Subcutaneous delivery of icariin via a gelatin methacryloyl (GelMA) hydrogel sustained-release system improves ovarian function in reproductively aged mice

Ovarian aging is characterized by a progressive decline in oocyte quality and quantity with age. Icariin (ICA), a flavonoid compound derived from <i>Epimedium</i> species, has demonstrated potential as an agent for ovarian restoration. In this study, a subcutaneous implantation system using gelatin methacryloyl (GelMA) hydrogel embedded with ICA was developed to restore ovarian function in aged female mice. Mice were assigned to receive subcutaneous implantation of GelMA alone (GelMA group), GelMA containing ICA (GelMA/ICA group), or a sham operation. Ovarian morphology, serum hormone levels, follicle counts across developmental stages, and reproductive outcomes were evaluated. <i>In vitro</i> fertilization (IVF) and embryo culture assays were performed to assess oocyte developmental potential, while a 10 day natural mating trial was conducted to determine fertility restoration. RNA sequencing (RNA-seq) and RT-qPCR were performed to elucidate the underlying molecular mechanisms. Results showed that GelMA/ICA treatment significantly increased ovarian index (0.19±0.01 vs. 0.13±0.01, <i>P</i><0.0001) and follicle numbers at all developmental stages, including primordial (383.33±151.65 vs. 107.14±32.26, <i>P</i><0.0001), primary (203.33±83.22 vs. 91.43±27.04, <i>P</i>=0.003), and secondary follicles (154.17±52.00 vs. 59.28±20.50, <i>P</i>=0.029) compared to the sham controls. Hormonal analyses revealed a significant reduction in serum follicle-stimulating hormone (FSH, 11.97±3.53 vs. 53.10±17.89 ng/mL, <i>P</i>=0.0008), accompanied by elevated anti-Müllerian hormone (AMH, 22.97±2.26 vs. 5.54±1.56 ng/mL, <i>P</i><0.0001) and estradiol (E ₂, 315.30±37.62 vs. 168.5±14.78 pg/mL, <i>P</i><0.0001). Oocyte yield and developmental potential improved significantly, as reflected by the increased number of superovulated MII oocytes (17.83±5.15 vs. 4.83±4.79, <i>P</i>=0.0002), and higher proportions of two-cell (85.90%±6.16% vs. 50.00%±10.00%, <i>P</i>=0.0009), four-cell (81.67%±9.76% vs. 50.00%±10.00%, <i>P</i>=0.0061), and blastocyst stage embryos (64.25%±10.55% vs. 23.33%±15.28%, <i>P</i>=0.0067). Live birth numbers were significantly increased following GelMA/ICA treatment (6.90±3.21 vs. 1.72±2.05, <i>P</i>=0.0001). Transcriptomic analysis revealed up-regulation of genes associated with cytoskeletal organization ( <i>Vil1</i>, <i>Tubb3</i>), lipid storage ( <i>Soat2</i>, <i>Plin4</i>), oocyte maturation ( <i>Oosp2</i>), and cytokine secretion ( <i>Cxcl12</i>). Collectively, these findings suggest that GelMA/ICA hydrogels effectively reverse key hallmarks of ovarian aging and restore reproductive function in aged mice, offering a promising platform for fertility preservation and a novel therapeutic for future investigations into ovarian aging.