关键词: 人卵巢组织, 新型玻璃化冷冻, 慢速冷冻, 血管内皮生长因子, 微血管密度

Abstract:

BACKGROUND: The cryopreservation of human ovarian tissue has become an attractive method to preserve female fertility. Human ovarian tissue experiences neovascularization after transplantation to recover blood supply, cryopreservation and resuscitation technique is a key for the neovascularization of human ovarian tissue following transplantation.
OBJECTIVE: To investigate vascular endothelial growth factor (VEGF) expression and microvessel density in human ovarian tissue following novel needle immersed vitrification (NIV) and slow-freezing, to explore the influence of two cryopreservation methods play in the neovascularization of human ovarian tissue after transplantation.
METHODS: Eight normal human ovarian tissues from patients with carcinoma of endometrium were cut into 12 fragments in the size of 1.5 mm × 1.5 mm × 1.0 mm, then randomly assigned to 3 groups: fresh control group, NIV group and slow-freezing group.
In the NIV group, pieces of ovarian tissue strips were dehydrated in an equilibration solution consisting of 7.5% ethylene glycol and 7.5% dimethyl sulfoxide in TCM-199 supplement with 20% fetal bovine serum and a vitrification solution consisting of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 mol/Lsucrose, then were plunged in liquid nitrogen directly and sealed in liquid nitrogen-filled cryovials. For thawing, the needles holding ovarian tissues were serially transferred into 1.0, 0.5, 0.25 mol/L sucrose solution and TCM-199 supplemented with 20% fetal bovine serum. In the slow-freezing group, pieces of human ovarian cortex fragments were placed in a 1.8-mL cryovial containing 1 mL of TCM-199 medium supplemented with 0.1 mol/L sucrose, 20% fetal bovine serum and 1.5 mol/L dimethyl sulfoxide, the cryovials were placed in the programmable freezer and cryopreserved by pre-set slow-cooling protocol. For thawing, the ovarian tissue strips were washed in a stepwise manner: 1.0 mol/L dimethyl sulfoxide + 0.1 mol/L sucrose, 0.5 mol/L dimethyl sulfoxide + 0.1 mol/L sucrose, 0.25 mol/L dimethyl sulfoxide + 0.1 mol/L sucrose and 0.1 mol/L sucrose. The frozen-thawed and fresh controlled human ovarian tissues were cultured in vitro. The expression of VEGF and CD34, as well as microvessel density, was analyzed by immunohistochemistry.
RESULTS AND CONCLUSION: There was patchy and mild expression of VEGF in the stromal cells of all the three groups before and after culture. The expression of VEGF increased and reached peak value after culture for 2 days, began to decrease after culture for 4 days and further attenuated after culture for 6 days in all the three groups. Compared with slow-freezing group, the expression of VEGF in NIV group was closer to that in fresh control group. Microvessel density of all the three groups increased and reached peak value after culture for 2 days, and the microvessel density of fresh control group and NIV group was significantly higher than that of slow-freezing group (P < 0.05). The microvessel density of slow-freezing group after culture for 4 days and that of all the three groups after culture for 6 days significantly decreased compared with after culture for 2 days (P < 0.05). NIV is superior to slow-freezing to preserve stromal cells and extracellular matrix of human ovarian tissue, and plays less influence in VEGF expression and angiogenesis in human ovarian tissue.