Clinical sample collection of FF

Clinical samples of FF were obtained from six patients with PCOS and six healthy individuals with normal ovarian function who were undergoing in vitro fertilization-embryo transfer in the Department of Reproductive Medicine. The PCOS group was subgrouped using the Rotterdam Consensus (2003) [23]. The exclusion criteria for both groups were as follows: age > 35 years; a history of ovarian surgery or treatment with radiation and chemotherapy, endometriosis, hyperprolactinemia, thyroid disorders, genetic abnormalities, congenital adrenal hyperplasia, Cushing’s syndrome, androgen-producing tumors, and other conditions that might affect follicular development; and patients who consumed drugs affecting hormone levels or glucolipid metabolism within 6 months before treatment. All analyses involving human samples were approved by the Clinical Ethics Review Committee of the Affiliated Hospital of Guilin Medical College. All methods were performed per the relevant guidelines and standardized methodology. All participants provided signed informed consent.

Isolation and characterization of FF-derived extracellular vesicles

All patients were treated for controlled ovarian hyperstimulation. Herein, 36 h after the injection of human chorionic gonadotropin (hCG 2000 U, Lizong Medicine Factory, Zhuhai, China), FF was collected by transvaginal ultrasound-guided aspiration of follicles (bilateral diameter > 14 mm). The FF was centrifuged at 1500 ×g for 15 min, and the supernatant (16 mL) was extracted by ultracentrifugation per the instructions of the Qiagen exoEasy Maxi kit (Qiagen, Hilden, Germany). Extracellular vesicle-specific antibodies, namely cluster of differentiation (CD)63(#R23327, 1:1,000 dilution; ZenBio, China) and CD9 (#ab92726, 1:1,000 dilution; Abcam USA) were used to detect the presence of extracellular vesicles by western blotting. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) of extracellular vesicles were performed at QIAGEN (Suzhou) Translational Medicine Co. Ltd. Particle size and the concentration of extracellular vesicles were analyzed through NTA. Brownian motion trajectories of extracellular vesicles were observed using the Nanosight NS300, and the diameter of extracellular vesicles was measured in NTA2.1 using Zetaview. The parameters in Naonosight were set as follows: laser type, blue laser (488 nm); particle size range, 10–1,000 nm; recommended concentration range, 106–109 particles/mL; video capture settings, sCMOS camera, 25 frames per second, 45-second video capture; measurement duration, five measurements; and optical settings, sensitivity threshold set at 5. The parameters in ZetaView were set as follows: sensitivity, 70; shutter, 70; minimum brightness, 20; maximum area, 1,000; and minimum area, 5.

For TEM, tweezers were used to hold the edge of the copper grid, paying attention to distinguish the front and back sides. The front side of the grid was coated with a carbon film, and the sample was added, namely 10 µL of extracellular vesicles, which was then allowed to settle for 1–2 min. Next, filter paper was used to remove the excess liquid, and 10 µL of 5% phosphotungstic acid solution was applied for negative staining of the sample for 1–2 min. After absorbing the excess liquid using filter paper, the grid was air-dried at room temperature for approximately 10 min and TEM imaging was performed at 100 kV (JEOL, Tokyo, Japan).

Extracellular vesicle miRNA sequencing (miRNA-seq) analysis

miRNA sequencing (miRNA-seq) analysis of FF extracellular vesicle samples was performed by Aksomics (Shanghai, China). Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and its quality was assessed by agarose gel electrophoresis. After measuring the RNA concentration using a NanoDrop ND-1000 spectrophotometer, a total of 1,000 ng of total RNA was used to enrich small RNAs using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and the RiboZero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, an Illumina Company). Next, the enriched small RNAs were used to construct small RNA libraries with the NEBNext Multiplex Small RNA Sample Prep Set for Illumina (NEB, Cat E7580), and their quality and quantity were evaluated using the Agilent 2100 Bioanalyzer and quantitative real-time polymerase chain reaction (qPCR) analysis. Raw amplification clusters were captured on an Illumina Flow Cell and cycled for sequencing on an Illumina NextSeq 500 Sequencer per the instructions of the manufacturer (miRNA-seq read length was 50 bp; single-ended sequencing). After quality control, intergroup differences were analyzed using EdgeR (R 4.2.3). For clustering analysis, differentially expressed genes were screened based on log2|fold change| ≥ 1 and p-value < 0.05, following which, the Database for Annotation, Visualization, and Integrative Discovery v6.7 was utilized to perform Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Cell culture and miRNA mimic/inhibitor transfection

The KGN cell line was obtained from Wuhan Punosai Life Science and Technology Co. It was cultured as the in vitro cellular model of PCOS in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum and 400 nM testosterone (#M6105, AbMole BioScience, Houston, USA) at 37 °C and 5% CO2 under humid conditions. After reaching 60–70% confluence, the cells were transiently transfected with 50 nM miR-34a-5p mimic, mimic negative control (NC), 100 nM miR-34a-5p inhibitor, and inhibitor NC using the riboFECT CP Transfection Kit (#C10511-05, RiboBio, Guangzhou, China) per the instructions of the manufacturer (RiboBio). At 24 h post-transfection, KGN cell messenger RNA (mRNA) was extracted for qPCR to verify the transfection effect. All operations are performed in a sterile environment and cells are regularly monitored for signs of bacterial or fungal contamination through microscopic examination and evaluation of cell morphology.

Dual-luciferase reporter assay

KGN cells were inoculated in 24-well plates and incubated for 24 h to achieve 60–70% fusion. Wild-type (WT) or mutant (MUT) mRNA fragments were then constructed in advance and inserted downstream of the psiCheck2 luciferase reporter gene. miR-34a-5p mimic (50 nM) and psiCHECK2 - LDHA- 3’UTR-WT (15 mg/L) or psiCHECK2 - LDHA − 3’UT R- MUT (15 mg/L) were co-transfected into KGN cells. After 36 h, the luciferase activities of the firefly and Renilla were detected by using the Dual-Luciferase Reporter Assay System (#E1910, Promega), and the relative luciferase activity was calculated by normalizing the Renilla luciferase activity to the firefly luciferase activity.

Cell proliferation assay

Cell proliferation assays were performed according to the instructions of Cell Counting Kit-8 (#BS350A, Biosharp). KGN cells were spread in a 96-well plate at a concentration of 5000 cells/well and incubated in a 37 °C, 5% CO2 cell culture incubator for 24 h. Then, 10 µL of the Cell Counting Kit-8 (CCK-8) solution was added to each well, and the cells were further incubated with CCK8 for another hour. Finally, the absorbance at 450 nm was measured using a microplate reader.

Flow cytometry

After 24 h of pretreatment, the cells were collected and diluted to a density of 1 × 105 and resuspended in binding buffer (10X Annexin v Binding Buffer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2. For a 1X working solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water). FITC Annexin V Apoptosis Detection Kit I was used according to the manufacturer’s instructions (BD Biosciences Pharmingen, San Diego, US). Fluorescence signals were sorted using a flow cytometer (FACSCalibur, BD Biosciences, USA) and the results were analyzed by using FlowJo software.

qPCR analysis

Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of RNA was reverse transcribed into complementary DNA per the instructions of the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop; #MR101-02, Vazyme). For qPCR, miRNA was amplified using miRNA Universal SYBR qPCR Master Mix (#MQ101-02, Vazyme) per the protocol of the manufacturer. The Homo-U6 gene served as the internal reference gene for miR. The cycle threshold values were determined using the 2−ΔΔCt method. The primer sequences are listed in Table 1.

Table 1 Primers for real-time quantitative PCRWestern blotting

Protein samples were extracted using the radioimmunoprecipitation assay lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors. Next, protein concentrations were quantified using the BCA Protein Assay Kit (Solarbio, China). The extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (The 60 V voltage lasts for 50 min, Then adjust the voltage to 120 V and continue electrophoresis for 60 min) and transferred to polyvinylidene fluoride, membranes. The membranes were then blocked with 5% skimmed milk for 2 h, washed with phosphate-buffered saline with Tween 20, and incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: CD63 (#R23327, 1:1,000 dilution; ZenBio), CD9 (#ab92726, 1:1,000 dilution; Abcam USA), β-actin (A5441, 1:10,000; Sigma-Aldrich), hexokinase 2 (HK2; A0994, 1:1,000 dilution; ABclonal, Wuhan, China), pyruvate kinase muscle isozyme M2 (PKM2; #4053, 1:1,000 dilution; Cell Signaling Technology, Massachusetts, USA), LDHA (#3558, 1:1,000 dilution; Cell Signaling Technology), BAX (RT40051, 1:1,000 dilution; Abmart), BCL-2 (13-8800, 1:1,000 dilution; Invitrogen), Caspase-3 (#9662, 1:1,000 dilution; Cell Signaling Technology), and β-Tubulin (#5568, 1:5,000 dilution; Cell Signaling Technology). Following this, the membranes were further incubated with horseradish peroxidase-conjugated affinipure goat anti-rabbit IgG (H + L) (SA00001-2, 1:5,000 dilution; Protein Tech Group Inc.) for 2 h at room temperature. Finally, the chemiluminescence of protein bands was detected by adding eECL (CW0049M, CWBIO) and using the Tanon-5500 Chemiluminescence Imaging System.

Determination of lactic acid and pyruvic acid content

The lactate and pyruvate contents in the culture medium were determined 36 h after the different treatments using a lactate test kit (#A019-2-1, Nanjing Jianjian Institute of Bioengineering, China) and a pyruvate assay kit (#A081-1-1, Nanjing Jianjian Institute of Bioengineering), respectively. The plates were read using a VersaMax microplate reader at 505 nm (pyruvate) and 530 nm (lactic acid) wavelengths.

Statistical analysis

Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, CA, USA) and presented as the mean ± standard deviation. Significant differences between/within groups were evaluated by the unpaired t-test or one-way ANOVA followed by Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.