Identification and characterization of ovarian extrachromosomal circular deoxyribonucleic acid in rats with polycystic ovary syndrome

Extrachromosomal circular deoxyribonucleic acids are increasingly recognized as dynamic genomic elements with potential regulatory functions, but their characteristics in polycystic ovary syndrome remain largely unknown. This study aimed to characterize ovarian extrachromosomal circular deoxyribonucleic acid profiles in a rat model of polycystic ovary syndrome and explore their genomic features, functional associations, and overlaps with non-coding ribonucleic acids, circular ribonucleic acids, and transposable elements. Female Sprague-Dawley rats were assigned to control and polycystic ovary syndrome groups, with polycystic ovary syndrome induced by dehydroepiandrosterone. Model establishment was confirmed by abnormal follicular development, altered sex hormone levels, and irregular estrous cycles. Circle sequencing identified 15,207 extrachromosomal circular deoxyribonucleic acid molecules representing 13,762 distinct types, with overall abundance reduced in polycystic ovary syndrome rat ovaries. A total of 263 circular deoxyribonucleic acids showed differential abundance between groups, including 260 small extrachromosomal circular deoxyribonucleic acids and 3 larger circular molecules. Chromosome 12 was the only chromosome showing significant differences in both chromosome-wise count distribution and chromosome-normalized density after Benjamini–Hochberg correction (both P < 0.001). Length distributions were bimodal in both groups, with peaks at 208 and 368 base pairs in controls and 212 and 359 base pairs in polycystic ovary syndrome. Junction analysis revealed frequent 4–20-base-pair direct repeats and inverted repeats. Genomic overlap analyses demonstrated intersections with annotated genes, non-coding ribonucleic acids, circular ribonucleic acids, and transposable elements. Functional enrichment highlighted synaptic structures, iron ion homeostasis, cell junction-related processes, and the T cell receptor signaling pathway. Transmission electron microscopy revealed ring-shaped circular deoxyribonucleic acid-like structures, while endpoint polymerase chain reaction confirmed relative abundance patterns of selected candidates consistent with the sequencing results. This study provides an initial characterization of eccDNA/ecDNA profiles in PCOS rat ovaries, revealing reduced abundance, altered genomic features, and enrichment of associated genes in T-cell receptor signaling. Genomic overlap with multiple ncRNAs, particularly lncRNAs, further suggests potential RNA-mediated regulatory mechanisms. These findings provide preliminary evidence for the involvement of eccDNA/ecDNA in PCOS pathophysiology and support further mechanistic and clinical investigation.