CN115029303B - 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 - Google Patents
一种用于coh周期中人未成熟卵母细胞ivm能量培养液 Download PDFInfo
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Abstract
本发明公开了一种基于NADH的用于COH周期中人未成熟卵母细胞IVM能量培养液，创造性地将卵裂培养液作为基础液与人血清按一定比例结合，附以溶入相关激素(FSH、hCG、17β‑estradiol)并在此基础上添加浓度为10‑6mol/L的NADH从而显著增加了人体外成熟卵母细胞内ATP的含量，提高了线粒体膜电位，保护了线粒体功能从而提高卵子质量，为卵子成熟、受精及后续的胚胎发育提供充足的能量。本发明旨在通过探索一种新颖的IVM培养液，优化IVM技术体系，从而提高COH周期中人卵子利用率，为高龄、卵子成熟障碍、反复多次试管失败等患者获得健康后代提供技术基础。
Description
技术领域
本发明涉及一种新颖的基于NADH的用于COH周期中人类未成熟卵母细胞IVM能量培养液，属于生殖医学工程领域。
背景技术
在辅助生殖技术治疗的控制性超促排卵(controlled ovarianhyperstimulation,COH)周期中，大约有15％-20％的卵母细胞(卵子)为未成熟卵子[1,2]，未成熟卵子没有极体，包括胞浆含有单个核仁的较大生殖泡GV(germinal vesicle)和无核的MI(metaphase I)。由于这些未成熟卵子发育潜能低，在临床上通常被视为废弃卵子而丢弃[3,4]。然而，人卵子是极其稀缺的生育资源，与精子不同，自出生时女性的卵泡数量就固定了，出生后不再产生新的卵泡，从青春期至更年期，每次月经周期有15-20个卵泡生长发育，但是一般只有一个优势卵泡能发育成熟并排卵。对于患有卵巢功能减退甚至衰竭疾病的女性患者，虽可通过卵子捐赠的方式获得子女，但其困难程度堪比器官移植。并且COH可能引起卵巢过度刺激综合征，这是一种严重而常见的并发症，对于遭受一次辅助生殖周期失败或卵巢过度刺激综合征高危的女性患者来说，再进行一次COH治疗无疑对其生理和心理造成巨大创伤。所以，能够获得并利用废弃的不成熟卵子培养成熟为患者本人使用或捐赠给需要的女性是生殖医学界的关注热点。
人卵子体外成熟(in vitro maturation,IVM)技术作为现代辅助生殖技术(assisted reproductive technology,ART)的重要组成部分，是通过体外培养获得成熟卵子的一项技术。自1935年Pincus等[5]观察到兔未成熟卵子可在体外自发成熟后，掀起了一股对卵子IVM技术的探索热潮，直至1983年，该项技术首次应用于临床并成功分娩婴儿[6]。但是目前的IVM技术仍面临着获得的卵子成熟率较低，卵子发育潜能较差等诸多挑战。如何进一步优化IVM技术体系，改善IVM卵子质量，并对优质卵子进行筛选是目前临床面临的亟需解决的难题。
线粒体是卵母细胞中最丰富的细胞器，具有双层膜结构，对维持细胞内代谢和能量产生具有重要作用，其在卵子成熟、着床前胚胎发育过程中经历动态变化，以支持关键的细胞发育事件[7-9]。人类卵子在生长发育过程中主要通过氧化磷酸化代谢丙酮酸提供能量，早期胚胎的代谢特点是低氧化代谢水平，利用丙酮酸、乳酸和氨基酸以支持发育[10]。而随着胚胎基因组的激活和囊胚形成的能量需求增加，囊胚期胚胎表现出高水平的糖酵解和氧气消耗[11]。在正常卵母细胞中，线粒体通过分裂与融合形成动态互连的网络，产生足够的ATP以维持卵母细胞成熟和植入前胚胎发育。然而在IVM过程中，诸多因素形成的活性氧(reactive oxygen species,ROS)会引起线粒体损伤，使线粒体功能紊乱，从而引起低成熟率，低受精率，卵子质量差等[12]，对后续胚胎发育产生不良影响。据研究报道证实：靶向性地提升线粒体生产ATP的能力可以改善IVM卵子发育潜力[13]。因此通过调控线粒体形态及功能从而提高未成熟卵子体外培养的成熟率及后期胚胎发育的能力，是提高IVM效果的关键所在。
NADH是烟酰胺腺嘌呤二核苷酸(NAD+)的还原态，还原型辅酶I，在维持细胞生长、分化和能量代谢中起重要作用。NADH产生于糖酵解和细胞呼吸作用中的三羧酸循环，并作为生物氢的载体和电子供体，参与线粒体内膜上的氧化磷酸化过程，其释放质子和电子，提供能量合成ATP，自身被氧化为NAD+[14]。NAD+是一种重要的辅助因子，可以直接或间接影响许多关键的细胞功能，包括细胞代谢、DNA修复、线粒体自噬、细胞衰老和免疫等[15-18]。在许多组织中NAD+水平随着年龄的增长而下降[16]。NADH与NAD+是一对氧化还原对，胞外增加NADH可以有效增加NAD+[19]。NADH的含量与ATP产生量直接相关，理论上，1分子NADH释放的能量可以合成3分子ATP，细胞中所含NADH越多，产生的能量越多。目前仍没有关于NADH作用于人类卵子的相关报道。
发明内容
本发明提供了一种新的用于COH周期中人未成熟卵母细胞IVM能量培养液，创造性地将卵裂培养液作为基础液与人血清按一定比例结合，附以溶入相关激素，并在此基础上添加理想浓度的NADH，充分发挥NADH自身抗氧化和促进线粒体产生ATP的特性，为人卵子的体外成熟及后期的胚胎发育提供充分的能量，创造性地提高COH周期中女性卵子利用率。
本发明的一种用于COH周期中人未成熟卵母细胞IVM能量培养液包含如下组分：
(70％-90％)V/V卵裂培养液、(10％-30％)V/V人血清、10-6mol/L NADH。
优选的，还可包含如下组分的至少一种：FSH、hCG、17β-estradiol。更有选的，包含如下组分的至少两种：FSH、hCG、17β-estradiol。最优选的，包含如下三种组分：FSH、hCG、17β-estradiol。其中，所述FSH、hCG、17β-estradiol的组成优选为：(0.07-0.08)IU/ml FSH+(0.4-0.6)IU/ml hCG、(0.06-0.2)mg/ml 17β-estradiol。
所述用于COH周期中人未成熟卵母细胞IVM能量培养液,优选的组成可以如下:
(78％-82％)V/V卵裂培养液+(18％-23％)V/V人血清+(0.073-0.076)IU/ml FSH+(0.46-0.52)IU/ml hCG+(0.08-0.13)mg/ml 17β-estradiol+10-6mol/L NADH。更优选的组成如下:
80％V/V卵裂培养液+20％V/V人血清+0.075IU/ml FSH+0.5IU/ml hCG+0.1mg/ml17β-estradiol+10-6mol/L NADH。其中，所述人血清可以为人自体血清或者人造血清，优选人自体血清。
本发明创造性地将卵裂培养液作为基础液与人血清按一定比例结合，附以溶入相关激素(FSH、hCG、17β-estradiol)并在此基础上添加浓度为10-6mol/L的NADH从而显著增加了人体外成熟卵母细胞内ATP的含量，提高了线粒体膜电位，保护了线粒体功能从而提高卵子质量，为卵子成熟、受精及后续的胚胎发育提供充足的能量。本发明旨在通过探索一种新颖的IVM培养液，优化IVM技术体系，从而提高COH周期中人卵子利用率，为高龄、卵子成熟障碍、反复多次试管失败等患者获得健康后代提供技术基础。
附图说明
图1：10-2mol/L浓度NADH培养的卵子凋亡。
图2：NADH对人体外成熟卵母细胞ATP的影响。Control组为不添加NADH组，NADH组为添加最优浓度10-6mol/L NADH组。*代表组间差异具有显著性(P<0.05)。
图3：NADH对人体外成熟卵母细胞线粒体膜电位(MMP)的影响。Control组为不添加NADH组，NADH组为添加最优浓度10-6mol/L NADH组。*代表组间差异具有显著性(P<0.05)。
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。
人血清提前56℃水浴30分钟灭活，卵裂培养液(COOK,Australia)与人血清按4：1的体积比配成混合液，以该配比的混合液来配制0.075IU/ml FSH，0.5IU/ml hCG，0.1mg/ml17β-estradiol和10-6mol/L NADH(EMD Millipore Corp,USA)的溶液即可。其中，雌激素：β-ESTRADIOL(17β-Estradiol)(雌激素),E-2758,250mg,批号：052K13705，公司SIGMA；FSH-促卵泡成熟激素，HCG-人绒毛膜促性腺激素，选用瑞士产Gonal-F/Profasi，Swithland；血清：选用患者自体血清；
收集在安徽医科大学第一附属医院生殖医学中心接受胞浆内单精子显微注射(Intracytoplasmic sperm injection,ICSI)治疗的COH周期中废弃的人类未成熟卵子(GV和MI)。
(1)卵子发育能力相关实验实施如下：
实施例采用上述IVM能量培养液，对比例仅调整NADH浓度分别为0,10-2mol/L，10- 4mol/L,10-6mol/L，10-8mol/L，其它条件不变。使用透明质酸酶去除卵子颗粒细胞后将卵子随机分到以上五组，体外培养24小时后挑选出成熟(MII)卵子进行ICSI受精，受精后转入卵裂液中培养三天，ICSI后16-18小时观察受精情况。在第三天将胚胎转入囊胚培养液继续培养到第五/六天。在倒置显微镜下对卵子受精、胚胎发育进行观察，统计成熟率、受精率、卵裂率、优质胚胎率、囊胚率和优质囊胚率。实验中发现10-2mol/L浓度NADH培养的卵子全部发黑凋亡，如图1所示，证明10-2mol/L的NADH浓度太高，对卵子产生了不利影响。故后期停止10-2mol/L NADH组的实验。
(2)线粒体功能相关实验实施如下：
2.1 MII卵子内ATP水平检测：
卵子ATP生成率测定使用ATP determination kit(BODIPY FL ATP,A12410,Invitrogen,Eugene,OR,USA)进行生物发光测定。准备工作：根据说明书制备标准液，提前将4％多聚甲醛、PBS预热；准备玻璃皿，用PBS做成微滴(～10μL)，覆盖矿物油。分别将对照组和实验组MII卵子在PBS中洗涤3次，用4％多聚甲醛37℃避光固定1h。固定后用PBS洗涤3次，将卵子置于标准液中室温染色1h。染色后再用PBS洗涤3次，将卵子转移到玻璃皿的微滴中进行共聚焦荧光显微镜拍摄,荧光强度越弱，ATP含量越低，拍摄图片用共聚焦显微镜图像处理系统分析。
2.2 MII卵子内MMP水平检测：
应用JC-1线粒体膜检测试剂盒(KTA4001，Abbkine)检测卵子线粒体膜电位水平变化。准备工作：配置3-5ml受精液，平衡12小时后准备受精皿1-2个置于孵箱中平衡；将试剂盒中的JC-1染置于室温并避光，ddH2O置于室温，CCCP置于4℃冰箱；用ddH2O稀释5×的AssayBuffer制备1×Assay Buffer备用，使用前预热至37℃；在开始检测前，在每1ml的AssayBuffer中混合4μl JC-1染料，加入Assay Buffer后立即将溶液涡旋混匀，使用前避光及预热至37℃；设置阳性对照：将10mM按照比例稀释成10μM，在2号皿中加入500μl的CCCP，并在37℃、5％平板孵箱中孵化20-30分钟；阴性对照：不做任何处理。实验步骤：(1)将配置好的JC-1染料工作液加入500μl至2号皿中央孔，外围加入500μl的PBS；(2)将MII的卵子置于染料染色，在37℃下避光染色15-30分钟；(3)检测：用激光共聚焦显微镜。正常线粒体内，JC-1聚集在线粒体基质中形成聚合物，聚合物发出强烈的红色荧光(Ex＝585nm,Em＝590nm)；不健康的线粒体由于膜电位的下降或丧失，JC-1只能以单体的形式存在于胞浆中，产生绿色荧光(Ex＝514nm,Em＝529nm)；(4)数据分析：健康细胞内，JC-1单体聚集形成聚合物，线粒体呈现强烈的红色荧光(激发波长550nm,发射波长600nm)。凋亡或坏死细胞内JC-1以单体形式存在，线粒体呈强烈的绿色荧光(激发波长485nm,发射波长535nm)。然后计算红色荧光信号与绿色荧光信号的比值，用来判断细胞健康程度。
目前，本发明相关实验已在安徽医科大学第一附属医院生殖医学中心完成，实验结果如下：
(1)发育结果：
表1各组卵子/胚胎发育情况
以上卵子和胚胎的发育结果显示，添加10-6mol/L的NADH卵子成熟度最高，并且获得优质胚胎率、囊胚率、优质囊胚率在各组中最高。IVM的最终目的是获得高质量的可移植囊胚，故认为10-6mol/L的NADH是IVM培养中的最优浓度。
(2)ATP结果：如图2所示，添加10-6mol/L NADH与不添加NADH的IVM液培养出来的成熟卵子内ATP含量具有显著差异(P<0.05),添加10-6mol/L NADH培养的体外成熟卵子ATP含量明显更高。
(3)MMP结果：如图3所示，添加10-6mol/L NADH培养的体外成熟卵子线粒体膜电位水平显著高于不添加NADH的IVM液培养出来的成熟卵子，且具有统计学意义(P<0.05)。
卵母细胞成熟是生命开始的关键过程，在此过程中，卵母细胞积聚大量的能量以支持后续的发育阶段，即受精和早期胚胎发育。卵母细胞成熟包括细胞核成熟和细胞质成熟，第一极体的出现标志着细胞核成熟，目前尚没有确切的指标来判断细胞质的成熟。在体内，核质成熟往往作为一个整体同步发生，但是在IVM过程中，则有可能会出现核质成熟不同步的现象，而成熟度不充分会降低卵母细胞质量，进而可能导致受精异常、胚胎发育阻滞和非整倍体等。在很多研究中，线粒体的相关特性被广泛用于评估卵母细胞质的成熟。以上结果表明，在IVM培养过程中，添加10-6mol/L NADH提高了人卵子内ATP含量及线粒体膜电位，保护了线粒体功能，为卵子成熟、受精及后续的胚胎发育提供充足的能量。
应当说明的是，本发明的上述所述之技术内容仅为使本领域技术人员能够获知本发明技术实质而进行的解释与阐明，故所述之技术内容并非用以限制本发明的实质保护范围。本发明的实质保护范围应以权利要求书所述之为准。本领域技术人员应当知晓，凡基于本发明的实质精神所作出的任何修改、等同替换和改进等，均应在本发明的实质保护范围之内。
参考文献
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Claims (3)
1.一种用于COH周期中人未成熟卵母细胞IVM能量培养液，组成如下：
（70%-90%）V/V卵裂培养液+（10%-30%）V/V人血清+（0.07-0.08） IU/ml FSH + （0.4-0.6 ）IU/ml hCG +（0.06-0.2）mg/ml 17β-estradiol+10-6mol/L NADH。
2.如权利要求1所述的用于COH周期中人未成熟卵母细胞IVM能量培养液，其特征在于，组成如下:
80%V/V卵裂培养液 + 20%V/V人血清 + 0.075 IU/ml FSH + 0.5 IU/ml hCG + 0.1mg/ml 17β-estradiol + 10-6mol/L NADH。
3.如权利要求1-2任一项所述的用于COH周期中人未成熟卵母细胞IVM能量培养液，其特征在于，所述人血清为人自体血清。
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