两种浓度玻璃化冷冻液对人卵巢组织的冷冻保护效果比较

Comparison of the Effect of Cryoprotective Vitrification Agent with Different Concentrations on Protecting Human Ovarian Tissue

目的比较两种不同浓度玻璃化冷冻保护剂对人卵巢组织的冷冻保存效果。方法收集2013年8月-2014年5月9例行妇科手术的患者卵巢组织,使用直接覆盖法(DCV)玻璃化冷冻法保存。玻璃化冷冻保护液采用高浓度和低浓度两组。高浓度组冷冻保护液为15%(V/V)二甲亚砜(DMSO)+15%(V/V)乙二醇+0.5 mol/L蔗糖组成;低浓度组冷冻保护液为12%DMSO+12%乙二醇+0.5 mol/L蔗糖组成,比较两组中始基卵泡形态正常率,始基卵泡及间质凋亡发生率。结果高浓度组中正常形态始基卵泡百分率同低浓度组相比,差异无统计学意义(P>0.05);低浓度组始基卵泡凋亡发生率为29.7%(58/195),高浓度组始基卵泡凋亡发生率为42.1%(69/164),差异有统计学意义(P<0.05)。低浓度组中卵巢间质细胞凋亡阳性率为30.2%(162/537),高浓度组中间质细胞凋亡阳性率为41.9%(206/492),差异有统计学意义(P<0.05)。结论 DCV玻璃化冷冻法中,低浓度冷冻保护液能通过减少细胞毒性,更好地保存始基卵泡及周围间质细胞,值得推广应用。

Objective To compare the effect of cryoprotective vitrification agent with different concentrations on protecting human ovarian tissue. Methods Human ovarian biopsy tissues were obtained from nine patients between August 2013 and May 2014. The cortical tissue pieces obtained from each patient were cryopreserved using direct cover vitrification （DCV） with two different concentrations. The vitrification solutions were divided into two groups： high concentration [15% （V/V） dimethyl sulphoxide （DMSO）＋15% （V/V） ethylene glycol （EG）＋0.5 mol/L sucrosel and low concentration group （12% DMSO＋12% EG＋0.5 mol/L sucrose）. The preservation effects in the two different concentration groups were compared by histologic evaluation using light microscope and apoptosis assessed by terminal dexoynucleotidyl transferase-mediated nick end labeling staining. Results There was no significant difference in the proportion of morphologically normal primordial follicles between high concentration group and low concentration group （P 〉 0.05）. The proportion of apoptotic primordial follicles in the low concentration group was 29.7% （58/195）, and was 42.1% （69/164） in the high concentration group, with a significant difference between the two groups （P 〈 0.05）. The proportion of apoptotic stromal cells in the low and high concentration group was 30.2% （162/537） and 41.9% （206/492） respectively with a significant difference （P 〈 0.05）. Conclusions Vitrification solutions with lower concentration can improve the preservation of the primordial follicles and stromal cells in human ovarian tissue. It is a method worth trying in order to improve the protective effect of vitrification by decreasing the toxiciw of vitrification solutions.