Patients who were diagnosed having EMs, requiring surgical treatment and referred to the general gynecology department of the Women’s Hospital, Zhejiang University School of Medicine, were included in the study. Written informed consent was obtained from each patient before study inclusion. A total of 205 women with EMs (110 cases) and without EMs (95 cases) were enrolled. Indications for surgery in the EMs group were as follows: pelvic mass, history of infertility, pelvic pain, and failed analgesics. Study inclusion criteria of the EMs group included: (1) Age ≤ 40 years; (2) histologically proven EMs; (3) restriction of samples to those of moderate or severe disease (stages 3 and 4) according to the r-ASRM Classification; (4) restriction to OMA or DIE phenotypes (SUP, OMA and DIE phenotypes are frequently associated with each other), with the final phenotypic diagnosis of EMs designated according to the worst lesion, as per a previous study (6). Exclusion criteria were: (1) women with only a SUP phenotype; (2) irregular menstrual cycles; (3) those with history of metabolite diseases such as diabetes, obesity, cardiovascular disease or thyroid disease etc; (4) those with a history of autoimmune or inflammatory diseases; (5) pregnancy; (6) those having had hormone treatment such as oral contraceptives, GnRH analogues or any other hormone treatment during the previous 3 months before the study.
Women ≤ 40 years old requiring surgical treatment, but without any evidence of EMs, were recruited as controls during the same period. Detailed history, a thorough physical examination of the abdominopelvic cavity and sonography screenings were performed by the designated experts for every patient. Control patients presenting with dysmenorrhea or tenderness in the pelvic area or a mass in ovary or those with history of metabolite diseases such as diabetes, obesity, cardiovascular disease or thyroid disease were excluded.
Measurement of serum metabolites
Weight and height was determined for all patients. The BMI was calculated as weight (kg) divided by the square of height (m2). Venous blood samples were obtained from each patient at baseline after an overnight fast of 12 hours. Cases and controls also received standard laboratory testing. The concentrations of serum fast blood glucose (FBG), TC, total TGs, HDL and LDL were measured using an enzymatic colorimetric assay. Lp(α), Serum ApoA1 and ApoB levels were measured using the immune turbidimetric method. Uric acid (UA) was measured using uric acid enzymatic methods. The metabolite profiles were performed on ABBOTT ARCHITEC c16000 (Chicago). The intra- and inter-assay coefficients of variation for all measurements were 5% and 10%, respectively. The ratios of these markers including the ratio of TG to HDL (R-TG/HDL), the ratio of TC to HDL (R-TC/HDL), the ratio of LDL to HDL (R-LDL/HDL), the ratio of ApoB to ApoA1 (R-ApoB/ApoA1) and the ratio of ApoA1 to ApoB (R-ApoA1/ApoB), were calculated.
Culture of human ectopic endometrial stromal cells
With permission of the patients, lesion tissues from the 8 patients whose intraoperative r-ASRM scores were all endometrial stage 3/4 were sampled under sterile conditions and kept in cold DMEM/F-12 (Gibco) with 1:100 Penicillin-Streptomycin Liquid (Beyotime) for subsequent cell culture. The tissues were digested with 0.2% type I collagenase (Solarbio) in 37OC for 1.5 hours and then hand filtered using a 70 µM cell filter (BD Falcon). Cells were cultured in DMEM/F-12 with 10% FBS (Gibco) and 1% Penicillin-Streptomycin Liquid in an incubator (Esco) at 37OC, 5% CO2. The cells were passaged when the density of primary cells had reached more than 75%. Resveratrol (Selleck) was initially dissolved in DMSO (Sangon) to make 20 mM and 8 mM mother fluids, and then diluted to the working concentrations of 100 µM and 40 µM, respectively.
Sample preparation and detection: Eight groups of the cultured primary HEcESCs were divided into two parts. One was treated with 100 µM resveratrol and the other was treated with solvent only. Culturing was for 48 hours where about 1 × 106 cells were collected and subjected for the following lipidomic analysis. All samples were prepared according to previously described techniques (22). An UHPLC system was used to coordinate an electrospray ion source using a Q Exactive-HF MS system (Thermo) which was used for lipid profiling (UPLC-MS). Chromatographic conditions: Flow rate was 0.26 mL/min while column temperature was 55OC. The mobile phases consisted of (A) 60% acetonitrile/H2O with 10 mM ammonium and (B) Isopropanol: acetonitrile = 9:1 (with 10 mM ammonium format). We applied positive and negative mode linear gradients to detect the subjects, respectively. Mass spectrometry was performed using a Thermo Q ExactiveTM benchtop Orbitrap mass spectrometer equipped with heated ESI source in ESI positive and negative modes (Thermo).
Data Processing: All assay raw data were collected using Xcalibur data acquisition software (Thermo). The data, including m/z-values, retention times, and peak areas, were extracted using LipidSearch software (Thermo). All of the detected lipids were quantified using the Thermo TraceFinderEFS software (version 3.2). The lipid molecules were named by reference to the LipidMaps website. We enabled One-MAP (www.5omics.com) software to support comprehensive metabolic data analysis. Multivariate statistical analysis was performed online. This included hierarchical clustering analysis, Pearson correlation heat maps, Z-score plot, Volcano plot, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), construction of a receiver operating characteristic curve (ROC) univariate, and the construction of a permutation plot and a variable importance in projection (VIP) plot.
Cell proliferation, invasiveness and apoptosis assays
Proliferation: Cultured HEcESCs, grown to the logarithmic growth stage, were digested with 0.25% Trypsin-EDTA and re-suspended. 100 µL of 4 × 104 cells/mL suspension was inoculated into 96-well plates (Corning) for 24 hours. Four experimental groups, each with triplets, were prepared as follows: blank (100 µL culture medium); control (DMSO); Re-40 µM (resveratrol at a concentration of 40 µM); Re-100 µM (resveratrol at a concentration of 100 µM). After 48 hours treatment, 10 µL CCK8 (Solarbio) solution was added and incubated for another 4 hours. The cell viability was measured with a BioTek Synergy 1 plate reader (BioTek) and calculated.
Invasiveness: The matrigel (Solarbio) was thawed at 4OC and diluted with 1: 12 in serum-free DMEM/F-12. The 8 µM upper chamber of the transwell plates (Corning) was coated and gelatinized for 1 hour in an incubator at 37OC. The cells were treated with DMSO, 40 µM resveratrol and 100 µM resveratrol for 48 hours and then digested. The upper chambers were filled with 2 × 104 cells in 1% FBS DMEM/F-12 medium and the lower chamber with 600 µL 10% FBS DMEM/F-12 medium. The triple transwell plates were placed at 37OC, in a 5% CO2 incubator for 48 hours. Transwell chambers were fixed with 95% ethanol and stained with 0.1% crystal violet for 30 minutes. Five visual fields (400×) were randomly selected under the microscope to count the cells that had crossed the matrigel.
Apoptosis: The cells were treated separately with DMSO, 40 µM resveratrol or 100 µM resveratrol for 48 hours, then digested and re-suspended using a binding buffer (Beyotime) to make a 1 × 106 cells/mL suspension. 100 µL cell suspension was added into a 5 mL flow tube and 5 µL Annexin V Alexa Fluor 488 was then added. The mixture was incubated in a dark room for 5 minutes and 10 µL PI, 200 µL PBS was then added. Cellular apoptosis was analyzed by NovoCyte Flow cytometer (ACEA).
Establishment of a rat model of EMs and medical treatment
Animals: Fifty female Sprague Dawley rats aged 8–10 weeks, weighing 200–250 g, were placed in a clean-level environment in the Zhejiang University Laboratory Animal Center with 12 hours light/dark cycles and regular feeding. Animal experimental methods and purposes were all in line with ethical standards and international practices.
Modeling: Prior to any surgery, the estrous cycle stages of female rats were examined using vaginal biopsy samples. Attrition cells, showing as irregular keratinocyte like cells and gathered together on the slides, was considered to be an indicator of a mature estrous stage for efficient EMs modeling. Rats having a 4–5 days estrous cycle and two consecutive estrus cycles were then selected for surgery. The animals were anesthetized using 45 mg/kg by intraperitoneal injection of 3% pentobarbital (BIOCAM) sodium and operated under strict aseptic conditions at a room temperature of 28-30OC. Rat estrus epithelial tissue with a 0.8 × 0.8 cm2 endometrium was auto-transplanted into the endometrial abdominal wall. Welfare nursing was provided after the operation. Ten rats were also selected for a placebo operation to serve as sham. The animals were fed regularly for 4 weeks.
Examination: A laparotomy was performed 4 weeks after the surgery. The rats were euthanized and the laparotomy was performed to measure size of the implant. Modeled rats were recorded and the lesion volume was calculated using the following formula: V = a × b2/2 (a represents the broadest transverse diameter of the lesion, b represents the vertical diameter line) and V ≥ 2 mm3 was considered as a successful model.
Resveratrol treatment and evaluation
Resveratrol was dissolved in 35% DMSO for intraperitoneal injection in rats, while the Sham group and EMs group were injected with the same amount of the solvent (0.9% NaCl + 35% DMSO). Thirty rats with successful modelling were divided into three groups randomly: EMs group (n = 10), Res-med group (n = 10, resveratrol dose = 15 mg/Kg/d), Res-high group (n = 10, resveratrol dose = 45 mg/Kg/d). The rats of four groups were administered continuously for 28 days. Lesions were examined (as above method). Lesion tissues and blood were sampled before and after resveratrol treatment for evaluation.
Lesion tissues were fixed in 10% formalin and dehydrated with a gradient of alcohol for paraffin slicing. The sections were processed according to a standard protocol for staining with Hematoxylin and Eosin (Solarbio). Images were taken under a light microscope (Nikon) and pathological features were analyzed.
Detection of serum TC, TG, HDL and LDL of rat models
Whole blood (500 µL) was collected and centrifuged to detect TC, TG, HDL and LDL and analyzed by fully automatic biochemical analyzer (Toshiba FR120). The following Detection kit (Beijian) were used: total cholesterol measurement kit (CHOD-PAP method); low-density lipoprotein cholesterol measurement kit (direct method-protective reagent method); high-density lipoprotein cholesterol measurement kit (Direct method-selective inhibition method); Triglyceride kit (GPO-PAP).
Total RNAs were extracted from tissues or cells using trizol (Sangon) and then reversely transcribed into cDNA using a reverse transcription kit (Vazyme). ChamQSYBRqPCR Master Mix (Vazyme) was applied for qRT-PCR using a real-time quantitative PCR machine HT faster 9600T (Biosystem). The following primers were used: β-actin-F: 5’-ATCCGTAAAGACCTCTATGC-3’, R: 5’-ACACAGAGTACTTGCGCTCA-3’; PPARα-F: 5’-GGCAATGCACTGAACATCGAG-3’, R: 5’-GAAAGCCGCTTGATAAGCCG-3’.
Total proteins were extracted using a standard protein lysis buffer. The protein concentration was determined using a BCA kit (Gene Ray). The standard curve was made according to the absorption value of standard liquid, and the concentration of protein was measured and calculated. Samples were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were immunoblotted with rabbit anti-PPARα (1:500, Proteintech) and mouse anti-Actin (1:1000, Goodhere Biotechnology Co, AB-M-M001). Detection of proteins was performed using the ChemiLucentTM ECL detection reagents (Millipore, WBKLS0500). Images were taken using the chemiluminescence imaging system (Clinx Science Instruments).
The open field assay
The open field experiment was carried out in a market equipment (open square box, 2 m × 2 m × 50 cm) which was equipped with an infrared camera (CCTVLENS). Experiments were performed in the standard manner. The rats were placed in the test room to acclimatize for 2 hours, and then each one placed in the same orientation when entering into the market. The test time of each rat was 5 minutes. The trajectory and movements of the rats was tracked using Video Track 3.10 software for subsequent analysis of parameters such as the movement time in the central area and the number of entrances into the central area. In between each experiment the field was cleaned with 70% ethanol to eliminate the odour of the previous rat.
Continuous characteristics were presented as Means ± SD for normal variables, and Median (Q1-Q3) for abnormal variables. The difference between control groups and the EMs group for different phenotypes was tested by ANOVA and T-test for normal variables, Kruskal-Wallis test and Wilcoxon test for abnormal variables. Categorical variables were given as N (%) and the chi-square test was applied to compare the distributions across different groups. Multivariable logistic models were applied to assess the association of Metabolite indicators with EMs in general, and with its two phonotypes more specifically.
As potential risk factors, all above interesting metabolite markers, plus general information including age, BMI, history of delivery and abortion, were entered into the initial model. A step-wise selection method, with a significance level of 0.05 required to allow a variable into the model (SLE = 0.05), and 0.10 for a variable to remain in the model (SLS = 0.10), was then adopted to identify the final model which contains the best subset of the potential risk factors. Finally, for each single variable, as well as for the union of variables in the final model, ROC curves were performed to determinate the diagnostic value where Youden index (sensitivity + specificity-1) was used to select best cut-off point. Statistical analyses were conducted using SAS, version 9.4 (SAS Institute, Cary, NC). The difference between the two groups was analyzed using a T test (Graphpad Prism5). Differences were considered significant at a p-value of < 0.05, marked *P < 0.05, **P < 0.01, ***P < 0.001.