Conditioned medium from healthy women's endometrial stem cells improve in�ammatory and stemness-expression genes in endometriosis women

Background. Endometriosis is a common, benign gynecological disease which is determined as an overspreading of endometrial tissue in exterior region of the uterine cavity. Evidence suggests that retrograde menstrual blood which contains mesenchymal stem cells with differential gene expression compared to healthy women may play a role in endometriosis creation. We aimed to identify whether the conditioned medium from Menstrual blood-derived stem cells (MenSCs) of healthy women can affect the expression level of in�ammatory and stemness genes of MenSCs from endometriosis women. Methods and Results. Endometriosis derived MenSCs (E-MenSCs) were treated with conditioned medium (CM) derived from healthy women’s MenSCs (NE-MenSCs). Some CD markers were analyzed by �ow cytometer before and after treatment compared with NE-MenSCs, and the expression level of in�ammatory and stemness genes was evaluated by real-time PCR. Results. E-MenSCs show different morphology in vitro culture in comparison with NE-MenSCs, which were changed in the presence of CM, into a morphology more similar to normal cells and showed signi�cant decrease expression of CD10 after CM treatment. In our results, the IL-1, COX-2, and HIF-1 α as an in�amaturay genes and OCT-4, NANOG, and SOX2 as a stemness genes showed signi�cantly different expression level in E-MenSCs after treating with CM. Conclusions. Our study indicates that the expression level of some inammatory-and stemness-related genes which have differential expression in E-MenSCs compared with NE-MenSCs, could be changed to normal status by using CM derived from NE-MenSCs.


Introduction
Endometriosis is a benign gynecological disease that affects approximately 5 to 10% of women worldwide during the years of reproduction [1,2] , and is described as the presence of endometrial tissues outside the uterine cavity, which leads to many complications such as chronic pelvic pain and infertility [3].Since endometriosis is a complex disease with a multifactorial etiology, generally accepted theories reported for the causes of endometriosis include retrograde menstruation, vascular dissemination, metaplasias in pelvic cavity, and immune dysfunction.For instance, many shreds of evidence offer that immune responses are activated in endometriosis that lead to cellular changes in menstrual blood-derived mesenchymal stem cells (MenSCs) [4,3].Regarding the differences observed in MenSCs of endometriosis women in comparison with healthy women, it seems very important to evaluate these cells in different aspects including surface markers and gene expression levels [2,4].Some genes are differentially expressed in MenSCs from endometriosis patients which can be the leading reasons for the disease complications.These gene expression differences can help to diagnosis and treatment of endometriosis [5].In ammatory genes are mostly overexpressed in these patients which gradually leads to chronic in ammation and immune responses in lesion sites and the whole pelvic cavity [6].Besides, the stemness genes, also known as pluripotency genes, are mostly expressed Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js differentially in MenSCs from endometriosis women which can result in angiogenesis, migration, and invasion [7,8].Since there is no proper and de nitive treatment for this disease, it could be important to know the basic principles that cause it, such as differentially expressed genes, to nd suitable treatment methods.
One of the main in ammatory actors is interleukin 1α (IL-1α).It is thought that IL-1α is a primary in ammatory factor in immune situations that stimulates prostaglandin synthesis and induces B cells to produce immunoglobulins.So, IL-1α can play a signi cant role in the pathogenesis of endometriosis [9].Also, cyclooxygenase-2 (COX-2) is an inducible enzyme that is produced by in ammatory mediators.In addition, this enzyme converts arachidonic acid to prostaglandins [10].It has been proved that expression of COX-2 enhances in women with endometriosis [10].Consequently, the high levels of COX-2 and Prostaglandins expression might be effective in endometriosis development [10].Furthermore, aberrant expression of tumor necrosis factor-α (TNF-α) is identi ed to promote the cell proliferation and adhesion in endometriosis, because high concentrations of TNF-α induce the peritoneal macrophages to secrete in ammatory factors [11].The in ammatory mediators such as IL-1β and TNF-α have key function in activating nuclear factor κB (NF-κB) and hypoxia-inducible factor (HIF-1α) signaling pathway.These factors regulate immune response and high expression of NF-κB and HIF-1α are observed in endometriosis [12].
On the other hand, stemness genes play an emerging role in endometrium regeneration in each female ovarian cycle.In addition, pluripotency genes promote cell migration in endometriosis.It was proved that the expression of stemness-related factors such as the Spalt-like protein 4 (SALL4), NANOG, octamerbinding transcription factor 4 (OCT4), and Sex determining region Y-box 2 (SOX2) are different in endometrial stem cells between endometriosis patients and healthy women [13][14][15].OCT4 is an important factor for the survival of cell pluripotency and is identi ed to be highly expressed in endometriosis women [16].In endometriosis, expression of the pluripotent transcription factor OCT4 encourages cell migration [7].NANOG expression in undifferentiated stem cells and it is a critical regulator in cells pluripotency and differentiation by interaction with other factors [17,18].Many studies revealed that SALL4 expression changes in numerous cancers and endometriosis [19,20].
Since there is no proper and de nitive treatment for endometriosis, it can be very important to know its etiology to nd suitable treatment methods.Extensive investigations in recent years on mesenchymal stem cells-derived conditioned medium (MSCs-CM) lead our mind to the dynamic role of endometrial stem/progenitor cells and their derivatives in the treatment of endometriosis.Growth factors, extracellular vesicles, and bioactive agents in the secretion released by stem cells into the in vitro culture medium, known as the conditioned medium, have been shown to induce many cell processes and changes in gene expression, and eventually, tissue repair and regeneration through paracrine mechanisms [21][22][23].
Regarding the various functions reported for MSCs-CM, it can be expected that the conditioned medium from MenSCs of healthy women can affect the surface markers and gene expression of MenSCs from endometriosis women.So, the aim of this study was to evaluate the expression of in ammatory and Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js stemness genes in endometriosis patients in the presence of conditioned medium from endometrial stem cells of healthy women.

Patient selection
This interventional experimental study was approved by ACECR biomedical research ethics committee (IR.IAU.QOM.REC.1399.016)and written informed consents were obtained from individuals before enrolling in the study.The patient group comprised endometriosis women (n = 3) (stages III-IV) undergoing laparoscopy for infertility or pain and the control group consisted of healthy women (nonendometriosis subjects) (n = 3).The inclusion criteria were as follows: (1) age ranging from 25 to 35 years; (2) history of ovulatory cycles with unregularly menstrual periods in endometriosis cases, (3) body mass index (BMI) of 18-28 kg/m2, (4) no hormonal treatments for at least the last 3 months, (5) no previous surgery for endometriosis women, (6) no history of malignancies or autoimmune diseases, and (7) evidence for deep endometriosis suggested by transvaginal ultrasound and magnetic resonance imaging (Fig. 1).

MenSCs Isolation and Culture
After selecting 3 proper cases in each groups, at least 2 ml menstrual blood was collected by pipelle catheter during the second or third day of menstruation and were immediately transferred to the laboratory.4 folds of blood sample was added EDTA 0.5 mM, an equal volume of blood sample was added to Ficoll-Paque media (Lymphodex, innotrain, Germany) carefully and centrifuged at 600 g for 30 min at room temperature.Following density gradient centrifugation, plasma and platelets in the upper layer were removed and mononuclear cell layer remained undisturbed at the interface.The mononuclear cell layer was transferred to a sterile centrifuge tube and washed twice with PBS.Cell pellets were grown in Dulbecco's modi ed Eagle's low glucose (DMEM-LG) medium supplemented with 10% FBS (Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, USA) and incubated in 97% humidity, and 5% CO2 at 37 ˚C.Media was changed after 24 h to remove all oating cells, followed by regular media changes every 3 days.Then Cells were trypsinized, and passaged when 70-80% con uency was achieved.The isolated menstrual bloodderived stem cells (MenSCs) from endometriosis and healthy women are de ned as E-MenSCs and NE-MenSCs, respectively.

Flow Cytometry for mesenchymal stem cell markers
To con rm the isolated cells as MenSCs, the expression level of positive cell surface markers (CD29, CD90, CD105, CD44, CD73, and CD10) and negative markers (CD34, CD45, CD133, CD38) for MenSCs were determined.FITC-conjugated monoclonal antibodies against CD34, CD38, CD90, CD10, CD44, and CD133, as well as PE-conjugated monoclonal antibodies for CD105, CD45, CD29 and CD73 were purchased from BD Biosciences (San Jose, CA, USA).PE-conjugated anti-CD105 and were from R&D Systems (Minneapolis, MN, USA).The entire corresponding isotype-matched control antibodies were Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js obtained from the same companies as their test antibodies.All the antibodies were used in ow cytometry experiments at the concentrations recommended by the manufacturers.Flow cytometry was performed using a FC500 ow cytometer (Beckman Coulter, Fullerton, CA) and analyzed using Beckman Coulter CXP software.

Preparation of conditioned medium and treatment
For preparation of conditioned medium, NE-MenSCs at passages 3-4 were seeded in T-75 asks at a density of 1 × 10 6 cells.Once the 70-80% con uency was obtained, the culture medium was collected after 3 days of culture.To remove cells and cell debris, culture medium was centrifuged and ltersterilized through a 0.22-m lter, and then it was termed as a conditioned medium of MenSCs (MenSCs-CM).The aliquoted MenSCs-CM was stored at −80°C till use.All in vitro experiments were performed using passages 3-5 of MenSCs-CM.
Then, E-MenSCs were treated in three experimental groups with NE-MenSCs derived conditioned medium for 2, 4 and 6 days.Before and after C.M treatment the cell lines was measured by Flow Cytometry assay and Real-time PCR.

Real-time Polymerase Chain Reaction and Gene Expression Analysis
Total RNA was isolated from treated cells using "Gene All Kit (Gene All Biotechnology, Seoul, Korea) according to the manufacturer's instructions.RNA purity and quantity were assessed using Nanodrop 2000 spectrophotometer (Thermo Fisher Scienti c, Wilmington, USA) at 260/280 nm.The reverse transcription was used to synthesize the rst-strand cDNA using transcription Kit (Yekta tajhiz, Iran)".
Quantitative real-time PCR assays were performed in triplicate to evaluate the expression of selected genes (table 1).For normalizing gene expression levels, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference.The 2 -∆∆Ct method was used to calculate the fold change of mRNA expressions for target genes.Real-time PCR was carried out using RealQ Plus Master Mix Green (AMPLIQONIII) following the manufacturer's instructions.Brie y, a mixture comprised of 10 µL SYBR green mix, 1 µL of cDNA (250 ng, 1 µL PCR forward primers and 1 µL PCR reverse primer in 5 pmol µL-1, and millipore water to achieve a nal volume of 20 µL was made.The sequences of primers are presented in Table1.The Threshold Cycle (CT) was determined manually for each run.Relative mRNA level was expressed as the relative fold change and calculated using the formula 2 -CT =2 -( CT(Sample)- CT(calibrator) ), where each CT = CT Target -CT GAPDH.One sample without any treatment from the control group was designated as a calibrator.The quanti cation of mRNA was performed as a value relative to an internal reference for GAPDH.

Statistical analysis
Experimental data are expressed as the mean±standard error (SE) and compared by ANOVA.ANOVA using a multiple comparisons test was used to determine statistical signi cance, followed by the Tukey Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js test.Statistical signi cance was set at p ≤0.05.All experiments were performed in triplicate for technical and biological repetitions.

Morphology of MenSCs
The MenSCs are morphologically similar to bone marrow-derived mesenchymal stem cells (BM-MSCs).In our study, microscopic observations showed that MenSCs from healthy women (NE-MenSCs) had broblast-like spindle-shaped morphology, while, E-MenSCs were less stretched and elongated (Fig .2A,   B).
The morphology of E-MenSCs was changed under treatment of CM derived from NE-MenSCs into a more elongated, spindle-shaped morphology (Fig. 2C).
To compare E-MenSCs and NE-MenSCs concerning the expression level of CD markers, we used the mean uorescence intensity (MFI).In our results, CD10 marker expression was signi cantly higher for E-MenSCs compared with that of NE-MenSCs.Also, after CM treatment, there was signi cantly decrease in CD10 expression for E-MenSCs (P ≤ 0.05) (Fig. 3B).

Pro-in ammatory genes expression under MenSCs-CM treatment
In this study E-MenSCs were treated with conditioned medium derived from NE-MenSCs of 3 rd passage, for 2, 4 and 6 days.According to the gene expression analysis, the best effect was related to 4 days of CM treatment and data from 2 and 6 days of treatment were omitted and not shown.Quantitative RT-PCR analysis of IL1β, COX-2, HIF, TNF-α and NF-kB genes was performed in E-MenSCs in before and after CM treatment, as well as in NE-MenSCs as control group.As shown in Figure 4, the expression of IL-1β in E-MenSCs was signi cantly higher compared to NE-MenSCs (1.00±0.1 vs. 0.008±0.003,P ≤0.05), that after CM treatment it was surprisingly decreased (0.013 vs. 1.00,P ≤0.05) so that was similar to NE-MenSCs (0.013±0.002 vs. 0.008±0.003,P=0.627).Similarly, the expression of COX2 in E-MenSCs was signi cantly higher compared to NE-MenSCs (

MenSCs-CM regulates stemness genes expression
As shown in Figure 5, the expression of some stemness genes was compared between E-MenSCs (before and after CM treatment) and NE-MenSCs.OCT4 expression showed a signi cant decrease in E-MenSCs compared to NE-MenSCs (1.00±

Discussion
Accumulating evidence showed that in ammation may be considered as a crucial mechanism in endometriosis.In addition, it was identi ed that stemness genes may be involved as key mediators in establish endometriosis [24][25][26][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][29][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 in ammatory and stemness genes of menstrual blood-derived stem cells of endometriosis patients (E-MenSCs).
According to our ndings, the interleukin-1 (IL-1) and cyclooxygenase-2 (COX-2) in ammatory 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 signi cant difference compared to NE-MenSCs as control group.
Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Besides, the tumor necrosis factor-alpha (TNF-α) expression did not change signi cantly after treatment with CM derived from NE-MenSCs and was still signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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 broblast-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 signi cant difference compared to control.
Moreover, considering in vitro ndings, it was identi ed that COX-2 expression could be increased in the eutopic and ectopic endometrium of endometriosis patients [12,[34][35][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-in ammatory cytokines via microRNA-302a [37].
In previous studies, it was observed that some stemness genes, including SOX-2 and NANOG demonstrate a signi cantly 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].
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 in uenced by the hypoxia inducible factor (HIF)-2-alpha transcription factor and HIF-2alpha regulates .
Furthermore, some studies showed that SALL4 is expressed aberrantly in some of tissues of endometriosis patients [15,43].Our ndings 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-in ammatory 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 uorescence 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 ndings 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 in ammatory and stemness genes.However, further in vitro and preclinical studies for its better validation in disease treatment are needed.
Understanding and identi cation 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.

Conclusion
Our study indicates that the expression level of some in ammatory-and stemness-related genes could be changed by use of CM derived from NE-MenSCs.Furthermore, the present study identi ed that conditioned medium derived from NE-MenSCs could be considered as a new candidate for ameliorating the endometriosis disease.After CM treatment, the cells changed to spindle-shaped morphology (C).

Figures
Figures

Figure 4 RealFigure 5 Loading
Figure 4 MenSCs in comparison with NE-MenSCs (1.00± 1.04 vs. 6.54±1.96, p= P ≤0.05).TNFα expression was increased after CM treatment but this change was not signi cant.There was no signi cant difference in NF-kB gene expression between groups.