抗氧化剂联合应用优化人卵巢组织冻融效果的研究

The improved protective efficacy of combined antioxidants on cryopreserved and thawed human ovarian tissue

目的以首都医科大学附属北京妇产医院生育力保护中心现有人卵巢组织冻存复苏方案为基础,研究抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)和T(taurine,T)对卵巢组织的保护效果,以最大限度保护卵泡和基质细胞活性,提高复苏后卵泡发育潜能。方法以本中心现有冻存复苏方案为对照组(C组),在此基础上加入5 mmol/L NAC(N组)或0.5 mmol/L牛磺酸(T组),或同时加入上述浓度的NAC和T(N+T组)。将16名患者的卵巢组织采用随机数字表法分配入以上4组,按照本中心标准化程序完成卵巢组织的处理和冻存。复苏后进行HE染色、钙黄绿素染色,进行形态学观察和卵泡活性检测,卵巢组织培养4 d后测定培养液中17β-雌二醇(17β-estradiol,E2)、抗苗勒管激素(anti-mullerian hormone,AMH),取培养后的卵巢组织测定细胞内活性氧类物质浓度(reactive oxygen species,ROS)。结果各组卵泡结构和活性均良好,组间比较差异无统计学意义(P>0.05);各组卵巢组织经培养4 d后分泌E2浓度差异无统计学意义(P>0.05);N+T组卵巢组织中卵泡分泌AMH浓度高于C组,差异有统计学意义(P<0.05);N组和N+T组细胞内ROS浓度低于C组,差异有统计学意义(P<0.05)。结论本中心现有冻存方案和在此基础上单独加入NAC或T的改良方案均可有效保护卵泡结构和卵泡活性,同时加入NAC和T可产生协同作用,在保存卵泡结构和活性的同时,提高复苏后卵泡分泌E2和AMH浓度,提高卵巢组织活性,减少细胞内ROS产生,降低氧化应激水平,提高复苏后的卵泡发育潜能。

Objective To investigate the protective efficacy of antioxidants N-acetyl-L-cysteine(NAC)and taurine(T)based on the currently used human ovarian tissue cryopreservation and thawing protocol in Beijing Obstetrics and Gynecology Hospital,Capital Medical University,in order to better preserve the viability of follicles and stroma cells,and improve the folliculogenesis potential.Methods The currently used cryopreservation and thawing protocol in our center was set as control group(group C),with addition of 5 mmol NAC(group N)or 0.5 mmol taurine(group T),or with addition of 5 mmol NAC and 0.5 mmol taurine(group N+T).Ovarian tissues from 16 patients were assigned into the 4 groups randomly.The biopsies were cryopreserved and thawed according to the standard operation procedure in our center.After thawing,HE staining and Calcein-AM staining were conducted to evaluate the morphological characteristics and follicle viability.After cultured in vitro for 4 days,the 17β-estradiol(E2)and anti-mullerian hormone(AMH)were measured,and the level of cellular reactive oxygen species(ROS)was also detected.Results All 4 groups obtained good preservation of follicle integrity and viability without statistical significance(P>0.05).After 4 days culture,E2 concentration in culture medium showed no significant difference in 4 groups(P>0.05),AMH concentration in group N+T were significantly higher than that in group C(P<0.05).Group N and Group N+T had lower ROS levels than group C(P<0.05).Conclusions The addition of NAC alone or taurine alone or combination of NAC and taurine could preserve the integrity and viability of the follicle.NAC and taurine could act synergistically to increase the level of E2 and AMH and improve the overall activity of ovarian tissues after thawing,decrease intracellular ROS production,lower the level of oxidative stress,and improve the potential development of follicles after ovarian tissue thawing.