Background/Purpose: MRI evidence of synovitis and bone marrow lesions, but not cartilage degeneration associate with pain in osteoarthritis (OA). In this work, we integrated clinical, histological, and transcriptomic analyses of synovial tissue from patients with knee OA to explore correlations of OA pain and sex related pain differences.

Methods: We enrolled 135 patients undergoing total knee arthroplasty who met 1986 ACR classification criteria for knee OA. Demographic, clinical data, patient-reported pain outcomes, and Osteoarthritis Research Society International (OARSI) cartilage scores and synovial histology features measured by board-certified pathologists were collected and analyzed (Table 1). Cell density and cell aggregate number and size were quantified using a previously established computer vision pipeline. Bulk RNA-seq was performed on RNAlater-preserved synovial tissues, using a KAPA-Stranded RNAseqKit with RiboErase for library preparation, and sequenced on a NovaSeq with 150 base pair paired-end reads targeting 100M reads per sample. Reads were pseudoaligned to Hg38 with Kallisto, and Limma was used to test for differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) was used to test for enrichment in GO pathways.

Results: Kellgren Lawrence (KL) grades, BMI, and symptom duration were similar between sexes. Females had higher Erythrocyte Sedimentation Rate (ESR) and reported worse Knee Injury and Osteoarthritis Outcome Score (KOOS) pain scores (p=0.01), particularly rest pain (at night, while in bed) (p=0.02) (Table 1). Rest pain was less common than mechanical pain (e.g., with twisting or stair use) but, when present, it correlated with higher total KOOS pain scores. Synovial histology revealed increased plasma cells, binucleate plasma cells, Russell bodies, and size and number of cellular aggregates in females. Females with synovial plasma cells reported increased rest pain. OARSI cartilage scores were similar in the most severely eroded femoral condyles between sexes, but females had significantly higher scores in the less eroded condyles (Figure 1). RNA-seq revealed 25 DEGs between sexes, and a pronounced enrichment in Xist escape genes in females. GSEA identified 132 pathways significantly enriched in females, including “adaptive immune response” and “complement activation”. Among females, genes associated with inflammation, fibrosis, and neurogenesis pathways were positively associated with rest pain, while genes associated with DNA damage response and progenitor cells were negatively associated with rest pain. There were no genes associated with rest pain in males.

Conclusion: Compared to males, females have significantly increased systemic and synovial tissue inflammation, along with increased multicompartmental cartilage degeneration. Among females, rest pain is positively associated with synovial inflammation, fibrosis, and neurogenesis pathway gene expression and negatively associated with DNA damage response and progenitor cell gene expression. This analysis provides insights into the molecular underpinnings of the clinical observation that MRI evidence of synovitis is associated with pain in knee OA.

Table 1. Clinical OA characteristics according to sex

Figure 1: Percentage age distribution across different age groups. Figure B: Sex differences in knee pain as measured by the KOOS pain subscale. Females reported significantly worse knee pain compared to males mean KOOS pain score. Figure C: KOOS pain scores reported by all patients for specific activities. Pain was highest during activities involving greater mechanical stress on the knee and progressively decreased during lower-stress activities such as sitting or lying down. A mechanical stress gradient is illustrated above, highlighting the inverse relationship between stress level and pain intensity. Figure D: KOOS pain scores reported by females and males across different activities. While no significant sex differences were found in most categories, females reported significantly greater pain “at night, in bed” compared to males. Figure E: The proportion of plasma cells (left) and Russell bodies (right) in synovial tissue from females and males. Females exhibited significantly higher levels of both plasma cells and Russell bodies compared to males. Figure F: The number of cells in the largest synovial cell aggregate (left) and the number of aggregates per tissue area (right) in females and males. Females exhibited significantly larger and more numerous synovial aggregates compared to males. Figure G: High-magnification image of Hematoxylin and eosin (H&E) stain synovial tissue with plasma cell infiltrates. Figure H: Low-magnification image of Hematoxylin and eosin (H&E) stain synovial tissue with aggregates within the synovium. Figure I: KOOS pain scores for individual activities in female patients with and without synovial plasma cell infiltration. Pain during low mechanical stress activities of sitting and at night in bed was significantly higher in females with plasma cells present in their synovial tissue. Figure J: OARSI cartilage scores for male and female patients in both severely degenerated (left) and relatively preserved (right) femoral condyles. No significant differences were observed in the severely degenerated regions, females exhibited significantly higher cartilage damage in the relatively preserved condyles compared to males. Figure K: Representative low-magnification images of Hematoxylin and eosin (H&E) stained cartilage of males (top) and females (bottom) described in Figure J

Figure 2A. Volcano plot of differential gene expression of rest pain (night pain in bed). Figure 2B. Gene Set Enrichment Analysis (GSEA) displaying pathway associated with rest pain

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-synovial-inflammation-xist-escape-gene-expression-and-knee-pain-in-females-with-osteoarthritis/