Inhibitory effect of resveratrol on the growth and angiogenesis of human endometrial tissue in an In Vitro three-dimensional model of endometriosis.

Endometriosis is a chronic estrogen-dependent disorder and one of the most common causes of infertility in women. Resveratrol (RES) is a polyphenolic and phytoestrogenic compound with anti-oxidative, anti-inflammatory, anti-angiogenic, and anti-estrogenic properties. The aim of the present study was to investigate the effect of different concentrations of RES on human endometrial growth and angiogenesis in an in vitro three-dimensional (3D) model of endometriosis.Human endometrial tissues of endometriosis (endometriotic) and normal (endometrial) subjects (n = 9/groups) were biopsied in sterile conditions and cut into 1 × 2 mm pieces. Tissue fragments of each biopsy were given concentrations of 0 (control), 10, 50, 100 and 200 µM RES for 21 days in 3D culture condition using fibrin as an extracellular matrix. Scoring methods were used for tissue changes, including; cellular invasion, monolayer formation and angiogenesis. Nitric oxide (NO) was measured using Griess's reaction, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the apoptotic gene expression.The mean of growth scores of endometriotic and endometrial tissue showed a significant dose dependent inhibition (P < 0.05). The levels of NO also significantly decreased in different groups. Apoptotic genes (P53, Bax, Bcl2 and caspase 3) and Sirt1 showed a significant increase in various concentrations of RES in both tissues (P < 0.05).RES exert dose- and time-dependent inhibitory effects on human endometrial tissue, and its higher doses suggested it as a natural supplement to inhibit the growth and treatment of endometriosis.

Noscapine Inhibiting the Growth and Angiogenesis of Human Eutopic Endometrium of Endometriosis Patients through Expression of Apoptotic Genes and Nitric Oxide Reduction in Three-Dimensional Culture Model.

Noscapine is a natural alkaloid with anti-angiogenesis activities. The aim of the present study was to examine the effect of noscapine on eutopic endometrium of endometriosis patients (EEE) and normal endometrium (NE) in a three-dimensional (3D) culture model. In this experimental in-vitro study, EEE (n = 8) and NE (n = 8) biopsies were taken from 16 reproductive aged women. The biopsies were cleared from blood and mucus. Each biopsy was cut into small fragments (1 × 1 mm) in a sterile condition. For 3D culture, the endometrial fragments were put between two layers of fibrin jell made of fibrinogen solution [3 mg/mL in Medium199 (M199) + thrombin]. Twenty-four wells of culture dish was divided into 5 groups for each biopsy: the control wells were treated with M199 containing 5% fetal bovine serum (FBS) while, the test wells were exposed to the same media containing one of the noscapine doses (10, 50, 100, and 200 µM). The expression of apoptotic genes, growth score, angiogenesis, and nitric oxide (NO) secretion were evaluated. The mean of growth score of groups exposed to 0, 10, 50, 100, and 200 µM were 2.2 ± 0.55, 1.7 ± 0.45, 1.44 ± 0.27, 0.29 ± 0.1, and 0.1 ± 0.08 in EEE, and also, 2.11 ± 0.6, 1.65 ± 0.5, 0.79 ± 0.41, 0.18 ± 0.1, and 0.1 ± 0.1 in NE, respectively, and the difference between the groups was significant (P < 0.05). The expression of apoptotic genes significantly increased while, the levels of Bcl-2 and Sirt1 reduced (P = 0.004). NO secretion reduced significantly (P < 0.05) in both EEE and NE groups. In conclusion, higher doses of noscapine showed inhibitory effect on growth and angiogenesis of EEE and NE.

Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine.

OBJECTIVES: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. MATERIALS AND METHODS: In this in vitro study, endometrial biopsies from endometriosis patients (n=9) were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml). Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0), 10, 25, 50 and 100 micromole/liter (µM) concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO) concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO)-Ethidium Bromide (EB) double staining and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Data were analyzed by one-way ANOVA. RESULTS: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P<0.05) and apoptotic index increased in 25, 50 and 100 µM noscapine concentrations in 48 hr significantly (P<0.05). Concentrations of NO didn't show a significant decrease. CONCLUSION: Noscapine increased endometriotic epithelial and stromal cell death and can be suggested as a treatment for endometriosis.

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression.

BACKGROUND: Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors. METHODS: In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48 or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student's t-test, and P<0.05 was considered statistically significant. RESULTS: Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05). CONCLUSIONS: Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.

Sildenafil Effect on Nitric Oxide Secretion by Normal Human Endometrial Epithelial Cells Cultured In vitro.

BACKGROUND: Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specific phosphodiesterase type 5. It increases intracellular nitric oxide (NO) production in some cells. There are reports on its positive effect on uterine circulation, endometrial thickness, and infertility improvement. Endometrial epithelial cells (EEC) play an important role in embryo attachment and implantation. The present work investigates the effect of sildenafil on human EEC and their NO secretion in vitro. MATERIALS AND METHODS: In this experimental in vitro study, endometrial biopsies (n=10) were washed in a phosphate buffered solution (PBS) and digested with collagenase I (2 mg/ml in DMEM/ F12 medium) at 37°C for 90 minutes. Epithelial glands were collected by sequential filtration through nylon meshes (70 and 40 µm pores), respectively. Epithelial glands were then treated with trypsin to obtain individual cells. The cells were counted and divided into four groups: control and 1, 10, and 20 µM sildenafil concentrations. Cells were cultured for 15 days at 37ºC and 5% CO2; the media were changed every 3 days, and their supernatants were collected for the NO assay. NO was measured by standard Greiss methods. Data were analyzed by one way ANOVA. RESULTS: There was no significant difference between groups in cell count and NO secretion, but the level of NO increased slightly in the experimental groups. The 10 µM dose showed the highest cell count. EEC morphology changed into long spindle cells in the case groups. CONCLUSION: Sildenafil (1, 10, and 20 µM) showed a mild proliferative effect on human EEC numbers, but no significant change was seen in NO production.