Zhang H, Yang Y, Ma W, Wu H, Zheng X, Hei C, Sun M, Ma W, Ma H, Chang Q, Wang H, Cai Y, Xie Yan, Zhao C, Pei X, Wang Y
Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia； Department of Histology and Embryology, Ningxia Medical University; Tissue Organ Bank and Tissue Engineering Centre, General Hospital of Ningxia Medical University, Ningxia, China.
Department of Orthopedics, The People No.3 Hospital of Anyang, Henan.
Cryo Letters. 2016 Mar-Apr;37(2):88-102.
Ovarian cryopreservation by vitrification is a very effective pathway for the preservation of female fertility during radiotherapy and chemotherapy. However, damage of follicles was triggered by cryo-injure during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. Appropriate FSH play important roles in anti-apoptosis and neoangiogenesis during ovarian follicle development.
Therefore, the purpose of this study was to investigate the effect of FSH on the revascularization and follicular survival of vitrified-warmed ovarian grafts.
Four-week-old C57BL/6J mice with diestrus were used and the ovaries were randomized into the following three groups: fresh control group (FCG), vitrified/warmed group (VCG) and vitrified/warmed group treated with 0.3 IU/mL FSH (FSH-VG) during ovarian vitrification. After warming, the ovaries of the three groups were allotransplanted into the renal capsule of receptor mice. Assessment of follicular quantity was performed by histological analysis. The angiogenesis factors, CD31 and MMP-2, and cell survival factors, PCNA, EdU and survivin were examined by immunohistochemistry and western blot analysis. Angiogenesis was detected by vascular perfusion with the fluorescent dye 2MD-FITC-Dextran.
The expression of CD31and MMP-2 were not significantly different in either VCG or FSH-VG compared with FCG, but when the ovaries were transplanted 48 hours later, the expression levels of CD31 and MMP-2 were lower for VCG than FCG (P < 0.05) and FSH-VG was not significantly different from FCG. Before transplantation, the expression levels of PCNA and survivin were lower for VCG and FSH-VG than FCG (p < 0.05), but FSH-VG was higher than VCG (p < 0.05). After 48 h of ovarian transplantation, the expression of survivin was lower for VCG than FCG (P < 0.05), but FSH-VG was not significantly different from FCG. In addition, these data were further supported by the results from detecting the 2MD-FITC-Dextran and EdU.
Taken together, supplementation with 0.3 IU/mL FSH during ovarian cryopreservation by vitrification increased the revascularization and follicular survival for mouse ovarian grafts through the up-regulated expression of angiogenesis and ovarian survival factors.
玻璃化冷冻保存卵巢是放疗和化疗期间保留女性生育能力的一种非常有效的途径。然而，在卵巢玻璃化过程中的冷冻损伤以及卵巢移植过程中的缺血/再灌注会引发卵泡损伤。适当的促卵泡生成素（FSH）在卵巢卵泡发育过程中的抗凋亡和新生血管形成中发挥重要作用。
因此，本研究旨在探讨FSH对玻璃化冷冻复苏卵巢移植物血管再生和卵泡存活的影响。
选用4周龄处于间情期的C57BL/6J小鼠，将其卵巢随机分为以下三组：新鲜对照组（FCG）、玻璃化冷冻/复苏组（VCG）和在卵巢玻璃化过程中用0.3IU/mL FSH处理的玻璃化冷冻/复苏组（FSH-VG）。复温后，将三组卵巢异体移植到受体小鼠的肾包膜下。通过组织学分析评估卵泡数量。采用免疫组织化学和蛋白质印迹分析检测血管生成因子CD31和基质金属蛋白酶-2（MMP-2）以及细胞存活因子增殖细胞核抗原（PCNA）、5-乙炔基-2'-脱氧尿苷（EdU）和生存素。通过用荧光染料2-甲氧基-2'-二甲基琥珀酰亚胺基-荧光素异硫氰酸酯-葡聚糖（2MD-FITC-Dextran）进行血管灌注检测血管生成。
与FCG相比，VCG或FSH-VG中CD31和MMP-2的表达均无显著差异，但在48小时后进行卵巢移植时，VCG中CD31和MMP-2的表达水平低于FCG（P<0.05），而FSH-VG与FCG无显著差异。移植前，VCG和FSH-VG中PCNA和生存素的表达水平低于FCG（P<0.05），但FSH-VG高于VCG（P<0.05）。卵巢移植48小时后，VCG中生存素的表达低于FCG（P<0.05），但FSH-VG与FCG无显著差异。此外，检测2MD-FITC-Dextran和EdU的结果进一步支持了这些数据。
综上所述，在玻璃化冷冻保存卵巢过程中补充0.3IU/mL FSH可通过上调血管生成和卵巢存活因子的表达来增加小鼠卵巢移植物的血管再生和卵泡存活。
