Patients
This study was approved by the ethics committee of Sir Run Run Shaw Hospital (Hangzhou, China). The patient group consisted of women with endometriosis (stage III– IV) undergoing laparoscopy for pain or infertility (n = 6). The control group consisted of women with no endometriosis undergoing laparoscopy for tubal disease (n = 6). Human SP was obtained from male partners [(n = 15), aged 25 to 39 years] of women undergoing treatment for female-factor infertility at Sir Run Run Shaw Hospital in China and healthy fertile men. None of the donors had received hormonal therapy for at least 6 months prior to surgery. Participants had regular menstrual cycles (27–32 days). Additional clinical characteristics of patients are listed in Supplementary Tab. 1. Prior to enrolling in the study, informed written consent (procedure approved by the local Ethics Committee) was obtained from each patient. All female patients exhibited stage III or IV endometriosis, as classified by the American Society for Reproductive Medicine. Sperm analyses of male patients were conducted according to recommendations of the World Health Organization (WHO) 17. Samples were included if all parameters were consistent with normal values in the WHO Laboratory Manual for the Examination and Processing of Human Semen17.
Isolation of MSCs
To isolate and generate MSC lines from the endometrium of women with endometriosis (E-MSCs) and without endometriosis (NE-MSCs), endometrial tissue biopsies were obtained just prior to surgery using an endometrial suction catheter (Lilycleaner, Ningbo, China). Cells were isolated from biopsy specimens of the endometrium and seeded in triplicate at low density (approximately 200 cells per 100-mm dish) in Dulbecco’s Modified Eagle’s Medium with F12 Supplement (DMEM/F12; Gino167 Biological, Hangzhou, China). Large colonies were isolated and trypsinized into single-cell suspensions following incubation for 21 days. The resulting cell suspensions were diluted and seeded into 96-well plates at a density of approximately one cell per well. After 14 days in culture, clonally derived proliferating colonies were trypsinized and individually cultured in individual 100-mm dishes. Cells were allowed to grow in DMEM/F12 with 10% fetal bovine serum (FBS), and adherent cells were cultured until they reached 80%–90% confluence. Cells were trypsinized, subcultured, and used for experiments during passages two to four.
Multipotent differentiation
The multipotency of endometrial MSCs for osteogenesis and adipogenesis was determined as previously described 18. Briefly, to induce osteogenesis, MSCs were plated at a density of 5 × 103 cells per cm2 and treated with 10 mm β-glycerol phosphate, 0.1 μM dexamethasone, and 50 μg/ml ascorbic acid (all from Sigma-Aldrich, St. Louis, MO, USA) for 2 weeks. On Day 14, cells were stained with Alizarin Red and alkaline phosphatase. To induce adipogenic differentiation, MSCs were cultured in DMEM supplemented with adipogenic supplements (Stem Cell Technologies, Vancouver, Canada). On Day 14, lipid vacuoles within cells were stained with Oil Red O.
SP Collection
Semen samples obtained from 15 donors were allowed to liquefy for 2 hours. After liquefaction, each sample was centrifuged at 700 × g for 10 minutes at room temperature within 2 hours of ejaculation. The supernatant was centrifuged at 10,000 × g for 30 minutes to remove spermatozoa. The collected samples were pooled, filtered and concentrated using Centricon Plus-20 centrifugal concentration tubes (3,000 NMWL; Millipore, Cork, Ireland) (Supplementary Fig. S1A). Acid activation of cytokines in SP was achieved by treating concentrated SP with 1 N HCl, followed by incubation at room temperature for 10 minutes. SP was then neutralized with 1.2 N NaOH/0.5 M HEPES before resuspending in DMEM/F12. The treated SP was aspirated and frozen at -80 °C. Prior to use, concentrated SP was thawed on ice, resuspended, and diluted 1:100 with DMEM/F12 for in vitro studies, or 1:500 with sterile-filtered phosphate-buffered saline (PBS) for in vivo studies.
Treatment of cells
Cells were seeded into 96-well plates at a density of 2 × 103 cells per well for proliferation analyses, and 5 × 104 cells per 60-mm dish for western blotting (WB). For cell proliferation analyses, cells were cultured at 37 °C for 1 day in DMEM/F12 containing 10% FBS, followed by incubation with control (DMEM/F12 + 2% FBS) or SP containing 2% FBS for the indicated time. For foci formation assay, flow cytometry analysis, and WB, cells were cultured at 37 °C for 1 day and then incubated for 1 day in serum-free culture media. Subsequently, culture media were replaced and cells were treated with vehicle control or SP. Foci formation assay was performed after 2 days of culture. WB analysis was performed after 2 days of culture or at specified time (0, 1, 2 and 6 hours).
Flow cytometry analysis
Endometrial MSCs (1 × 106) were resuspended in PBS and incubated with 1 μg of fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat monoclonal antibodies for 1 hour at 4 °C in 100 ml of PBS containing 0.1% bovine serum albumin (BSA). The following antibodies were used: anti-CD44, CD90, CD176, CD73, CD29, CD117, CD 34, CD45, HLA-DR, and FITC-conjugated isotype-matched immunoglobulin G (1:50; all from Becton Dickinson, Franklin Lakes, NJ). Cells were washed twice with PBS before analysis using an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Cell-cycle parameters were determined by flow cytometry of propidium iodide (PI)-stained endometrial MSCs 19. Briefly, MSCs (2 × 106) were fixed in 70% ethanol at 4 °C overnight. The ethanol was discarded and MSCs were resuspended in PBS. Cell pellets were stained with 50 µg/ml PI and 100 μg/ml RNase A in PBS for 30 minutes at room temperature. After washing with PBS, MSCs were analyzed by an Epics XL flow cytometer.
Flow cytometric analysis of apoptosis markers was performed. Endometrial MSCs were washed in cold PBS and incubated with FITC-conjugated Annexin and PI solution (Invitrogen, Carlsbad, CA, USA) at room temperature for 15 minutes in the dark. Stained cells were analyzed using an Epics XL flow cytometer.
Cell proliferation assay
Cell proliferation was determined using a Cell Counting Kit 8 (CCK-8) assay. Endometrial MSCs were seeded into 96-well plates at a density of 2 × 103 cells per well. After cell attachment, control (phenol red-free DMEM/F12 containing 2% FBS) or resuspended SP containing 2% FBS was added to cells. To evaluate changes in cell proliferation, cell counting was performed using a CCK-8 kit (Tojindo, Shanghai, China) according to the manufacturer’s instructions.
Colony-forming assay
MSCs were seeded into six-well culture plates at a density of 1 × 102 cells per well and incubated at 37 °C for 14 days. Subsequently, MSCs were stained with a crystal violet solution and imaged with a microscope equipped with a digital camera.
Antibodies and western blotting
Cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resulting proteins were transferred onto polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline containing Tween-20 and 5% BSA, and then probed with the following primary antibodies: proliferating cell nuclear antigen (PCNA; 1:500; Abcam, Cambridge, UK), B-cell lymphoma-2 (Bcl-2; 1:500; Abcam), β-Tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:3000; Abcam), total Akt, p-Akt-Ser473, p-Akt-Thr308, p-p42/44, total p42/44, CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E2 (1:1000, Cell Signaling Technology, Beverly, MA, USA). Anti-rabbit and anti-mouse antibodies (1:1000; Cell Signaling Technology) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA) were used to visualize immune-positive bands. Protein expression levels were normalized to β-Tubulin or GAPDH. ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij) was used to evaluate protein band densities.
Mouse model of endometriosis
Mature female athymic nude mice (6 weeks old) purchased from Shanghai Laboratory Animal Co. Ltd. (Shanghai, China) were housed in a pathogen-free and climate-controlled environment (23–25 °C) with regulated 12-hour light/dark cycles. One week was allowed for acclimatization prior to experimental proceedings. Three days prior to endometrial transplantation, mice received a daily intraperitoneal injection of 200 µg/kg 17β-estradiol. The nude mouse model of endometriosis was established as previously described 18,20. Proliferative-phase human endometrial fragments (1–2 mm3) from endometriosis patients were washed with sterile serum-free DMEM/F-12 culture medium. Human endometrial fragments were implanted subcutaneously into mice under general anesthesia. To achieve higher local concentrations, subcutaneous injection was employed as the administration route based on previous animal experiments 18,20,21 (Supplementary Fig. S1B). Subcutaneous injections of TGF-β1 (40 ng/g), SP, or the TGF-β receptor inhibitor SB431542 (10 ug/g) were employed. Daily intralesional injections were started on the first day after endometrial tissue implantation and continued for 14 days. Mice were then randomly divided into four groups: control, TGF-β1, SP, and SP+SB431542. Endometrial tissues were harvested and measured after 14 days of intralesion injections commencing from day 1 post-implantation. Treatment dosages used were determined by preliminary experiments. Endometriotic implants were collected, fixed in 10% formalin-acetic acid, and embedded in paraffin for histopathological examination. Immunohistochemical staining was performed according to well-established protocols 1. Anti-human leukocyte antigen (HLA) antibody (1:100; Abcam) was used to identify human cells within xenograft tissue. Tissue sections were also incubated with a polyclonal antibody for the proliferative marker Ki67 (1:100; Abcam). Staining scores and parameters were calculated using a computerized image analysis system, as previously described 18. Areas of positive staining were calculated. In addition, the whole area of the endometriotic implant in each section was analyzed.
Data analysis and statistics
SPSS version 16.0 (SPSS, Chicago, IL) was used to analyze complete datasets. Student’s t test, one-way analysis of variance, or Scheffe’s general linear model of repeated measures method was used to compare differences between treatment groups. Statistical significance was defined as P < 0.05.