不同玻璃液冻存人卵巢组织的近期异种移植效果的比较

Comparison of short-term transplantation effect of different vitrification solution on human ovarian tissue

目的通过研究不同的玻璃液对人卵巢组织冻存后的近期异种移植效果,以筛选出较优的玻璃化冷冻方案。方法收集2018年3月至2019年12月期间在北京大学深圳医院因妇科疾病行手术治疗的4例患者卵巢组织,将卵巢组织分割成5~10 mm×10 mm×1 mm的小块,分为新鲜对照组和4个处理组,4个处理组分别放入G1、G2、G3、G4不同的玻璃液中,经过4组玻璃液预平衡和平衡后冻存4周后复苏,复苏后的组织移植到胚龄10 d的鸡卵绒毛尿囊膜(chorioallantoic membrane,CAM)上培养,培养5 d后取出移植于CAM的卵巢组织,HE染色比较移植后4组卵巢组织的正常原始卵泡率,免疫组织化学法检测CD105标记的4组卵巢组织的新生血管并比较其微血管密度;Western blotting法检测并比较4组卵巢组织Bcl-2和Bax蛋白的表达。结果经过G1、G2、G3、G4玻璃液冻融并移植于鸡胚5 d后的卵巢组织,正常原始卵泡率分别为63.2%(24/38)、68.3%(28/41)、61.9%(26/42)、69.6%(32/46),新鲜对照组的正常卵泡率为82.8%(82/99),5组间的差异有统计学意义(P=0.044),进一步两两比较,各处理组间正常原始卵泡率均低于新鲜对照组(均P<0.05),而各处理组间的正常卵泡率差异无统计学意义(P>0.05)。G1组、G2组、G3组和G4组鸡胚移植后的卵巢组织均可见CD105表达量增加,CD105标记的微血管密度(microvascular density,MVD)在新鲜对照组和G1组、G2组、G3组、G4组分别为6.51±1.30/mm^(2)、11.10±1.62/mm^(2)、13.04±1.84/mm^(2)、9.11±1.09/mm^(2)、11.28±1.62/mm^(2),各处理组的MVD均高于新鲜对照组,差异有统计学意义(P<0.001,P<0.001,P=0.022,P<0.001),G1组、G2组和G4组的MVD均高于G3组(P=0.024,P<0.001,P=0.034)。Western blotting检测结果显示G1组、G2组、G3组、G4组和新鲜对照组的Bcl-2/Bax的比值分别为0.71±0.37、0.84±0.29、0.45±0.18、0.84±0.29和0.44±0.21,差异有统计学意义(P=0.013),G2组Bcl-2/Bax的比值明显高于新鲜对照组和G3组(P=0.025,P=0.038)。结论玻璃化冷冻保存的人卵巢组织,在移植早期原始卵泡丢失较多。经过G2和G4玻璃液冷冻复融后的人卵巢组织,在移植5 d后血管化程度较高,抗凋亡能力强,是适用于人卵巢组织冷冻的较优的玻璃化冷冻方案。

Objective To investigate the short-term transplantation effect of different vitrification sollutions in order to optimize the cryopreservation protocol on human ovarian tissue.Methods Ovarian tissues came from 4 patients who were treated surgically due to gynecological diseases in Peking University Shenzhen Hospital from March 2018 to December 2019.The size of 5-10 mm×10 mm×1 mm ovarian tissues were distributed to fresh control group and four experimental groups(G1,G2,G3 and G4).After equilibrium and thawing,the frozen ovarian tissues were grafted into chorioallantoic membrane(CAM).The transplanted ovarian tissues were collected 5 d after transplantation.The morphology of follicles was assessed by HE,the expression of CD105 protein was detected by immunohistochemistry and the microvascular density was recorded.The expression of Bcl-2 and Bax proteins was detected by Western blotting.Results There was a significantly difference in the percentage of morphologically normal primordial follicles(MNPF)among G1[63.2%(24/38)],G2[68.3%(28/41)],G3[61.9%(26/42)],G4[69.6%(32/46)]and fresh control groups[82.8%(82/99),P=0.044].Compared with fresh control group,the percentages of MNPF were statistically lower in the treatment groups(all P<0.05).The CD105-microvascular density(MVD)in fresh control group,G1,G2,G3 and G4 groups were 6.51±1.30/mm^(2),11.10±1.62/mm^(2),13.04±1.84/mm^(2),9.11±1.09/mm^(2),11.28±1.62/mm^(2),respectively.The CD105-MVD in four treatment groups after transplantation was significantly higher than that of fresh control group(P<0.001,P<0.001,P=0.022,P<0.001),and the CD105-MVD of G1,G2 and G4 groups was higher than that of G3 group(P=0.024,P<0.001,P=0.034).The ratio of Bcl-2/Bax in G1,G2,G3,G4 groups and fresh control group were 0.71±0.37,0.84±0.29,0.45±0.18,0.84±0.29 and 0.44±0.21,respectively.The ratio of Bcl-2/Bax of G2 group was statistically higher than that of fresh control group and G3 group(P=0.025,P=0.038).Conclusion After 5 d of transplantation into CAM,the percentage of normal primordial follicles is lower.Acorrding to the higher vascularization and anti-apoptotic ability,G2 and G4 are the optimal vitrification protocol for human ovarian tissue cryopreservation.