P-37: Vitrification of Mouse Ovary in Presence of Zinc in Vitrification Medium: Histological Evaluation
Whole ovary cryopreservation has been proved to be a feasible technique in fertility preservation. The normal ovarian cortex contains a large population of primordial follicles, this characteristic make them far more tolerant to cryoinjury. Researchers have used different cryoprotectants and various techniques to improve cryopreservation. It is plausible that inclusion of an antioxidant in the cryopreservation solutions may help in maintaining embryo viability after cryopreservation by reducing the effects of harmful oxygen radicals during the cryopreservation procedure. Zinc is an essential trace element . It has been known that a non adequate level of zinc can alter not only gene expression but also a variety of cellular functions .The present study was designed to determine the effects of different zinc concentrations in the ovary vitrification solutions on follicle morphology as assessed by light microscopy .
Materials and methods
In present study, the ovaries of 2-4 week-old NMRI mice dissected and randomly assigned to following groups :V0,V1,V2 ,V3 (vitrified warmed ovaries with 0, 100,150 and 200 μg/dl zinc concentration in vitrification solution). Ovaries were vitrified sequentially by immersion into two vitrification solutions VS1: 7.5% ethylene glycol (EG) and 7.5% DMSO in α-MEM supplemented with FBS 20% for 7 minutes and VS2: 15% EG + 15% DMSO in α-MEM supplemented with 20% FBS and 0.5 M sucrose for 3 minutes. They were placed in the 0.25 ml straw with a minimum volume of vitrification medium and then plunged into liquid nitrogen for 1 week. Warming was performed in α-MEM including 20% FBS that supplemented with descending concentrations of sucrose (1, 0.5, 0.25 M) at room temperature for 5 minutes. The recovered vitrified ovaries were fixed in 10% buffered formalin, embedded in paraffin wax, serially sectioned at 5 μm, stained with haematoxylin and eosin and analyzed under light microscope .
Most histological features of vitrified samples were normal. The cytoplasm of the oocytes was clear and all granulosa layers and theca were intact and firmly attached to the related basement membranes. Stromal cells were normal and with distinct boundaries, prominent nucleus, and pinkish cytoplasm. However in some follicles, stromal cells had distinct margins with foamy cytoplasm, detachment of granulosa layers from the basement membrane and deformity of oocytes were also seen . After vitrification-warming the least damage was observed in the small follicles. The rate of normal preantral follicles in the group V3 was statistically higher than V0 group (94.61 ± 50% vs. 77.42 ± 17% ANOVA, P-value<0.05). The proportions of normal follicles in cryopreserved groups V0, 1, 2, 3 were 83.36 ± 3.6%, 94.35 ± 48%, 92.55 ± 11% and 94.52 ± 05% respectively (ANOVA, P value<0.05). Conclusion: Our study showed ovary vitrification
Our study showed ovary vitrification with optimal cryoprotectant solutions such as EG plus DMSO is the most effective for preserving the structural efficiency of ovarian follicles. Vitrification didnt not cause significant changes to the morphology of follicles. Also results demonstrate that supplementation of vitrification medium in a dose manner improved the rate of normal follicles.