Subcutaneous implantation of Icariin with gelatin methacryloyl (GelMA) hydrogel sustained-release system improves ovarian function in reproductive aged mice

Ovarian aging is characterized by a decline in the quality and quantity of oocytes as age increases. Icariin (ICA) has demonstrated potential in restoring ovarian function. In this context, we introduce a subcutaneous implantation of a gelatin methacryloyl (GelMA) hydrogel containing ICA to enhance the ovarian function of aged mice. Aged female mice were treated with subcutaneous implantation of GelMA hydrogel (GelMA group) or GelMA hydrogel containing ICA (GelMA/ICA group), while the sham group underwent a sham operation. Serum hormone levels, ovarian weight, and follicle count were assessed. In vitro fertilization (IVF) and embryo culture were performed to evaluate oocyte developmental potential, and a 10-day mating test was conducted to assess fertility. RNA sequencing and qRT-PCR were used to explore the molecular mechanisms. GelMA/ICA treatment significantly increased ovarian index (1.87±0.13 vs. 1.31±0.14, P<0.0001) and follicle numbers at all developmental stages, including primordial (383.33±151.65 vs. 107.14±32.26, P<0.0001), primary (203.33±83.22 vs. 91.43±27.04, P=0.003), and secondary follicles (154.17±52.00 vs. 59.28±20.50, P=0.029). Serum FSH was significantly reduced (11.97±3.53 vs. 53.10±17.89 ng/mL, P=0.0008), while AMH (22.97±2.26 vs. 5.54±1.56 ng/mL, P<0.0001) and E2 (315.30±37.62 vs. 168.5±14.78 pg/mL, P<0.0001) were elevated. The number of superovulated MII oocytes (17.83±5.15 vs. 4.83±4.79, P=0.0002), two-cell (85.90%±6.16% vs. 50.00%±10.00%, P=0.0009), four-cell (81.67%±9.76% vs. 50.00%±10.00%, P=0.0061), and blastocyst rates (64.25%±10.55% vs. 23.33%±15.28%, P=0.0067) were significantly improved. The number of live births increased (6.90±3.21 vs. 1.72±2.05, P=0.0001). RNA-seq and qRT-PCR revealed upregulation of genes related to cytoskeleton (Vil1, Tubb3), lipid storage (Soat2, Plin4), oocyte maturation (Oosp2), and cytokine secretion (Cxcl12). These findings suggest that GelMA/ICA hydrogel enhances ovarian function and fertility in aged mice. We believe that the subcutaneous implantation of GelMA/ICA could provide a novel therapeutic concept for future investigations into ovarian aging.