The experimental design and overall workflow as shown in Figure 1. To ensure the comparability of results, we established detailed inclusion and exclusion criteria for both the patient and control groups.

This study finally included 3 patients with OAPS (OAPS group) and 3 healthy controls (normal control, NC group). All participants received care at Shandong Provincial Hospital. Peripheral whole blood samples (8 mL) were collected from all subjects in the morning after overnight fasting using EDTA anticoagulant-coated tubes. The blood sample was quickly transferred to the anticoagulant containing heparin and mixed by vortexing or inversion 5 to 10 times.

The study cohort comprised patients meeting the following inclusion criteria (20, 21): (1) diagnosis of antiphospholipid syndrome according to international consensus criteria (Sydney classification), confirmed by persistent positivity (≥12 weeks apart) of lupus anticoagulant, anti-cardiolipin antibodies (IgG/IgM), or anti-β2-glycoprotein I antibodies (IgG/IgM) via standardized laboratory assays; (2) age 20–40 years; (3) at 11–14 weeks of pregnancy, the diagnosis of single-child intrauterine pregnancy was confirmed by transvaginal ultrasound and the number of weeks of pregnancy was checked for the patient. Exclusion criteria comprised: (1) clinically significant uterine anomalies (e.g., fibroids ≥4 cm) or adnexal pathology (e.g., ovarian cysts >5 cm); (2) active systemic infections, endocrine disorders requiring pharmacological management (e.g., uncontrolled diabetes mellitus, thyroid dysfunction), or hematological comorbidities (e.g., inherited thrombophilia, coagulation factor deficiencies); (3) pre-existing autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis) or cardiovascular/renal/hepatic dysfunction; (4) multifetal gestation or nonviable pregnancy; (5) non-criteria (atypical) OAPS cases; (6) patients with other pregnancy-related complications or comorbidities(e.g., preeclampsia, gestational hypertension, gestational diabetes).

The NC group inclusion criteria comprised: 1) individuals aged 20–40 years; 2) at 11–14 weeks of pregnancy, the diagnosis of single-child intrauterine pregnancy was confirmed by transvaginal ultrasound, and the number of weeks of pregnancy was checked for the patient; 3) absence of a documented history of spontaneous abortion, pre-eclampsia, eclampsia, or preterm birth. Exclusion criteria involved: 1) concurrent diagnoses of gynecological disorders (uterine fibroids, ovarian cysts), endocrine dysfunction, or active infectious conditions; 2) laboratory-confirmed antiphospholipid antibody positivity through standardized immunoassay testing. 3) any autoimmune, hematologic, cardiovascular, renal, or hepatic disorders; 4) multiple or nonviable pregnancies; 5) any pregnancy-related complications or comorbidities identified during the current gestation. This selection protocol ensured the exclusion of potential confounding medical factors while maintaining gestational age homogeneity across the study cohort.

Exosomes were isolated from plasma samples using a sequential centrifugation protocol as follows (22): First, samples underwent initial centrifugation at 1,500 × g for 10 min at 4°C to eliminate cellular debris and large particulates. The supernatant was then subjected to a second centrifugation at 10,000 × g for 30 minutes at 4°C to eliminate smaller particles using by Thermo Scientific Sorvall Legend X1R(Thermo Fisher Scientific, USA). The resulting supernatant was transferred to clean centrifuge tubes and filtered through a 0.22-μm pore-size (Millipore, USA) membrane to remove any remaining large vesicles. The filtrate was then ultracentrifuged (Beckman Coulter, Optima L-100XP) at 100,000 × g for 60 minutes at 4°C using a fixed-angle rotor to pellet the exosomes (23, 24). Following careful supernatant removal, the exosome-containing pellet was resuspended in 1× phosphate-buffered saline (PBS) and stored at −80 °C until further analysis. This optimized protocol ensures high-purity exosome isolation through a combination of differential centrifugation and size-based filtration (25).

Exosome isolation and validation were performed through three complementary analytical approaches: nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) protein profiling.

Cryopreserved exosome samples were thawed in a 25 °C water bath and immediately placed on ice to preserve vesicle integrity. For particle size distribution analysis, the exosome suspensions were diluted in 1× phosphate-buffered saline (PBS) (Thermo Fisher Scientific, USA) at an appropriate concentration and directly analyzed using NTA with the NanoSight NS300 system (Malvern Panalytical). All measurements were performed in triplicate at a controlled temperature of 25 °C, with the camera level adjusted to optimize particle visualization.

Exosome morphology was assessed by negative staining TEM. Briefly, 10 μL of purified exosome suspension was placed onto Formvar/carbon-coated copper grids (200 mesh) and allowed to adsorb for 1 minute. Excess liquid was carefully removed with filter paper. Samples were subsequently stained with 10 μL of 2% uranyl acetate for 1 minute and air-dried at room temperature. Imaging was performed using a transmission electron microscope operating at 80 kV (JEM-1400, JEOL Ltd).

WB analysis was conducted to verify the presence of exosomal marker proteins CD81 and TSG101, and to confirm the absence of the endoplasmic reticulum marker calnexin, indicating minimal cellular contamination. Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific, USA). Equal amounts of total exosomal protein (20 μg per sample per lane) were loaded onto 12.5% SDS–PAGE gels (Epizyme, Shanghai, China), separated, and transferred onto polyvinylidene difluoride (PVDF) membranes (0.22 μm pore size (Millipore, USA)) using a wet transfer system to ensure consistent protein loading across samples. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: rabbit monoclonal anti-CD81 (1:1,000; Abcam, ab109201), anti-TSG101 (1:1,000; Abmart, T55985), and anti-calnexin (1:1,000; Cell Signaling, 2679S). After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; Sabbiotech, L3012) for 1 hour at room temperature. Protein bands were detected using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and imaged with a ChemiDoc XRS+ system (Bio-Rad).

Immunoblotting results showed strong positive bands for exosomal markers CD81 (~24 kDa) and TSG101 (~44 kDa) in the isolated exosome samples, with signal intensities comparable to those in the positive control group (CL). A positive control group was included using exosomes previously isolated and characterized by our laboratory. These exosomes had been confirmed to express typical exosomal markers (TSG101, CD81) and were used to verify the specificity of antibody detection and the accuracy of exosome identification. In contrast, calnexin (~90 kDa), a negative marker not present in exosomes, was undetectable in the exosome preparations. This expression pattern confirms the successful isolation of exosomes with minimal contamination from the endoplasmic reticulum, following established standards for exosome characterization. This multi-modal characterization approach provides comprehensive validation of exosome identity, morphology, and protein composition, following the MISEV2018 guidelines for extracellular vesicle research (26).

RNA Extraction and Purification: Total RNA was extracted from the exosome samples using the Norgen Exosome RNA Purification Kit (N-51000) according to the manufacturer’s protocol. The concentration and integrity of the extracted RNA were evaluated using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), respectively. The total RNA was further purified using the RNeasy Mini Kit (QIAGEN, 74106).

Microarray Hybridization and Data Processing: Total RNA (50 ng) was mixed with Spike-In controls (Agilent 5188-5282) and labeled using the Low Input Quick-Amp Labeling Kit, One-Color (Agilent 5190-2305) following manufacturer protocols. Briefly, RNA underwent reverse transcription to synthesize cDNA, which was then amplified through T7 RNA polymerase-mediated in vitro transcription to produce complementary RNA (cRNA) labeled with Cyanine-3-CTP. Labeled cRNA was purified using the RNeasy Mini Kit (QIAGEN 74106).

The purified and labeled cRNA (1.65 μg) was mixed with 10X Blocking Agent and 25X Fragmentation Buffer (Gene Expression Hybridization Kit, Agilent 5188-5242), incubated at 60°C for 30 minutes, and then hybridized to the LC Human ceRNA Array V1.0 (4x180K, Design ID: 085202) at 65°C for 17 hours. After washing, the array was scanned using an Agilent Scanner G5761A (Agilent Technologies, Santa Clara, CA, USA).

Scanned microarray images were analyzed using Feature Extraction software (version 12.0.3.1; Agilent Technologies, Santa Clara, CA, USA) to extract the raw data. The row data were normalized using the quantile normalization method and further processed with GeneSpring software (version 14.8; Agilent Technologies, Santa Clara, CA, USA). Probes that were detected in at least 80% of the samples within each group were retained for subsequent analysis.

Microarray data analysis was performed using R statistical software (version 3.6.3). Differentially expressed genes (DEGs) between the OAPS group and control group exosome samples were identified through a Student’s t-test. Genes meeting the threshold of adjusted p-value < 0.05 and absolute log2 fold change (|log₂FC|) ≥ 1 were classified as statistically significant DEGs. Unsupervised hierarchical clustering of the identified DEGs was performed using the hierarchical clustering algorithm in the stats package, and the expression patterns were visualized as a heatmap via the pheatmap package (version 1.0.12). Furthermore, a volcano plot was generated using the ggplot2 package (version 3.3.3) to illustrate the distribution of DEGs based on their statistical significance and fold change.

To explore the biological significance of the identified DEGs, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses were executed using the OmicStudio online platform (https://www.omicstudio.cn/tool), which applies robust statistical methods to annotate gene functions and identify signaling pathways significantly enriched among the DEGs.

Target binding relationships between miRNAs and their corresponding mRNAs, lncRNAs, and circRNAs were predicted using TargetScan (version 5.0) and miRanda (version 3.3a). The prediction thresholds were set to TargetScan score ≥ 50 and miRanda energy < –10. Only the intersection results from both tools were retained for further analysis. Finally, high-confidence interaction pairs with a TargetScan score ≥ 95 were selected to construct competing endogenous RNA (ceRNA) relationship pairs based on shared miRNA targets. The resulting ceRNA network was visualized using Cytoscape software.

A total of three patients with OAPS (OAPS group) and three healthy controls (NC group) were enrolled in this study, all of whom received care at Shandong Provincial Hospital (Table 1). Written informed consent was obtained from all patients. This study was carried out according to the World Medical Association Declaration of Helsinki and was approved by the ethics committee of Shandong Provincial Hospital affiliated to Shandong University.

Peripheral blood samples were collected from all participants in the morning after overnight fasting. The blood was quickly transferred into tubes containing heparin as an anticoagulant and mixed gently by vortexing or inverting 5 to 10 times to ensure proper mixing.

The average peak size of exosomes from control and OAPS blood samples was 159.8 ± 2.8 nm and 140.3 ± 0.4 nm, respectively (Figure 2A), consistent with the known size range of exosomes. TEM showed that the extracted substance was nearly round or cup-shaped with an approximate diameter of 100 nm (Figure 2B). Western blot analysis confirmed the presence of exosome-specific markers CD81 and TSG101, while the endoplasmic reticulum marker calnexin, used as a negative control, was absent (Figure 2C).

Collectively, these findings indicate that exosomes were successfully isolated from the blood samples of both healthy controls and OAPS patients.

Differentially expressed mRNAs (DE-mRNAs) were subjected to cluster analysis (Figure 3A) based on |log₂FC| ≥ 1 and P < 0.05. The results showed that these DE-mRNAs (Figures 3A–C) could effectively distinguish between control and OAPS samples. As shown in the bar plot (Figure 3B) and volcano plot (Figure 3C), a total of 28 genes were significantly upregulated and 15 genes were downregulated in the OAPS group compared to the NC group.

To further investigate the functional implications of the differentially expressed (DE) mRNAs, Gene Ontology (GO) enrichment analysis was performed. The top 20 enriched GO terms were identified, involving 14 genes, including ERBB4, SIM2, DRD2, SLC29A1, SPTLC2, TNFSF15, CD86, EGR2, PRPH2, NOC2L, MYL2, SLC30A8, P3H4, and SUV39H1. These genes were mainly associated with biological processes such as “embryonic pattern specification,” “positive regulation of receptor signaling pathway via JAK-STAT,” and “cytokine production” (Figures 3D, F). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted, and the top 20 KEGG pathways were summarized. These pathways involved 18 genes, including ERBB4, HSPA1L, MVB12B, AGAP1, HLA_DQB1, CD86, CELA3A, ATP1B1, MYL2, DGKK, PLA2G6, OR3A2, OR2L2, OR8B4, CD19, CPT1C, CAB39L, and RFC3. Notably enriched pathways included “Allograft rejection,” “Protein digestion and absorption,” and the “AMPK signaling pathway” (Figures 3E, G).

Differentially expressed long non-coding RNAs (DE-lncRNAs) were identified using the criteria of |log₂FC| ≥ 1 and P < 0.05, and subjected to hierarchical clustering analysis (Figure 4A). The results demonstrated that these DE-lncRNAs could differentiate between the control and OAPS samples (Figures 4A–C). The bar plot (Figure 4B) and volcano plot (Figure 4C) showed that, compared to the NC group, there were 34 significantly upregulated genes and 21 downregulated genes in OAPS.

The top 20 GO terms were summarized; there were biological processes such as “protein localization to plasma membrane”, “ATPase binding”, “neurotransmitter secretion”, “post-embryonic development”, and “c tRNA (guanine-N2-)-methyltransferase activity” (Figures 4D, F).

The KEGG pathways were summarized, and pathways such as “Synaptic vesicle cycle”, “Insulin secretion”, “Platelet activation”, “Rap1 signaling pathway”, and “Neuroactive ligand-receptor interaction” were included (Figures 4E).

Differentially expressed circular RNAs (DE-circRNAs) were subjected to cluster analysis (Figure 5A) based on |log₂FC| ≥ 1 and P < 0.05. The results showed that these DE-circRNAs (Figures 5A–C) could effectively distinguish between control and OAPS samples. The bar plot (Figure 5B) and volcano plot (Figure 5C) showed that, compared to the NC group, there were 33 significantly upregulated genes and 39 downregulated genes in OAPS.

Enrichment analysis was performed on the DE-circRNAs. The top 20 GO terms were summarized; among them, there were biological processes such as “diacylglycerol binding”, “RNA (guanine-N7)-methylation”, “regulation of cell migration”, “ATP-dependent diacylglycerol kinase activity”, and “negative regulation of mitochondrial membrane potential” (Figures 5D, F).

The top 20 KEGG pathways were summarized. Pathways such as “Glycerolipid metabolism”, “Phosphatidylinositol signaling system”, “Biotin metabolism “, “Phospholipase D signaling pathway”, and “Toll-like receptor signaling pathway” were included (Figures 5E, G).

Differentially expressed microRNAs (DE-miRNAs) were subjected to cluster analysis (Figure 6A) based on |log₂FC| ≥ 1 and P < 0.05. The results showed that these DE-miRNAs (Figures 6A–C) could effectively distinguish between control and OAPS samples. The bar plot (Figure 6B) and volcano plot (Figure 6C) showed that, compared to NC, there were 6 significantly upregulated genes and 13 downregulated genes in OAPS.

For the screened DE miRNAs, biological processes of GO terms and KEGG signaling pathways were analyzed, and with P < 0.05 as the enrichment significance threshold, 20 GO terms of biological processes and 20 KEGG pathways were significantly enriched. The enriched GO terms included “protein binding,” “metal ion binding,” “transferase activity,” “nucleotide binding,” and “positive regulation of transcription by RNA polymerase II” (Figures 6D, F). The significantly enriched KEGG pathways included the “Ras signaling pathway,” “Regulation of actin cytoskeleton,” “Hippo signaling pathway,” “ErbB signaling pathway,” “Phospholipase D signaling pathway,” and “AMPK signaling pathway” (Figures 6E, G).

A total of 31 miRNAs, 15 mRNAs,18 lncRNAs, and 101 circRNAs were obtained through screening relationships. TargetScan (5.0) and Miranda (3.3a) software were used to predict the targeting binding relationship between miRNA and mRNA, and lincRNA, respectively, and the intersection of the two software was taken. A ceRNA regulatory network composed of 15 miRNAs, 14 lncRNAs, 15 mRNAs, and 68 circRNAs was constructed based on the common miRNA combination. Cytoscape software was used to visualize the network (Figure 7A).

Subsequently, the genes involved in the ceRNA network were subjected to GO term and KEGG pathway enrichment analyses. (Figures 7B-E). A total of 20 GO biological processes and 7 KEGG pathways were significantly enriched (P < 0.05). As shown in Figure 7D, “ negative regulation of dopamine secretion”, “ uridine transport”, “ cardiac muscle tissue regeneration”, “ positive regulation of receptor signaling pathway via JAK-STAT”, and “ pancreas morphogenesis” were significantly enriched.

In addition, the 7 KEGG pathways include the “ AMPK signaling pathway”, “ mTOR signaling pathway”, “ ErbB signaling pathway”, “ Antigen processing and presentation “, “ Cocaine addiction”, “ Nucleocytoplasmic transport “, and “Alcoholism”. (Figure 7E).