Data resource

PCOS gene expression profiling data were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), including four datasets, GSE155489, GSE138518, GSE193123, and GSE216609. The GSE155489 dataset encompasses 6 samples of oocytes from PCOS patients and 6 samples from healthy individuals. Additionally, it comprises 4 samples of GCs from PCOS patients and 4 samples from healthy individuals. Furthermore, the sample types of the other datasets are GCs, Supplementary Table S1 presents more details concerning the above datasets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) and the Reactome pathway database (https://reactome.org/) were employed to find the list of PD-1/PD-L1 signaling pathway and PI3K/AKT signaling pathway genes.

Identification of differentially expressed genes (DEGs)

DEGs identification was performed using the GEO2R (www.ncbi.nlm.nih.gov/geo/geo2r) online tool in GEO, DEGs (PCOS vs. control) were determined with p < 0.05 and |logFC|≥0.5 as filter criteria. To investigate the role of the PD-1/PD-L1 pathway in oocytes and GCs in PCOS, heatmaps and volcano plots were generated to display the differentially expressed PD-1/PD-L1 pathway genes (DEPPGs). The results of the DEPPGs were visualized using R software (version 4.1.1), with volcano plots and heatmaps created using the ggplot2 package (version 3.3.6).

Functional enrichment analysis of DEPPGs

Genes were annotated using the org.Hs.eg.db package (v.3.10.0) from R (version 4.1.1), and package cluster Profiler (v.4.4.4) was subsequently used to perform Gene Ontology (GO) and KEGG pathway analysis of DEPPGs in PCOS patients. A p-value < 0.05 and count ≥ 2 were selected as cut-off values for the enrichment analysis. Bubble plots and chord plots were drawn to visualize the enrichment results.

Protein–protein interaction (PPI) network construction and hub module analysis

The STRING 12 online database [17] (https://string-db.org/) was used to establish a protein-protein interaction network (PPI) of differentially expressed PI3K/AKT pathway genes (DEPAGs), a confidence interaction score set at 0.400 was regarded as significant standards. The results were then imported into Cytoscape 3.8.0 software for visualization [18]. Key modules were identified from the PPI network using Molecular Complex Detection (MCODE). The degree of each protein node was evaluated using 10 algorithms in the cytoHubba plugin [19]. The hub genes were selected based on the intersection of the top 10 genes from each algorithm in the cytoHubba and MCODE.

Animal model of PCOS induced by DHEA

Sixteen female BALB/c mice (21 days old) were purchased from Xinjiang Medical University [SCXK (XIN) 2018-0002]. All animal studies were approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Serial Number: 20220309-163). The mice were maintained in a pathogen-free facility with standard environmental conditions (21–25 ℃, 12-hour light-dark cycle) and had free access to rodent feed and water according to the institutional guidelines.

All the animals were acclimatized for 5 days. Mice were randomly assigned to two groups (n = 8 per group). One group was injected subcutaneously with DHEA (6 mg/100 g body weight) dissolved in 0.2 mL sesame oil once a day for continuous 21 days to establish an animal model of PCOS. The vehicle control group was injected with 0.2 mL sesame oil daily for 21 consecutive days.

Assessment of estrous cycle

Analysis of vaginal smears was performed daily from the 11th day after the first injection of DHEA or vehicle. The stages of the estrous cycle were determined daily based on direct examination (“wet smear”) technique. Mouse secretions were collected from the vagina using a cotton swab dipped in PBS, then spread evenly on the slide. Meanwhile, cell morphology and estrous cycles were observed by microscopy (Nikon, Tokyo, Japan). Predominant nucleated epithelial cells and some cornified epithelial cells indicated the proestrous stage, predominant cornified squamous epithelial cells indicated the estrous stage, both cornified squamous epithelial cells and leukocytes indicated the metaestrous stage, and predominant leukocytes indicated the diestrous stage.

Analysis of serum hormone concentrations

Blood for the determination of hormone levels was collected from the inner canthus of the mice at the end of the experiment. Then, the blood was put into 1.5 ml tubes for 2 h at room temperature to clot and centrifuged at 2000 rpm for 15 min. The serum was stored at −80℃ until analysis. The levels of serum gonadal hormones, including Luteinizing Hormone (LH), testosterone (T), Estradiol (E2), anti-Mullerian hormone (AMH) and Follicle-Stimulating Hormone (FSH) were determined using commercial ELISA kits (Jiangsu Baolai Biotechnology Co., Ltd., China) according to the manufacturer’s instructions.

Ovarian morphology

The mice were sacrificed by cervical vertebra dislocation and their ovaries were isolated, weighed, and fixed in 4% paraformaldehyde solution. The samples were then dehydrated, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), and imaged under light microscope (Nikon, Tokyo, Japan) to assess changes in ovarian tissue. Total numbers of cystic follicles (fluid-filled cysts with an oocyte lacking connection to GCs, and an attenuated GC layer), and corpora lutea were classified and counted.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay

Ovarian apoptosis levels were determined using a One-step TUNEL In Situ Apoptosis Kit. (Elabscience, China) following the manufacturer’s instructions. Briefly, ovarian tissue sections were dewaxed with xylene and washed with ethanol. The proteinase K working solution (20 µg/mL) was added to the samples and incubated for 20 min at room temperature. TdT labeling buffer was employed to incubate these sections at 37 ℃ for 60 min in a humid chamber. 4′,6-diamidino-2-phenylindole (DAPI) was then added to restore the cell nucleus for 5 min. Subsequently, the sections were sealed using a fluorescence-quenching sealer. The sections were viewed under a fluorescence microscope. Images were acquired with apoptotic cell nuclei in red and non-apoptotic nuclei in blue. The percentage of TUNEL-positive cells in the total cells was determined using ImageJ.

Isolation and identification of GCs from mouse ovarian tissue

Three-week‐old female mice were intraperitoneally injected with 5 IU of Pregnant Mare Serum Gonadotropin (PMSG) for 24 h and then sacrificed by neck dislocation. The ovary was collected, and the fat and oviducts were dissected from the ovaries. The follicles were pierced with a 1 mL syringe needle after peeling off under a stereomicroscope. Collect GCs and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) (BI, Kibbutz, Israel) and 1% penicillin/streptomycin in a humidified incubator at 37℃ with 5% CO2 and use immunofluorescence [20] to detect the specific marker of GCs, follicle-stimulating hormone receptor (FSHR) protein on the surface of GCs.

PD-L1 intervention in GCs

To investigate the function of PD-L1 on GCs, the isolated GCs were cultured in different groups: GCs treated with PD-L1 Fc (20 µg/mL), Anti-PD-L1 antibody (ab210931; abcam, MA, USA) (20 µg/mL), PD-L1 Fc (20 µg/mL) + Anti-PD-L1 antibody (20 µg/mL), and GCs without treatment for 48 h. Cells were then harvested for subsequent qRT-PCR or Western blot analysis.

Western blot analysis

Proteins were extracted from GCs after intervention by RIPA lysis buffer containing protease inhibitors. Protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific, CA, USA). A total of 20 µg of each protein extract was separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST) for 2 h at room temperature. Subsequently, primary antibodies against CYP19A1 (AF6231; Beyotime, Shanghai, China), caspase-3 (AF1213; Beyotime, Shanghai, China), Bcl-2 (AF0060; Beyotime, Shanghai, China), Bax (AF1270; Beyotime, Shanghai, China), PI3K (BM4107; Boster, Wuhan, China), p-PI3K (bs-5570R; Bioss, Beijing, China), AKT (AF1789; Beyotime, Shanghai, China), p-AKT (BM4721; Boster, Wuhan, China), and β-actin (AF5003; Beyotime, Shanghai, China) were incubated overnight at 4℃. After washing three times with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The membranes were visualized using a chemiluminescence Western detection system (Bio-Rad, Hercules, CA, USA) and the band density was quantified using ImageJ software. β-actin was used as an internal reference for detecting relative expression levels.

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech Co., Ltd., Beijing, China) and QuantiNova SYBR Green Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. Primer sequences are listed in Table 1. β-actin was used as a reference gene to normalize gene expression. The relative gene expression levels reported in this study were analyzed with the 2−ΔΔCt method.

Table 1 Primer sequences for the qRT-PCRPatient selection and GCs separation

The subjects in this study consisted of 12 PCOS and 13 healthy women who visited the Reproductive Medicine Center of the First Affiliated Hospital of Xinjiang Medical University. PCOS was diagnosed according to the Rotterdam criteria [21]. The control subjects were selected from women attending the clinic on account of male azoospermia. All of the controls had regular menstrual cycles and normal ovarian morphology, and none exhibited clinical or biochemical hyperandrogenism. The study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ethical Approval Number K202305-02). Informed consent was obtained from all participants.

Patients subjected to in vitro fertilization (IVF) procedures were the source of GCs in our study. Follicular fluid aspirated from follicles of individuals during oocyte retrieval was pooled and GCs were isolated from the follicular fluid as previously described [22]. Briefly, follicular fluid was centrifuged at 300 × g for 10 min to precipitate cells. The pellet was suspended in erythrocyte lysis buffer for 10 min, the mixture was centrifuged at 350 × g for 15 min at 4℃. GCs were isolated from the interface layer, washed once with PBS, after centrifugation at 350 × g for 15 min, GCs were collected and used for RNA extraction.

Statistical analysis

Statistical analysis was performed with GraphPad Prism v9.0 and SPSS v26.0. Normally distributed quantitative data are represented as means ± SEM and analyzed using Student’s t test. Non-normally distributed data are represented as medians (quartile range) and analyzed using Mann-Whitney U test. One-way ANOVA followed by Tukey’s multiple comparisons test was used for comparison between multiple groups. A p-value < 0.05 was considered statistically significant.