The study population was obtained from Tongji Hospital, Huazhong University of Science and Technology, China. The participants included were 11 women with an uncomplicated first-term pregnancy and 10 pregnant women diagnosed with RSA. The inclusion criteria for both groups were: The couples and embryos had normal chromosomes and no family genetic history, absence of anatomical deformity of reproductive organs, normal endocrine profile, normal coagulation and autoimmune function as well as no vaginal infections. No women received any treatment that affected the results of these examinations in the past 3 months before being enrolled in the study. The diagnostic criteria for RSA was two or more consecutive spontaneous abortions with the same sexual partner. There was no statistical difference in ages, gestational ages and menstrual cycle between the two groups. The protocol was approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology (2020-S150) and informed consent of all women was obtained. Villous and decidual tissues were collected during uterine curettage. The collected samples were washed immediately with normal saline and divided into two parts: one was fixed with polyformaldehyde for paraffin embedding and the other was stored at -80 ℃.
The trophoblast cell line HTR-8/SVneo was cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. The macrophage cell line RAW264.7 was cultured in DMEM with 10% FBS, but no antibiotics, which might cause macrophage polarization. The cell was also placed in the cell incubator at 37 ℃ with 5% CO2 in humidified air.
The HTR-8/SVneo Cells were seeded in a six-well plate with a density of between 40 and 60% for transfection. Transfection was performed using lipo3000 reagent (Invitrogen) in OPTI-MEM medium (Gibco) and siRNA (RiboBio), targeting the coding region of G-CSF (50 nM) and negative control siRNA (50 nM), according to the manufacturer's instructions. After 48 to 72 h of culture, the cells were collected for detection.
The HTR-8/SVneo cells were planted in 10 cm petri dishes and cultured. The cells were then expanded in large quantities and culture medium was collected. The medium used to extract exosome was supercentrifuged overnight to remove its own exosome before cell culture. The medium was centrifuged at 300 g for 10 min, 2000 g for 30 min and 12000 g for 45 min to remove whole cells and debris. The resulting supernatant was passed through a 0.22 µm sterile filter and then ultra-centrifuged at 120000 g for 2 h. The exosome-containing pellets were washed in PBS, and ultra-centrifuged again at 120000 g for 2 h . The final precipitates were dissolved in PBS and stored at − 80°C. The size and morphology of exosomes were identified through TEM and nanoparticle tracking analysis NTA.
Macrophage phagocytosis assay
The exosomes were incubated with PKH26 dye (Sigma) and the staining was stopped by serum, followed by ultra-centrifuged at 120000 g for 2 h. The obtained exosomes were added to the RAW264.7 culture medium at a concentration of 10 µg/mL. After 24 h of culture, the macrophages were fixed with paraformaldehyde, stained with DAPI, sealed with anti-fluorescence quenching agent and observed under fluorescence microscope as well as laser scanning confocal microscope.
Female CBA/J, male DBA/2J and male BALB/c mice (6 weeks old) were acquired from Wuhan SLB Biotech CO., LTD for the animal studies. All the studies were performed in the Laboratory Animal Centre, Tongji Hospital. The experimental protocols of this study were approved by the Animal Ethics Committee of Tongji Hospital (TJH-202009005). Mice were reared under controlled temperature (21 ℃ -22 ℃), humidity, and 12 h of a light/dark cycle with water and food ad libitum for 2 weeks. Female and male mice were mated in the ration of 2:1 at 20:00 hrs and checked at 6:00 hrs the next day for vaginal plugs. The presence of a vaginal plug was designated as day 0.5 of pregnancy. Pregnant mice received daily intraperitoneal injections of recombinant G-CSF or saline from day 4.5 to 13.5. CBA/J females that mated with BALB/c were considered as normal group and received 200 µl saline (n = 6), and CBA/J females that mated with DBA/2 were randomized and divided into the following groups: RSA group received 200 µl saline (n = 6) and TREAT group received 200 µl of 50 µg/kg recombinant G-CSF [34–36]. Pregnant females were killed at day 14.5, the total number of resorbing and healthy embryos were recorded, the foetal and placenta weight were also determined and the placentas were fixed with polyformaldehyde for paraffin embedding. Abortion sites were identified by their small size accompanied by a necrotic, haemorrhagic appearance compared to normal embryos and placentas. The resorption rate (%) was calculated as the number of total abortion divided by the number of total implantation and multiplied by100.
Patients and mice paraffin sections were deparaffinised and rehydrated. Slides were then incubated in citric buffer for antigen retrieval. The sections were treated with 3% hydrogen peroxide to suppress endogenous peroxidase activity. After incubation with 10% donkey serum, primary rabbit antibodies (G-CSF, abcam, diluted 1:200; G-CSFR, GeneTex, diluted 1:200; NLRP3, abcam, diluted 1:200) were applied overnight at 4°C. The second goat-anti-rabbit antibody was incubated after washing with PBS for three times. Diaminobenzidine was used as a chromogen and a light counterstaining was performed with haematoxylin. After dehydration and drying, the sections were sealed with neutral resin and observed under an optical microscope. The images were analysed semi-quantitatively using imageJ software and MOD = intDen/Area.
RNA isolation and quantitative real-time polymerase chain reaction (RTPCR)
Total RNA was extracted from tissues or cells using TRIzol reagent. Two µg RNA of every sample was converted to cDNA through reverse transcription. Quantitative real-time PCR analysis of genes was done using primers described in an additional file (Additional file 6: Table S1) and detected using the CFX96TM real-time system. Gene expression data was normalized to the mRNA levels of housekeeping gene GAPDH.
Total proteins from tissues or cells were lysed using RIPA buffer. Twenty µg of proteins was loaded on a 10% Bis-Tris SDS gel and run for approximately 2 h at 120 V and then transferred to the PVDF membranes. The membranes were then blocked in 5% BSA and immunoblotted with primary antibodies (G-CSF, abcam, diluted 1:1000; G-CSFR, GeneTex, diluted 1:1000; CD9, abcam, diluted 1:1000; TSG101, abcam, diluted 1:1000; calnexin, abcam, diluted 1:1000; NLRP3, abcam, diluted 1:1000; GAPDH, abcam, diluted 1:1000) overnight at 4°C. Membranes were washed with TBST and incubated in the goat anti-rabbit or anti-mouse secondary antibodies (Servicebio, diluted 1:5000) for 1 h at room temperature. After three washes with TBST, the membranes were coloured by an ECL system. The images were then analysed semi-quantitatively using imageJ statistical software. Lastly, the data on protein expression were normalized to the levels of GAPDH.
Cell migration was evaluated using a Transwell chamber. Cells suspended in serum-free media were seeded in the upper chambers at a density of 2×104 cells per well. Complete DMEM (500 µL) with 20% FBS was added to the lower chambers. After 24 hours of culturing, the cells in the upper chamber were removed with a cotton swab and the cells on the lower surface were fixed and stained with purple crystal. Excised and mounted filter membranes were photographed using an optical microscope and cells in four random fields per sample were counted and recorded.
Cell proliferation was determined through CCK8 assay. Cells were plated in 96-well plates at a density of 4×103 cells per well. The CCK8 mixture was prepared according to DMEM:CCK8 = 9:1 and added to each well at 0 h, 12 h, 24 h, 48 h and 72 h after cell intervention. Absorbance was measured at 450 nm using an enzyme-labelled instrument after 2 h of culture.
SPSS version 22.0 software was used for data analysis. Results were presented as mean ± SEM. One-way analysis of variance (ANOVA) was used to assess statistical significance among various experiment groups followed by Bonferroni post hoc analysis. Statistically significant difference were set at p < 0.05.