Clinical samples, ovarian stimulation
The clinical samples were obtained from 66 PCOS patients and 80 healthy controls undergoing in vitro fertilization (IVF) at the Department of Reproductive Medicine Center, the Fourth of Shijiazhuang Maternity Hospital between April 2020 and September 2021. PCOS was diagnosed in strict accordance with the 2003 Rotterdam criteria, excluding Cushing’s syndrome, androgen secreting tumors, thyroid dysfunction, endometriosis, hyperprolactinemia and other endocrine diseases. All analysis performed in studies involving human samples were approved by the Clinical Ethics Review Board of the Obstetrics and Gynecology Hospital of Shijiazhuang (approval number: 20210080). All methods were performed in accordance with the relevant guidelines and regulatory methods section. All participants signed informed consent. Meanwhile, this study was conducted in accordance with the 1964 Declaration of Helsinki.
Ovarian hormonal stimulation was performed according to a short-acting gonadotropin-releasing hormone agonist (GnRH-a) long protocol.
Isolation and identification of follicular fluid exosomes
Exosomes were isolated from healthy controls patients (female tubal factors) and PCOS patients’ follicular fluids using the exoEasy Maxi Kit (Qiagen, Germany) according to the manufacturer’s instructions. Finally, the pellets containing exosomes were resuspended in XE buffer and stored at -80°C for subsequent experiments.
Characterization of FF-exosomes was confirmed by transmission electron microscope (TEM): 5-10 µL exosomes were dropped onto the carbon-coated copper grids at room temperature for 3-5 min, and then absorb the excess liquid with absorbent paper. Then10 µL 2% phosphotungstic acid was pipetted on the grids for staining for 2-3 min, the excess fluid was removed, and the grid was dried at room temperature. Finally, the copper grid was detected under TEM at 80KV (HITACHI). Exosome-specific marker TSG101 and EV-associated protein marker HSP70 were analyzed by western blotting.
Isolation and culture of granulosa cells from follicular fluids
On the day of oocyte retrieval, the first tube of serum-free follicular fluid was collected, and then centrifuged at 2000 g for 10 min, the supernatant was stored at -80°C for further experiments. The cell pellets were resuspended in phosphate-buffered saline (PBS) and 1:1 added onto ficoll solution, then centrifuged for 15 min at 1500 g. Cells at the interface were removed and washed twice with PBS, the cell pellets were resuspened in DMEM/F-12 (Hyclone, logan, UT, USA) medium containing 10% Fetal Bovine Serum (FBS) (Gibco, Carlsbad, CA, USA), supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in 5% CO2 cell culture incubator.
Exosome uptake assay
The purified exosomes were labeled with PKH67 green Fluorescent Cell Liner Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, 20 µg exosome-XE was added to 0.5mL Dilution C, 1µL PKH67 was added to 0.5mL Dilution C, then fully mixed and incubated with each other at room temperature for 3 min. 1mL 1% bovine serum albumin was then added to neutralize the excess dye. Then exosome-PKH67 was extracted with exoEasy Maxi Kit following the manufacturer’s instructions (Qiagen, Germany). The labeled exosomes were then co-incubated with KGN cells for 24 h. Then the cells were fixed with 4% paraformaldehyde at room temperature for 10min, washed twice with PBS, the nuclei were stained with DAPI. Finally, the signal was observed under confocal microscope.
RNA extraction, reverse transcription and RT-PCR analysis
Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then 1µg of RNA was reverse-transcribed into cDNA. QRT-PCR was performed using the SYBR Premix Ex Taq kit in accordance with the manufacturer’s protocol. The exosomal RNA was extracted using exoRNeasy Serum/Plasma Starter Kit (Qiagen, Germany). miRNA was then reverse-transcribed into cDNA by Bulge-LoopTM miRNA RT Primer (RiboBio Co., Ltd Guangzhou, China). U6 small nuclear RNA was used as the internal reference of miRNAs. Ct values were indicated by using 2−ΔΔCt method.
To quantify apoptosis related gene Bax and Bcl-2 mRNA expression, we used oligo d(T) 18 primers to reverse transcribe total RNA into cDNA. Then, qRT-PCR was performed by using SYBR Green dye and specific primers for Bax, Bcl-2 and β-actin. The primer sequences we used are listed in Table S1.
Western blotting
SDS lysis buffer freshly mixed with a protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, Cambridge, MA) was used to isolate proteins from cells. Western blotting assay was performed as described previously33. All the antibodies we used are listed in Table S2.
Apoptosis and proliferation assay
Cell apoptosis assay was conducted as described PE AnnexinV Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The cells were pretreated with Exosomes or miR-143-3p mimic or inhibitor (RiboBio Co., Ltd Guangzhou, China), and then 48 h after treatment, cells were collected and diluted into the density of 1x 105 which resuspended in binding buffer, then 5 µL PE AnnexinV and 7-AAD were introduced. The cells were incubated at room temperature in darkness for 15 min. Apoptosis cells were observed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
The cell proliferation assay was conducted according to manufacturer’s instructions. KGN cells were plated in 96-well plates at a concentration of 5000 cells/well. After 24 h, 48 h and 72 h incubation, cells were incubated with 10µLCell Counting Kit-8 (CCK8) (Yeasen biotech Co., Ltd. Shanghai, China) for another 3 h at 37°C in 5% CO2 cell culture incubator. Finally, the absorbance at 450 nm was measured by Microplate reader.
Vector construction and dual-luciferase reporter assay
MiRNAs that target BMPR1A were predicted by online software programs, TargetScan (TargetScan Human 7.1). The full-length of 3’UTR BMPR1A containing the predicted wild-type or mut-type miR-143-3p binding sites were amplified by PCR and then cloned into the PGL3-Basic reporter vector. PGL-3-BMPR1A-wt plasmids and PGL-3-BMPR1A-mut plasmids co-transfected with miR-143-3p mimic into the KGN cells along with pRL-TK vector using lipo2000 reagent (Invitrogen, Carlsbad, CA, USA). After 24 h of transfection, luciferase activity was determined using dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol, the Renilla luciferase activity was regarded as normalization.
Statistical analysis
Clinical data was analyzed by SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). The measured data was presented as average and standard deviation of three independent experiments. Quantitative data analysis was performed using Graph Pad Prism software version 7.0. The differences of statistics significance between different treatments were evaluated by one-way ANOVA or Student t-test. P values lower than 0.05 was considered statistically significant.