Frozen-Thawed ovarian autografts treated with scaffold-based melatonin delivery in rats

Cryopreserved ovarian tissue transplantation is the only option for for fertility preservation in children undergoing cytotoxic treatments. Among antioxidant substances, melatonin has been increasingly investigated to improve the avascular ovarian graft. However, little is known about its effect when applied through an absorbable sponge matrix - a local and non-invasive application approach. Thirty adult Wistar rats were divided into three experimental groups (n = 10 each): control (CG), Gelfoam® (GF), and Gelfoam® + melatonin (GFM). The animals underwent oophorectomy and both ovaries were subjected to a slow cryopreservation protocol and stored in liquid nitrogen for 24 h. After thawing, the whole ovaries were transplanted into the retroperitoneum without vascular anastomosis and fixed to each side of the psoas muscle with a single non-absorbable suture. Immediately after transplantation, ovarian grafts were treated with Gelfoam® soaked either in vehicle (GF) or in melatonin at 10⁻⁷ M (GFM). In all groups, the applied vehicle volume was 15 µl. No treatments were administered to the control group. The grafts were recovered after 30 days, and the animals were euthanized with a lethal dose of anesthetic. Histological analyses (follicle and corpus luteum density), fibrosis assessment (collagen fiber types I and II), and immunohistochemistry for endothelial cells (von Willebrand factor), apoptosis (TUNEL), and cell proliferation (Ki67) were performed. Ovarian follicles at different maturation stages and intact and functional corpora lutea were observed in all groups. Melatonin promoted an increase in endothelial cells (p < 0.05, GFM vs. CG and GF) and reduced cellular proliferation within the ovarian follicles (p < 0.05, CG vs. GFM; Fig. 3) without alter this parameter in the corpora lutea (p > 0.05). Animals in the GF group showed increased cell proliferation in the corpora lutea, which was attenuated by melatonin treatment (GF vs. CG and GFM; p < 0.05). Apoptosis in the corpora lutea was reduced by melatonin treatment (p < 0.05, CG vs. GFM). No differences were observed among the groups in the number of leukocytes, corpora lutea, viable and atretic ovarian follicles, quantification of type I and III collagen fibers or apoptosis in ovarian follicles. Melatonin delivered via an absorbable sponge improved the viability of cryopreserved ovarian grafts in rats by enhancing endothelial cells and reducing apoptosis and cell proliferation, without inducing an inflammation response. Future studies should evaluate its effects on human tissues, either alone or in combination with other cytoprotective agents, to optimize ovarian graft survival.