Patients and sample collection
This study was a retrospective case-control study and was approved by the Ethics Committee of the Second Hospital of Shandong University. From December 2016 to December 2018, the subjects were enrolled from the Second Hospital of Shandong University. Pregnant women were subjected to a 75-gram oral glucose tolerance test (OGTT) during the 24th to 28th weeks of pregnancy. GDM was diagnosed in patients with one or more of the abnormal values, including OGTT fasting ≥ 5.1mmol/L, 1h ≥ 10.0 mmol/L and 2h ≥ 8.5 mmol/L, according to the recommendations of the International Association of Diabetes and Pregnancy Study Groups. Women with other pregnancy-related diseases, such as pre-pregnancy diabetes, chronic hypertension, multiple pregnancy, and pregnancy-induced hypertension were excluded. The GDM patients and age paired normal glucose tolerant (NGT) subjects were included in this study. The blood samples were collected from three cohorts with informed consent, including third trimester (36-41 weeks) group (GDM, n=46; NGT, n=47), second trimester (24-26 weeks) group (GDM, n=58; NGT, n=56), and first trimester (11-13 weeks) group (GDM, n=24; NGT, n=43). The peripheral venous blood specimens were obtained from women 48 h before and 48 h after delivery in third trimester group [31]. The blood samples were collected from pregnant women during Down's screening in the first trimester group and those underwent OGTT in the second trimester.
Exosome isolation
The blood samples (5 mL) were centrifuged at 3,000 rpm for 10 min at room temperature for plasma isolation. Then, the plasma samples were mixed with 0.2 volume of total Exosome Isolation Reagent (Thermo Scientific, Worcester, MA, USA), and incubated at 4°C for 60 min. Subsequently, exosome-containing pellets were collected by centrifugation at 10,000 rpm for 10 min at 4°C and suspended in appropriate amount of phosphate buffer saline (PBS).
CircRNA microarray
Three GDM patients with mean age of 32.33 ± 6.11 years during the three-trimester pregnancy were included. The basic information of the three patients were listed in supplemental Table 1. Three paired plasma exosome samples extracted from subjects before and after delivery were used for microarray analysis. The circRNA microarray analysis was performed by the Sinotech Genomics Corporation., Shanghai, China. Briefly, the exosome RNA was extracted and purified by using a RNeasy Mini Kit (Qiagen, GmBH, Germany). The integrity and concentration of RNA were checked by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). The qualified RNAs were used for cRNA synthesis and labeling with the application of Sino Human ceRNA array V3.0. Upon hybridization, the array was imaged and scanned by the Agilent Microarray Scanner (Agilent technologies, Santa Clara, CA, US). The raw data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US), and normalized by limma package in R based on Quantile algorithm.
Reverse transcription (RT) quantitative PCR (qPCR) analysis
Exosome RNA (0.5 μg) was reversely transcribed to cDNA using M-MuL V reverse transcriptase (Fermentas, Hanover, MD, USA), as per the manufacturer’s procedures. The primers for each circRNA were designed by Primer 5 (Table 2). PCR reaction was performed by using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific, Foster City, CA, USA) with the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Conditions for PCR analysis were listed as follows: denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 60 s, and 72°C for 30s. The qPCR data were analyzed by using 2-ΔΔCt method and normalized to GAPDH. The same procedure was repeated for 3 times.
Western blotting
The exosomal maker proteins of CD63 and Tumor Suspectibility Gene 101 (TSG101) were analyzed by western blot assay. Total protein extraction of exosomes was achieved by using the lysis buffer Pro-Prep (iNtRON Biotechnology, South Korea). Proteins (20 μg) were subjected to separation by using a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) system, followed by polyvinylidene fluoride (PVDF) membranes transferring. Subsequently, membranes were incubated with primary antibodies, namely anti-CD63 (1:2,000, ab59479; Abcam), and anti-TSG101 (1:3,000, ab125011; Abcam) and then the secondary antibody of horseradish peroxidase-conjugated anti-rabbit (1:5,000, Novus Biologicals, Littleton, CO, USA). The protein blots were visualized by Western Blotting Luminol Reagent (Bio-Rad, Hercules, CA, USA) incubation and a X-ray film exposure.
Transmission electron microscopy (TEM)
Exosomes suspended in PBS were fixed by using 1% glutaraldehyde, and then placed on a copper grid coated with formaldehyde/carbon. Exosomes were stained with 1% uranyl acetate and observed under a JEOL 1200EX electron microscope (JEOL Co., Ltd., Tokyo, Japan).
Nanoparticle analysis
The size distribution profile of exosomes was analyzed by nanoparticle tracking analysis (NTA). The exosome sample at 1×108 particles/mL was diluted by PBS and subjected to NTA for 3 cycles at 11 positions. and the particle size and concentration were analyzed by using the built-in ZetaView.
Statistical analyses
Statistical analysis of the data was performed using the SPSS Statistics 22.0 software (University of Waikato, Hillcrest, NZ). The Shapiro-Wilk test was applied to evaluate the normality of the acquired data. Data with a non-normal distribution was expressed as median and quartile intervals, while the normally distributed data was expressed as mean ± SD. If the data conformed to normal distribution, paired T-test was used to analyze the differences between two groups, otherwise the Wilcoxon test was applied. Pearson’s chi-squared test was used to analyze categorical variables which were represented as numbers and percentages. Correlation between circRNAs and OGTT glucose levels were analyzed by Pearson correlation analysis and ROC curve was constructed to evaluate the diagnostic value of the circRNAs. The significance level for all analyses was set at a p-value of 0.05.