Decidual tissue collection
This study was approved by the Ethics Committee of Renji Hospital (No. 2018033004). Human decidual tissues were obtained from women with clinically normal pregnancies (terminated for nonmedical reasons, n= 20) and women with unexplained RM (n = 20). All the participants were Han ethnicity. The average age of women with normal pregnancies used as healthy controls (HC) was 29.0 ± 3.4 years, while the average age of patients with RM was 28.1 ± 4.5 years. The gestational ages when pregnancies were terminated in the HC group and RM group were 9.2 ± 1.3 weeks and 8.2 ± 2.3 weeks, respectively.
The exclusion criteria were as follows: (1) uterine anatomical malformation according to pelvic examination and ultrasound; (2) genetic abnormalities; (3) endocrine or metabolic diseases; (4) infection; and (5) other known causes. Written consent was obtained from all subjects before the collection of decidual tissues according to the guidelines of the Ministry of Public Health of China. Decidual tissues were collected immediately after suction and curettage, and blood was removed by scrubbing the decidua on cotton gauze. Collected tissues were then quickly frozen in liquid nitrogen and subsequently stored at -80°C before use.
Western blot analysis
Briefly, total protein extracts were prepared from homogenized tissues or cultured cells with RIPA buffer containing protease inhibitors and a phosphatase inhibitor (cOmplete & PhosSTOP, Roche, Basel, Switzerland). Protein was quantified with an Enhanced BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated by SDS-PAGE before wet transfer onto nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked with 5% nonfat milk for 2 h at room temperature. After blocking, the membranes were incubated at 4°C overnight with primary antibodies against SKP2 (1:1000; ab183039, Abcam, Cambridge, UK), GLUT1 (1:1000; 66290-1, Proteintech, Rosemont, IL, USA), or β-actin (1:5000; sc47778, Santa Cruz, AR, USA), the latter of which was used as an internal control. Protein complexes were developed and observed with a chemiluminescent detection system (Millipore, Waltham, MA, USA) and a G-Box Chemiluminescence image capture system (Syngene, Cambridge, UK). The resulting bands were analyzed using Gel-Pro Analyzer software (Media Cybernetics).
Quantitative PCR analysis
Total RNA was extracted from cells or tissues using an animal total RNA isolation kit (Foregene, Chengdu, China) according to the manufacturer’s instructions. The concentration and purity of RNA were assessed using a spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (500 ng) was reverse transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), and the resulting cDNA was used as the template for qPCR. Gene expression quantitation was performed in triplicate using SYBR GREEN PCR Master Mix (Toyobo, Osaka, Japan) in an Applied Biosystem Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). ACTB was used as an internal control. The results were analyzed using the ΔΔCt method, and the sequences of the primers for PCR are listed in Table 1.
Immunohistochemistry was performed as previously described[24, 25]. Briefly, paraffin sections (5 µm) of human decidua from early pregnancy were dehydrated in a graded ethanol series and incubated with 3% hydrogen peroxide to block endogenous peroxidase. Subsequently, after antigen retrieval, the sections were incubated with 0.3% Triton X-100 for 20 min. The sections were then blocked with immunoglobulin G and incubated with a primary antibody against SKP2 overnight at 4°C (1:50, ab183039, Abcam, Cambridge, UK). The sections were overlaid with peroxidase-conjugated goat anti-rabbit IgG (PV-9000, ZSGB-BIO, Beijing, China); then, the reaction was developed with 3,3′-diaminobenzidine and counterstained with hematoxylin. Negative controls were treated with nonimmune serum instead of the primary antibody.
HESC culture and treatment
HESCs kindly donated by Dr. Haibin Wang (Xiamen University, Xiamen, China) were cultured in Phenol Red-free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 plus 10% charcoal-stripped fetal bovine serum (Biological Industries) at 37°C in a 5% CO2 atmosphere. To induce decidualization in vitro, HESCs were incubated with 1 μmol/L medroxyprogesterone-17-acetate (MPA) (Sigma-Aldrich, St. Louis, MS, USA) and 0.5 mmol/L N6,20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma-Aldrich, St. Louis, MS, USA). The medium was replaced every two days.
Primary endometrial stromal cell (ESC) and primary decidual stromal cell (DSC) isolation
Proliferative endometrial tissues were obtained from women with normal menstrual cycles via endometrial biopsy at the time of diagnostic hysteroscopy. Primary ESCs were isolated according to previously described methods. Briefly, endometrial tissues were fully washed with PBS and then minced in DMEM/F12. Minced tissues were digested with collagenase type I (c0130; Sigma-Aldrich, St. Louis, MS, USA) for 70 min at 37°C and sequentially digested with deoxyribonuclease (DN25; Sigma-Aldrich, St. Louis, MS, USA) for 20 min at 37°C. The suspension was filtered through 180 μm and 40 μm sterile griddles and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the cell pellet was suspended and inoculated in 6-well plates (106 cells per well). Decidualization was induced according to the methods employed for HESCs.
Primary DSCs were isolated according to previously described methods. Briefly, decidual tissues were obtained from women whose healthy pregnancies were artificially terminated by personal choice, then the samples were washed in PBS and minced in DMEM/F12.
Cultured cells, including HESCs and isolated primary ESCs, were transfected with siRNA using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). For in vitro-induced decidualization, cAMP and MPA were added after 24 h of transfection. The medium was removed and replaced every 2 days.
All experiments were independently repeated at least three times. All data were analyzed using SPSS statistical software (Version 22.0, IBM, Chicago, Illinois, USA). Differences between two groups were analyzed by one-way ANOVA or Student’s t-test. All data are presented as the mean ± SEM. Differences were considered statistically significant at P < 0.05.