show a significant difference compared to control.
In the previous studies, it was demonstrated that some pro-inflammatory genes including IL-1 and COX-2 can demonstrate a significantly dysregulated expression in some of tissues of endometriosis patients [33,12,34-36]. For instance, Chalpe et al. (2015) demonstrated that pro-inflammatory genes, including IL-1β induce differential expressions in endometrial stromal cells [33].
Moreover, considering in vitro findings, it was identified that COX-2 expression could be increased in the eutopic and ectopic endometrium of endometriosis patients [12,34-36]. These studies were in consistent with our study results. Besides, it was cleared that COX-2 upregulation could be observed in endometriotic stromal cells due to the suppression of COUPTFII in these stem cells by pro-inflammatory cytokines via microRNA-302a [37].
In previous studies, it was observed that some stemness genes, including SOX-2 and NANOG demonstrate a significantly higher expression in some tissues of endometriosis patients [14,15,7]. For example, a study revealed that the SOX-2 stemness gene has an aberrant expression in endometriosis [38]. Furthermore, Song et al. (2014) reported that SOX-2 and NANOG genes were overexpressed in ovarian endometriosis [14].
In addition, aberrant expression of NANOG and OCT-4 stemness genes has been verified in endometriotic tissues [15,7]. The results from our study are consistent with several mentioned studies. SOX-2, OCT-4, and NANOG has been found to cooperatively activate and regulate other stem cells related genes [39,40].
Additionally, our study outcomes are in agreement with part of the results reported by Proestling et al. (2016) that some of the genes related to stemness, including OCT-4 gene show upregulation in epithelial and stromal cells of endometriotic tissue [13]. Also, a research study showed that OCT-4 and NANOG genes have an increase expression in endometriotic mesenchymal stem cells that was consistent with our study [41]. The expression of OCT-4 is influenced by the hypoxia inducible factor (HIF)-2-alpha transcription factor and HIF-2alpha regulates OCT-4 [42].
Furthermore, some studies showed that SALL4 is expressed aberrantly in some of tissues of endometriosis patients [15,43]. Our findings are in agreement with some reports which reveal that SALL4 stemness gene have upregulated expression in endometriosis disease [15,43].
Evidence demonstrated that increases in COX-2 production regulates cell survival, migration and invasion of extrauterine endometriotic tissues. It seems that the reason for this increase in COX-2 gene expression in stromal cells may be due to the regulation of its expression by pro-inflammatory cytokines such as IL-1β or due to the high stability of COX-2 mRNA [44].
On the other hand, in a research study, the mean fluorescence intensity was used to evaluate the expression of CD10. The results obtained from this study indicated that CD10 expression was considerably higher in stromal stem cells from endometriosis compared with that of stromal stem cells from non-endometriosis [45]. Our findings are in agreement with results obtained by this study [45].
Altogether, the present study outcomes recommend that CM derived from NE-MenSCs could be exerted as an effective strategy to improve the gene expression related to endometriosis formation, such as inflammatory and stemness genes. However, further in vitro and preclinical studies for its better validation in disease treatment are needed.
Understanding and identification of the most important contents (extracellular vesicles, microRNAs, etc.) of the NE-MenSCs-derived CM which has the greatest impact on the promotion of cellular processes and changes in gene expression levels in MenSCs from endometriosis women, is an essential question that should be answered in the future research studies.