家兔和人卵巢组织冷冻复苏及异种移植的实验研究
摘要
近年来,随着社会的发展,生活水平的提高,人们对自身健康及生殖质量日益关注,对生殖能力的保存提出了越来越高的要求,由此极大地促进了学者们对人类卵巢组织冻存技术的研究和临床应用的探索。2008年,WHO调查发现140万例新增肿瘤患者中近70万是女性,<40岁育龄妇女占8%。放、化疗及手术等治疗有效地治疗了患者的疾病并延长了生命,但大剂量放疗、化疗和卵巢手术可导致年轻女性生殖力的丧失。保存女性生殖能力的方法有：取卵并行体外受精后胚胎冷冻、取卵后卵子冷冻、卵巢组织冷冻。因胚胎冷冻及卵子冷冻需要进行促排卵、取卵等较长周期的治疗,影响和延误恶性肿瘤患者的治疗,且只适用于已婚女性,因此应用受到限制。而卵巢组织冷冻保存不需要对卵巢进行药物刺激促排卵,在手术及化疗前直接切除卵巢组织进行冷冻,不影响患者自身原有恶性肿瘤及疾病的化疗及放疗,对女性生育储备的生殖内分泌功能保存有着重要的意义,为此类患者提供了一种补偿方法。20世纪50年代初,Parkes将大鼠卵巢组织切片慢速冷冻保存于-79℃的甘油盐水混合液中,快速解冻复苏后自体移植,之后观察到移植的卵巢组织出现卵泡生长发育,大鼠恢复了内分泌功能。20世纪90年代,卵巢组织冷冻随着冷冻科学技术的发展和改进以及各种抗冻剂的出现,发展非常迅速,不断有小鼠、绵羊和非人类哺乳类动物卵巢组织冷冻实验研究报道,并有冻融卵巢组织移植成功的案例。绵羊与人类卵巢无论是卵巢组织结构及排卵周期均较为相似,因此绵羊卵巢组织冻融并移植后获得成功的妊娠,为人类卵巢组织冻融技术的发展和应用提供了有意义的模型,并奠定了坚实的实验基础。Donnez J在腹腔镜下对一例何杰金氏患者自体原位移植了冻存6年的卵巢组织,随访发现该患者术后半年就恢复了月‘经周期,并有周期性排卵及黄体生成,术后11个月HCG测定和B超证实患者成功妊娠,并顺利分娩。我国开展卵巢组织冷冻技术还处于摸索阶段,陈子江等研究冷冻人卵巢皮质,观察卵巢皮质内各级卵泡形态及卵巢组织体外培养获得了一定的经验。
卵巢组织冷冻保存的优势在于其可冷冻卵巢皮质原位未成熟的始基卵泡,因其具有相对静止、体积小、缺乏透明带及皮质颗粒的特性,容易耐受冷冻及复苏造成的损伤,成功的卵巢组织冷冻和复苏,可保存卵巢组织中大量结构完整、具有生存活力及发育能力的原始卵泡,从而开辟了一条卵巢早衰和恶性肿瘤患者的治疗新途径。有效的卵巢冷冻保存及复苏技术是建立卵子库的基本前提,复苏后卵巢组织的成功移植对女性生育力的保存及恢复具有重要意义。为了拯救面临威胁和濒危动物,保存基因多样性,世界各地基因组储存中心正在收集和冷冻来自这些动物的卵巢组织。
卵巢冷冻保存及复苏还存在许多有待解决的问题,虽然在鼠类和大型灵长类动物中卵巢组织冷冻保存技术已有后代出生并获得了阶段性的研究成果,但用于人类卵巢组织的冷冻保存及复苏移植技术还存在许多问题,难以充分有效地的运用于临床,其关键难点包括确立最适宜的卵巢组织冷冻保存方案,因为冷冻过程中各个环节均有许多因素会影响卵巢组织的冻融效果,如组织切块的大小,所选择的冷冻方法、冷冻保护剂的种类及浓度、冷冻和复苏过程、操作人员的技术水平、硬件设备条件等,目前尚无定论的减少冷冻损伤的最佳冷冻方案。
卵巢组织复苏移植后的成活情况是决定生殖能力能否恢复的关键。影响移植成功的因素有很多,首先是移植后局部灌注损伤：Aubard等学者在羊卵巢组织自体移植模型实验中发现,只有5%的原始卵泡在移植后能够存活,分析原因可能是移植后的卵巢未行血管吻合,自身无血供,而移植局部的血管再生至少需要48小时,在此期间由于血供不足可能对卵巢造成了不可逆的损伤,如何保护移植组织不受缺血缺氧损害是卵巢组织冻融后移植亟待解决的一个问题；其次是最适宜移植部位的确立：迄今为止,国内外研究尚无最适宜卵巢组织移植的最佳部位定论,但能确认的是最适宜的移植部位必须具备能为组织的植入和成活发育提供良好条件、并且方便卵泡的检测和穿刺取卵的部位；最后是卵巢组织同种自体移植后的安全性问题的解决：卵巢组织的冷冻和移植的不断进步,为癌症患者恢复其自身卵巢功能和保存生育能力带来了新的希望。但卵巢组织移植仍然存在着一定的风险。冻融卵巢组织同种移植多数是为了对恶性肿瘤患者的生育能力进行保存,因此有学者质疑,移植恶性肿瘤患者的冷冻卵巢组织中是否存在微小癌灶,从而导致移植后肿瘤细胞发生播散或者再复发。MuellerA等实验研究发现大鼠卵巢经同种异位移植后,发生性索间质瘤的几率达100%。关于移植组织安全性的监测,Dror等研究表明采用RT-PCR(?)邕够敏感的检测到癌症患者卵巢组织内残存的微小病灶,从而提高移植的安全性。因此,在决定移植前,采用先进的检测手段如聚合酶链反应(PCR、FISH、基因芯片等技术,检测移植卵巢组织内残存的微小病灶,以降低卵巢移植所带来的肿瘤细胞转移风险,有利于提高卵巢组织移植的有效性和安全性。
关于卵巢组织冷冻、复苏、移植研究所采用的标本,人类卵巢组织冻存研究标本大多来自于因卵巢疾患而需要进行妇科手术的病人,由于她们年龄一般都比较大,卵巢中某些区域整群卵泡已经耗尽,同时合并有子宫内膜异位囊肿等卵巢病变,所以很难获得足够数量的卵泡标本,而且人类卵巢组织的冷冻、复苏、移植涉及诸多伦理问题,所以实验标本的获得非常珍贵而有限,实验数据不够充沛。有研究显示种属间卵巢原始卵泡没有明显差异,本实验前期选择家兔卵巢组织来进行研究,是由于其低廉的价格,广阔的来源,较短的繁殖周期,且人类与家兔卵巢始基卵泡中卵母细胞的大小及进入减数分裂的卵母细胞微细结构基本相似。在前期家兔卵巢冻存研究基础上,我们又对来自卵巢手术剥离的人类卵巢组织标本进行了冷冻、复苏和移植的研究。
本研究分四个部分,即家兔卵巢组织冷冻复苏的实验研究、家兔卵巢组织异种异位移植的实验研究、人卵巢组织冷冻复苏的实验研究、人卵巢组织异种异位移植的实验研究四个阶段。通过采用不同冷冻程序(慢速程序化法、快速玻璃化法)、不同冷冻载体(麦管法、微滴法、坚硬表面法)、不同冷冻试剂及配伍方法(二甲基亚砜DMSO、乙二醇EG、丙二醇PROH)等分别对家兔及人卵巢组织进行了冷冻、复苏、体外培养、异体移植的系统研究,以期寻找适宜的卵巢组织冷冻复苏方案及移植部位,为临床开展人类卵巢组织冷冻及移植提供实验数据参考,为建立卵巢组织冷冻库奠定基础。
第一部分：家兔卵巢组织冷冻复苏的实验研究
研究目的
采用不同冷冻保护剂(PROH、DMSO及EG)单独或联合应用于程序化或玻璃化法对家兔卵巢组织进行冷冻并复苏,观察复苏后卵巢组织中各级卵泡形态、卵巢组织Ⅱ型跨膜酪氨酸激酶受体(c-kit)和增殖细胞核抗原(ki67)以及主要组织相容性复合体类抗原(MHC-2)表达的变化,从而判断各种冷冻方案对家兔卵巢组织形态学、细胞增殖活性、卵巢组织抗原性的影响,以探讨相对适用于卵巢组织的冷冻复苏方案。
研究方法及结果
1四种不同冷冻复苏方法对家兔卵巢组织形态学的影响
1.1研究对象及分组：选择清洁级性成熟雌性日本大耳白兔12只,随机分为四组,麻醉后开腹取其卵巢组织并切成lxlxlmm3块,按随机方法分组将家兔卵巢组织块分别进行慢速程序化(分A2及B2两个亚组,冷冻剂分别采用PROH、 DMSO)及快速玻璃化(分C2及D2两个亚组,冷冻剂分别采用DMSO+PROH、 DMSO+EG)冷冻并复苏；冷冻前每组各取少量新鲜卵巢组织块做为对照组(A1、B1、C1、D1)。
1.2研究内容：通过卵巢组织学检测,观察新鲜对照组及各冷冻复苏组卵巢组织内各级卵泡比例、形态结构及正常形态率等变化。
1.3研究结果
1.3.1新鲜及冷冻复苏家兔卵巢组织形态学特点：来自各组家兔的新鲜卵巢组织切片中,显微镜下共计数各级卵泡1236枚,其中始基卵泡所占比率(88.2%)及其正常形态率(91.4%)均最高,初级卵泡及次级卵泡比率分别为8.4%和3.4%；计数来自三组冷冻复苏卵巢组织切片中的卵泡共954枚,其中始其卵泡857枚,占89.8%,初级卵泡占7.3%,次级卵泡仅占2.8%。
1.3.2采用不同冷冻程序和冷冻剂处理后家兔卵巢组织各级卵泡形态学的变化：冷冻组中,以PROH慢速程序化冷冻组始基卵泡的形态正常率最高,达80.1%,明显高于DMSO程序化组(70.5%)及两组玻璃化组(DMSO+PROH及DMSO+EG组分别为67.6%,69.7%),差异有统计学意义；将各冷冻组所观察到的各级卵泡合并计算,其中始基卵泡形态正常率(72.7%),明显高于初级卵泡的形态正常率(55.7%),因次级卵泡总数极少,所以未行统计学分析。
1.3.3冷冻复苏前后家兔卵巢组织结构变化：镜下观察冷冻前家兔卵巢组织内可见排裂规则的卵巢间质细胞紧密包裹的各级卵泡。所有冷冻复苏组卵巢组织均可见以初级卵泡和次级卵泡为主的受损情况,这些卵泡与周围间质细胞分离,但始基卵泡受影响较小；各冷冻组卵巢间质细胞排裂紊乱、连接疏松,形成裂隙。
2程序化及玻璃化冷冻对家兔卵巢抗原性及卵泡细胞增殖活性的影响
2.1研究对象及分组：选择9只4-5月龄清洁级性成熟雌性日本大耳白兔,随机分为3组(新鲜对照组、PROH慢速程序化冷冻组、DMSO+EG'决速玻璃化冷冻组),获取卵巢组织方法如前所述,切块待用。
2.2研究内容
2.2.1家兔卵巢组织相容性抗原(MHC-2)的表达：应用SP免疫组化法检测上述新鲜组及经两种冷冻方法处理后家兔卵巢组织MHC-2抗原表达的变化,通过观察MHC-2抗原的平均吸光度值,判断冷冻对卵巢组织抗原性的影响。
2.2.2冷冻复苏对各级卵泡细胞增殖活性的影响：分离新鲜对照组及冷冻复苏组卵巢组织内大、小卵泡(直径分别为100-150μm及300-500μm),对其进行体外培养后,采用3H标记的胸腺嘧啶核苷掺入试验(3H-TdR),移入液闪瓶中上机测其脉冲信号数(cpm),判断冷冻复苏对各级卵泡细胞增殖活性的影响。
2.3研究结果
2.3.1PROH程序化与DMSO+EG玻璃化冷冻法对家兔卵巢MHC-2表达的影响：免疫组化结果显示MHC-2主要表达于卵巢间质细胞和次级卵泡卵母细胞的胞膜,而始基卵泡及初级卵泡的卵母细胞和颗粒细胞均无表达；玻璃化冷冻组较程序化组和新鲜对照组卵巢组织MHC-2抗原的平均吸光度值明显降低；程序化组与新鲜对照组平均吸光度值无明显差异。
2.3.2PROH程序化与DMSO+EG玻璃化冷冻法对家兔卵泡细胞增殖活性的影响：通过3H-TdR掺入试验检测各组卵泡细胞增殖活性发现,程序化及玻璃化冷冻组与新鲜卵巢组小卵泡的cpm值差异无统计学意义,而冷冻组大卵泡的cpm值明显降低；两冷冻组间差异无统计学意义。
3.不同玻璃化冷冻载体对家兔卵巢组织c-kit和W67表达的影响
3.1研究对象及分组：选择12只4-5月龄清洁级性成熟雌性日本大耳白兔,按是否冷冻及玻璃化冷冻所选择的载体不同随机分为4组：新鲜对照组(A组)、麦管法玻璃化冷冻组(B组)、微滴法玻璃化冷冻组(C组)、坚硬表面法(SSV)玻璃化冷冻组(D组)。玻璃化冷冻试剂采用EG+DMSO;获取家兔卵巢组织方法如前所述,将卵巢组织切块待用。
3.2研究内容：通过对冻融卵巢组织的组织学观察,并采用免疫组化法检测冻融卵巢组织Ⅱ型跨膜酪氨酸激酶受体(c-kit)和增殖细胞核抗原(Ki67)表达,判断冷冻对卵巢组织增殖活性及发育潜能的影响。
3.3研究结果：采用免疫组化检测家兔新鲜及冻融卵巢组织,结果显示各级卵泡内卵母细胞的胞膜和/或胞浆中ki67蛋白均出现呈棕黄或棕褐色的阳性表达；与新鲜组相比较,经麦管法、微滴法、SSV法冷冻处理后Ki67和c-kit阳性率表达率差异无统计学意义。
结论
1. PROH慢速程序化冷冻方法用于家兔卵巢组织冷冻获得了较高的始基卵泡正常形态率及细胞增殖活性,可能是一种较好的冷冻方法。
2.玻璃化方法虽使始基卵泡形态正常率降低,但也能使始基及初级卵泡卵母细胞保持良好的增殖活性,并可降低家兔卵巢组织抗原性,可能会减轻复苏后卵巢组织移植的免疫排斥,加之该技术操作简便,费用低廉,可能会有良好的应用前景。
3.程序化冷冻及玻璃化冷冻对形态结构完整的小卵泡细胞增殖活力影响不明显,而大卵泡增殖活性明显下降。
第二部分：家兔卵巢组织异体异位移植的实验研究
研究目的
在前期研究基础上,选择低浓度配伍比的DMSO+EG做为冷冻保护剂,玻璃化冷冻家兔卵巢组织,将新鲜及冷冻复苏后卵巢组织块分别移植于去势裸鼠颈部皮下,观察接受移植后裸鼠动情周期、激素分泌、子宫形态变化及移植物变化,以评价冷冻效果。
研究方法
1研究对象及分组
选择清洁级性成熟雌性日本大耳白兔4只,取其卵巢组织处理后切块,分三组用于新鲜组织观察、新鲜移植、冷冻复苏移植；移植宿主采用8-10周龄的SPF级性成熟雌性Nu/Nu裸鼠36只,分4组(每组9只),除正常对照组外,其余三组均切除双侧卵巢去势,之后分别用于新鲜组织移植、冻融组织移植及去势对照组。
2研究方法
2.1卵巢组织冷冻复苏方法：采用低浓度比二甲基亚砜(DMSO)+乙二醇(EG)作为冷冻保护液,通过坚硬表面铜块(SSV)法对卵巢组织进行玻璃化冷冻后快速复苏。
2.2卵巢组织移植：麻醉裸鼠后,消毒并切开裸鼠左侧颈部皮肤,眼科镊分别夹取新鲜或冻融家兔卵巢组织多点移植于颈部皮下,缝合并标记移植部位。
2.3家兔卵巢组织冷冻复苏后移植的效果评价
2.3.1裸鼠阴道脱落细胞学观察：每天观察各组裸鼠阴道涂片上皮细胞变化以判断裸鼠动情周期恢复及持续时间。
2.3.2裸鼠血中雌激素测定：全部正常对照组及部分移植组裸鼠有动情周期出现；去势未移植组无动情周期。分别测定对照组、去势组及移植后出现动情周期的裸鼠血清雌激素(E2)水平,判定卵巢功能情况。
2.3.3裸鼠子宫形态学观察及移植物观察：移植35天后,切除各组裸鼠完整的子宫,分别于肉眼及镜下观察其形态学变化；切开新鲜及冷冻卵巢移植组裸鼠颈部皮肤,取出移植物,进行组织学观察。
研究结果
1裸鼠动情周期情况
正常对照组裸鼠全部有规律的动情周期；新鲜移植组9个裸鼠中7个出现动情周期,冷冻移植组9个中6个出现动情周期,两移植组裸鼠恢复动情周期天数无差异,正常及移植组动情周期持续天数无差异。
2裸鼠血中雌激素水平测定
测定全部出现动情周期的裸鼠和去势裸鼠血中E2水平,两移植组恢复动情周期的裸鼠血中E2水平与正常对照组无明显差异,均明显高于去势组。
3裸鼠子宫形态学观察
去势组裸鼠子宫明显有别于有动情周期的裸鼠子宫,呈萎缩、苍白状,内膜薄,腺体少,表面被覆立方或者单层扁平上皮细胞；而正常对照及移植组裸鼠子宫色泽红润而有弹性,形态饱满,内膜厚且腺体粗大、量多,腺细胞胞浆丰富,内膜表面被覆单层高柱状或假复层上皮细胞。
4卵巢组织移植物形态学观察
取出裸鼠颈部皮下移植物进行观察,新鲜移植组与冷冻移植组移植物肉眼可见大部分长成淡黄色圆形或椭圆体,体积增大明显,移植物柔软,部分移植组织块表面出现凹凸不平的串珠状突起。存活的移植物镜下可见毛细血管生成,皮质内可见到各级卵泡及闭锁卵泡。
结论
采用低浓度DMSO+EG玻璃化冷冻法能够较好地保存卵巢组织的结构和功能,新鲜及冻融后的家兔卵巢组织裸鼠异种异位移植后能够存活,并具有内分泌功能。
第三部分：人卵巢组织冷冻复苏的实验研究
研究目的
以不同浓度配伍的DMSO+EG做为玻璃化冷冻保护剂,分别以麦管法、微滴法及SSV法对人卵巢组织进行玻璃化冷冻,通过观察冷冻前后人卵巢组织中卵泡形态学变化、细胞凋亡率检测及复苏后卵巢组织体外培养上清液E2测定,评价不同冷冻复苏方案的效果。
研究方法
1研究对象
2008.7-12月共收集20例因卵巢疾病而行腹腔镜手术的患者剥离的卵巢囊肿壁上残留组织,利用患者手术切除的废弃组织进行研究,该研究通过伦理委员会审核,并经患者知情同意。将收集到的卵巢组织切成约lxlxlmm3的组织块待用。
2研究内容
2.1卵巢组织冷冻及复苏：通过采用不同浓度的DMSO和EG制备两组玻璃化试剂(低浓度Ⅰ组,高浓度Ⅱ组)。将卵巢组织块分别在平衡液中和两种玻璃化冷冻液中浸泡暴露一定的时间后,再分别将卵巢组织块通过微滴法、麦管法和SSV法进行玻璃化冷冻,快速复温后进行组织学检查及体外培养。
2.2复苏后人卵巢组织体外培养：定期收集组织培养上清液进行E2测定,14d后捞出卵巢组织块,固定染色行组织学检测。
2.3人卵巢组织冷冻复苏效果判断：
2.3.1冷冻前后卵巢组织中卵泡形态学分析：在卵巢组织块冷冻前后和体外培养14d后,观察各组卵巢组织结构及卵泡形态变化,计数正常形态和异常改变的卵泡比率,检测卵巢皮质内卵泡密度与分布以及各级卵泡的比例变化。
2.3.2冻融卵巢组织各种细胞凋亡情况：采用DNA断裂原位末端标记法检测(Tunel)试剂盒,从DNA水平上检测冻融后卵巢组织内各种细胞凋亡情况。
2.3.3卵巢组织体外培养上清液中雌二醇的检测：每48小时采集卵巢组织体外培养上清液,离心后保存在-80℃的冰箱中,以RIA法测定上清液中E2水平。
结果
1冷冻前后人卵巢组织中卵泡形态及各级卵泡比例变化
DMSO+EG高浓度配伍Ⅱ组原始卵泡正常形态率(43.7%)较新鲜对照组(75.5%)及低浓度Ⅰ组(69.4%)明显下降,三组间各级卵泡所占比例无明显差异,均以始基卵泡比例最高。
2冷冻复苏后人卵巢组织结构形态学变化
新鲜人卵巢组织间质细胞排裂致密而规则,卵巢皮质中间质细胞紧密包裹各级卵泡。经两种不同配伍比例的玻璃化冷冻液处理并分别采用三种载体冷冻保存的人卵巢组织复苏后对始基卵泡影响不明显,但可见卵巢组织内部分间质细胞排列紊乱,疏松甚至出现分离。
3人卵巢组织各类细胞冷冻复苏后凋亡情况
冻融后人卵巢组织中各级卵泡中都没有出现阳性表达的棕色或棕褐色凋亡细胞；而卵巢组织内间质细胞的细胞核内发现有凋亡细胞。经单因素方差分析,玻璃化冷冻各组卵巢组织内间质细胞凋亡率与新鲜对照组相比,差异无统计学意义。
4各冷冻组复苏后卵巢组织体外培养液中E2的变化
低浓度配伍Ⅰ组各载体组卵巢组织培养液中E2的分泌水平均高于高浓度配伍组Ⅱ各载体组,其中SSV法载体间差异更显著,而各冷冻组内载体间差异无统计学意义。
结论
1.人卵巢组织内始基卵泡对冷冻损伤的耐受性高于初级卵泡。
2.低浓度的冷冻保护剂配合SSV法玻璃化冷冻方案对保存人卵巢组织的效果较佳。
第四部分：人卵巢组织异种异位移植的实验研究
研究目的
在前期研究基础上,采用低浓度比DMSO+EG做为冷冻保护剂,通过SSV法对人卵巢组织进行玻璃化冷冻复苏,并将人卵巢复苏组织块多点移植至去势裸鼠颈部皮下,观察接受移植的裸鼠阴道脱落细胞学、血E2分泌及子宫形态学变化,以判断冷冻效果。
研究方法
1研究对象及分组
2009.11～2010.3,收集20例因卵巢疾病而行腹腔镜手术的患者剥离的卵巢囊肿壁上残留组织,利用患者手术切除的废弃组织进行冷冻复苏及异体移植研究,并在短期内处死移植裸鼠进行观察,对冷冻及移植效果进行评价,未对患者造成任何伤害,该研究通过伦理委员会审核,并经患者知情同意。将收集到的卵巢组织切成约1×1×1mm3的小块待用。移植宿主采用健康性成熟Nu/Nu裸鼠,按是否移植分为四组(正常对照组、新鲜移植组、冻存移植组、去势组),后三组裸鼠切除双侧卵巢去势。
2研究内容
2.1冷冻及复苏：采用低浓度比DMSO+EG通过SSV法对卵巢组织进行玻璃化冷冻,快速复温法进行复苏。
2.2卵巢组织移植方法：分别将新鲜及冻融复苏后人卵巢组织多点移植于相应组别裸鼠左侧颈部皮下。
2.3观察指标：
2.3.1裸鼠阴道脱落细胞学观察：移植术后第5天起每日对裸鼠阴道细胞学检测观察动情周期。
2.3.2裸鼠血雌激素水平测定：分别于动情期采集移植组裸鼠血测定血清E2水平,判定卵巢功能情况；去势裸鼠于处死前采血检测。
2.3.3裸鼠子宫形态观察及移植物收集：甲醛固定切除的各组裸鼠子宫,切片染色后于光镜下观察子宫内膜腺体和间质的形态；切开移植组裸鼠颈部移植部位寻找卵巢组织移植物。
结果
1移植后裸鼠动情周期恢复情况
正常对照组阴道细胞呈持续规律的动情周期性变化,新鲜移植组中4/9裸鼠恢复动情周期,冷冻移植组3/9裸鼠恢复动情周期；去势组阴道有核上皮细胞和角化细胞逐渐消失,白细胞大量增加,脱落细胞未见周期性变化。新鲜移植组裸鼠首次恢复动情周期时间[(19.0±1.6)d]稍早于冷冻移植裸鼠[(22.3±1.2)d],但两组动情周期持续天数[(5.0±0.8)、(5.8±1.2)d]与正常对照组[(4.8±0.7)d]比较,差异无显著性意义。
2移植组裸鼠子宫形态学变化
移植组小鼠子宫增粗红润,弹性好,与正常对照组相似。移植组与正常组裸鼠子宫湿重明显重于去势对照组。光镜下观察,正常对照组及移植组裸鼠子宫内膜间质疏松,内膜腺体粗大、量多,腺细胞呈高柱状,胞浆丰富,有假复层上皮细胞。与之相反,去势组裸鼠子宫变细变薄,外观苍白,内膜萎缩变薄,间质致密,腺体数量少,腺腔较小,腺细胞呈静止期表现。
恢复动情周期的移植组裸鼠虽有动情周期出现并有与正常对照组相近的E2分泌水平,但颈部移植部位却未寻找到明显移植物。
3裸鼠血清平均E2水平
新鲜与冷冻移植组有动情周期的裸鼠血清平均E2水平与正常对照组无明显差异,均显著高于去势裸鼠组。
结论
1.采用低浓度比DMSO+EG冷冻剂SSV法玻璃化冷冻人卵巢组织,复苏后行异体异位移植可获得与新鲜卵巢组织移植相同的效果。
2.人卵巢组织异体异位裸鼠颈部皮下无血管吻合移植能够存活并恢复裸鼠动情周期及内分泌功能。
With the development of society, the improvement of people's living standards and increasing concerning of their own health, much more critical demands on the preservation of women's reproductive capacity are proposed recently. This has greatly promotes the development and deeper of the study on the cryopreserved technology of human ovary tissue and clinical application. A study of World Health Organization (WHO) found that700thousand people of1.4million newly increased cancer patients were women in2008. And it will lead to the loss of young women's reproductive by large dose of radiotherapy, chemotherapy and ovarian surgery. However, cryopreserved ovarian tissue can provide a kind of compensation method for those patients, because of keeping large number of follicles. In early1950s, Parkes and other scholars using the method of slow freezing to make the rats ovarian tissue slice stored in-79℃glycerol mixture, and carried on the autologous transplantation after rapid thaw recovery. Then the growth and development of follicle in the transplantation ovarian tissue was observed. In1990s, along with the development and improvement of science and technology as well as a variety of antifreeze, the freezing technology of ovarian tissue has been developing rapidly, Many experimental study about ovarian tissue frozen of mice, sheep and non-human mammals was reported Constantly, as well as successful reports about freezing and thawing ovary tissue transplant. Sheep ovaries is similar with mankind no matter in the ovarian tissue structure or the ovulation cycle. Therefore the successful pregnancy after freezing thawing and transplantation of ovary tissue in sheep provide a meaningful model for technology development and application of human ovarian tissue freezing and thawing, and laid the solid experimental basis. Donnez J carried on autologous transplantation by laparoscopic surgery for a Hodgkin's lymphoma patient whose ovarian tissue has been frozen for six years,after six months, the patient restored the menstrual cycle, cyclical ovulation and corpus luteum generation.11months after surgery, HCG determination and ultrasonic confirmed that patients with successful pregnancy and a smooth delivery. Ovarian tissue freezing technology in our country is still in the exploratory stage, Zijiang Chen has gained some experience in frozening human ovarian cortex, studing at follicle morphology and ovarian cortex tissue in vitro culture.
The advantage of ovarian cryopreservation technology is freezing the immature cortical primordium follicles, which have the character of relatively quiet, small in size and lack oolemma and cortical granule, also tolerate the damage of freeze and recovery. Cryopreserved ovarian tissue has abundant primate follicles, complete structure, energy and viability. Successful cryopreservation and reviving will open up a new treatment way for the patiens with premature ovarian failure and malignancy. In order to survive threatened and endangered animals and to maintain gene diversity, gene group stock centers around the world are now collecting and freezing the ovarian tissue from these animals.
About human ovarian cryopreservation and recovery, there are still many problems to be resolved, although ovarian tissue cryopreservation technologies in rodents and large primates have already born offspring and achieved stage research. The application of this technology in clinical is difficult. The main difficulty is to establish the best frozen measure. Many factors will influence the effect of ovarian cryopreservation technology, including cryoprotectant choose, seeding temperature, thickness of tissue slice and process of freeze and thaw, the skill level of the operator, hardware conditions, etc. There are no best appropriate frozen measure for less injury now.
The key of recovery reproductive capacity is survival situation of ovarian tissue after transplantation. Firstly, partial reperfusion injury after transplantation. In the autologous transplantation model experiments of sheep ovarian tissue, Aubard and some scholars found there was only5%primordial follicle alive after transplantation in their experiment. It is likely because ovary lack vascular anastomosis and has no its own blood supply after transplantation. It needs at least48h to revascularization. During this time it has caused irreversible damage to ovarian, so how to protect the transplanted tissue out of hypoxic-ischemic damage after freezing and thawing is a problem which should be solved quickly. Secondly, to find the best transplantation site. So far, it is unknown where is the best site, however what should be clear is that the site should has fine live condition for tissue and be easy for follicle check and puncture oocyte retrieval. At last, solution of the problem of security after the transplantation of ovary tissue with autologous. With the development of frozen and transplant of ovary tissue, it brings new hope to the cancer patients who want to restore its own ovarian function and preservation of fertility, but it still has a certain risk about the transplantation of ovary tissue. Freezing and thawing ovarian tissue allograft mainly in order to save the fertility of cancer patients, some scholars have questioned that transplant the frozen ovarian tissue containing the tiny cancer lesions will lead the patiens to the occurrence of disseminated tumor cells after transplantation or recurrence. The experimental study by Mueller A,etc. found that the rat after heterotopic transplantation, the occurrence of a few of the sex cord-stromal tumors rate of100%. Dror and other studies have shown that using RT-PCR can detection the ovarian tissue remnants of small lesions of cancer patients sensitively, thereby enhancing the safety of the transplant. Before transplantation, using advanced detection methods, such as polymerase chain reaction (PCR), FISH technology, gene chip technology, detect the ovarian tissue remnants of small lesions to reduce tumor cell metastasis risk of ovarian transplant, benefit to improve the effectiveness and safety of ovarian tissue transplant.
The samples of ovarian tissue we used to study cry op reservation, recovery and transplantation are mostly obtained from the patients who need to carry on gynecological operation because of ovary disease. Because of their age are generally large, whole flock ovarian follicle in some region of ovary was exhausted and combined with ovary pathological changes such as endometrial implantation cyst, sufficient samples are hardly to obtain. Moreover ovarian cryopreservation, revive and transplantation all related to ethic problems, so the number of the samples were limited. It is proved that there are no obvious difference between species about primordial follicles. This article chose the ovary tissue of rabbit to study in the early stage, because of their popular prices, broad sources, short breeding cycle and the similar size and the minuteness structure of the meiosis ovary. Basic to the early study, this article did a research on the human ovarian cryopreservation, revive and transplantation which come off the ovary operation.
Our research study includes four stages:The experimental studies of the frozen and thawed of rabbit ovarian tissue; The experimental study of heterologous ectopic transplantation of rabbit ovarian tissue; The experimental study of frozen and thawed of human ovarian; The experimental study of heterologous ectopic transplantation of human ovarian tissue. We used different frozen procedure (including rapid vitrification, slow-freezing protocols), different frozen carrier (vasculature, microsphere and hard surface method), different frozen reagent and compatibility (DMSO, EG, PROH) to study ovary tissue of human and rabbit in frozen-thawed, in vitro culture, allotransplantation etc. In order to find appropriate ovary tissue frozen-thawed technology and transplantation site, to provide experimental data for developing human ovarian tissue frozen and transplantation, to make basement for founding ovarian tissue frozen bank.
Part I:The experimental studies of the frozen and thawed of rabbit ovarian tissue
Purpose
Application PROH, DMSO and EG alone or compatibility in slow programmed freezing or vitrification to frozen and recovery the rabbit ovarian tissue, then study the follicle shape of ovarian tissue, c-kit, ki67and MHC-Ⅱ expression, in order to determine a variety of frozen rabbit ovarian tissue morphology, cell proliferation activity, ovarian tissue antigenicity and immunogenicity to explore relatively suitable for freezing ovarian tissue freezing recovery program.
Methods and results
1The effect of4different frozen methods on rabbit ovarian morphology
1.1Objects and groups:Choose12healthy female Japanese white rabbits with big ear. The rabbits are randomly divided into4groups, laparotomy after anesthesia to take ovarian tissue and diced, according to group to take rabbits ovarian tissue blocks with slowly programmed freezing recovery (two subgroups, respectively using PROH and DMSO as frozen liquid, labeled A2and B2)), and rapid vitrification frozen recovery (two subgroups, respectively DMSO+PROH, labeled C2and DMSO+EG, labeled D2), frozen and thawed ovarian tissue; Prior to freezing, take a small amount blocks of fresh ovarian tissue from each group, as controls group.
1.2Contents:By histological examination of the ovarian tissue, observing ovarian tissue follicles proportion morphological structure and normal rate in the fresh control and recovery.
1.3Results
1.3.1The characteristics of fresh ovarian tissue of rabbits morphological:Slices of fresh ovarian tissue blocks from rabbits, the total number of follicles are1236under the microscope in the slices of fresh ovarian tissue blocks from rabbits, which accounted for88.2%of primordial follicles and the normal form rate of primordial follicles are all the highest91.4%,, the primary follicles8.4%, the secondary follicles accounted for only33.4%. Follicles are954which accounting from the three set of frozen-thawed ovarian tissue slice,857of them are primordial follicles account for89.8%, the primary follicles7.3%, the secondary follicles accounted for only2.8%.
1.3.2Follicles morphological changes in rabbit ovarian tissue after different freezing program and refrigerant:In frozen group, slowly programmed freezing PROH group of primordial follicles has the highest normal rate of80.1%, significantly higher than the programmed DMSO group (70.5%), rapid vitrification DMSO+PROH group (67.6%) and rapid vitrification DMSO+EG group (69.7%), the difference has statistically significant; make a calculation about all frozen groups at all levels of follicle, primordial follicles with normal morphology (72.7%) was significantly higher than the primary follicles morphologically normal rate (55.7%), due to the total number of secondary follicles is so exile, statistical analysis isn't be implemented.
1.3.3The effect of frozen to rabbit ovarian tissue structure:Observing the rabbit fresh ovarian tissue under the microscope to observe the rabbit fresh ovarian tissue,we can see the follicles packed by row close rules ovarian stromal cells tightly (Fig1-1). All the frozen resuscitation group ovarian tissue can visible the damage which mainly are primary follicles and secondary follicles. These follicles are isolated with surrounding stromal cells,but primordial follicles are less affected; interstitial cells in various frozen ovaries are disorders and the connection is loose and formation of cracks (Fig.1-2～1-6)
2Effects of slow-frozen method and vitrification of value-added activity of rabbit ovarian follicle cells
2.1Objects and groups:9healthy female Japanese white rabbits aged4-5month were chosed. The rabbits are randomly divided into3groups (fresh control group, PROH slow-freezing-thawing, DMSO+EG Rapid vitrification group). Get ovarian tissue as previously described, diced standby.
2.2Contents
2.2.1The expression of rabbit ovarian tissue MHC-2antigen:the variation of the two sets of frozen rabbit ovarian tissue MHC-2antigen expression was detected by SP immunohistochemical and judge the impact of frozen to the ovarian tissue antigen through observe the average absorbance value of the MHC-2antigen.
2.2.2Effect of freezing and thawing to Proliferation activity of all follicle cells: Separation the follicles in the fresh control and freezing/thawing ovarian tissue, cultured in vitro, then transferred to a liquid scintillation vial machine to measure cpm to observe the effect of freezing to all levels of follicle cells proliferative activity.
2.3Results
2.3.1The effect of procedures and vitrification to MHC-2expression of rabbit ovarian tissue:Immunohistochemistry results show that MHC-2mainly expressed in ovarian interstitial cells, secondary follicles oocytes, oocytes and granulosa cells which in primordial follicles and primary follicles had no expression; Vitrification DMSO+EG group compared with program PROH group and the control group-of ovarian tissue MHC-antigen,average absorbance values significantly lower; PROH group has no statistically significant in absorbance value compared to the control group.
2.3.1The effect to proliferative activity of rabbits follicle cells:After culture of follicle cells in vitro with program PROH and vitrification DMSO+EG frozen program, use thymidine marked by3H incorporation test to study the proliferative activity and found that DMSO+EG vitrification frozen program and program PROH has no statistically significant in cpm value differences with fresh group of small OGC, and then the cpm value of the big OGC significantly lower; there are no statistically significant between them.
3The effect of different vitrification carriers to c-kit and ki67expression of rabbit ovarian tissue
3.1Objects and Group:12healthy female Japanese white rabbits were chose,,according to different vitrification frozen carriers randomly divided into4groups:Control group(A), straw method group(B), microsphere group(C), SSV group(D). Taking ovary tissue as what mentioned before, make the ovarian tissue diced stand.
3.2Contents:According to observe the frozen-thawed ovarian tissue histology and test the expression of frozen-thawed ovary tissue c-kit and ki67which adopt immunohistochemical method, to judge the impact of frozen to ovarian tissue proliferation activity and development potential.
3.3Results:The effect of the expression of c-kit and Ki67in rabbit ovary tissue with different vitrification frozen carrier, the immunohistochemistry results demonstrated that the ki67protein in all levels of follicular oocytes membrane and/or cytoplasm ki67protein in rabbit ovarian tissue both showed positive expression in brown or tan,. Be frozen by straw, microsphere, SSV method,the expression rate of Ki67and c-kit has no statistically significant compared with the fresh group.
Conclusion:
1. The method of PROH slow programmed freezing of rabbit ovarian tissue achieved the higher primordial follicles normal morphology and cell proliferative activity, may be a better freezing methods
2. Vitrification method can reduce the primordial follicles morphologically normal rate,make primordial follicles good proliferative activity, and rabbit ovarian tissue immunogenicity is decreased significantly. And the vitrification method is simple, economical and practical, possible be a future research directions
3. There were no significant influence in the form of the structural integrity of small follicles cell viability about the freezing of PROH programmed and DMSO+EG vitrification.
Part Ⅱ:The experimental study of heterologous ectopic transplantation of rabbit ovarian tissue
Purpose
After heterotopic transplantation of the fresh and vitrification ovarian tissue into the neck of the castrated nude mice subcutaneously, to observe the nude mice estrous cycle hormone secretion, changes of mice uterine morphology and transplanted tissues, in order to evaluate the effect of freezing.
Methods
1Material and groups
We choose4cleanly female Japanese big ear white rabbits,and cut their ovarian tissue into block, then divided into3groups to observe fresh tissue,fresh transplants, frozen and thawed transplant; Transplant host using8to10weeks of age, of SPF grade mature female Nu/Nu nude mice36.The mouse were divided to4groups which have9mouse. Except the control,3other groups were used for fresh tissue transplantation, freeze-thaw transplantation and castration control after castrated.
2Methods
2.1The freezing recovery method of ovarian tissue:DG+DMSO in lower concentration as cryoprotectant solution was used in the rapid recovery after ovarian tissue vitrification, SSV method be used as a carrier.
2.2Ovarian tissue transplant:After anesthetizing nude mice, we disinfected and cut the skin of nude mice on the left side of the neck, and forceps gripping transplanted rabbit ovarian tissue which was fresh multi-point or freezing and thawing subcutaneously, then suture and mark transplantation part.
2.3The evaluation of transplant effect of freezing recovery rabbit ovarian tissue
2.3.1The observation of vaginal exfoliative cytology about Nude mice:observing the changes about the vaginal smear epithelial cells of nude mice to determine estrous cycle and its duration.
2.3.2The detection of estrogens in nude mice's blood:Both normal control group and the two transplant groups of nude mice have estrous cycle, the castration that not transplant group don't have estrous cycle. Testing serum estrogen (E2) level of each group respectively to determine the function of ovary.
2.3.3Uterine morphological observation and ovarian graft observation of Nude mouse:observing morphological changes of uterus removed from each group by the naked eye and microscope respectively; Cut the neck skin of the fresh and frozen ovary transplantation group to remove graft, then have histological observation.
Results
1Estrous cycle conditions of Nude mice:
Normal control group have regular estrous cycle;7/9of fresh transplant group have estrous cycle, while the frozen transplant group is6/9. There are no difference between the two transplanted group in recovering estrous cycle days, as well estrous duration between three groups.
2The detection of estrogens in nude mice blood:
Determining the E2levels in blood of all nude mice and ovariectomized which occur estrous cycle, there is no significant difference about the E2levels in blood between the two transplant group and the normal control group, but they were significantly higher than castrated group.
3The observation of uterus morphology about nude mice
Ovariectomized group'uterus displayed some special characters, such as atrophy, pale, thin endometrium, less glands, surface covered with cubic or simple squamous epithelial cells, which were significantly different with the other groups; then control group and transplanted group had a ruddy uterus with full shap thickness of coarse glands, the amount is more abundant cytoplasm of the glandular cells were high columnar or pseudostratified layer of the surface coated with a single layer of high columnar, endometrial epithelial cells.
4Morphological observation of the ovarian tissue graft
Removing subcutaneous graft from the nude mouse to be observed, it can be seen that most grafts grew into a pale yellow round or oval body, and its volume increased significantly. the surface of part transplanted tissue blocks were covered uneven beaded protrusions. Surviving transplants have rich capillaries by objective lens and follicles within the cortex
Conclusions
The low concentration of DMSO+EG vitrification method can save the structure and function of the ovarian tissue, fresh and freeze-thaw nude heterotopic ovarian tissue can live and maintain endocrine function after transplantation.
Part Ⅲ:The experimental study of frozen and thawed of human ovarian
Purpose
With different concentration of compatibility DMSO+EG as vitrification frozen protective agent, vitrified freeze ovarian tissue separately using straw method, microsphere method, SSV method, then observeing follicles'morphological change of human ovary tissue before and after frozen, apoptosis rate detection and E2determination in supernate of ovary tissue blocks cultivate in vitro after recovery, which help to evaluate the influence of different frozen-thawed methods.
Methods
1Objects
We collected20residual organization from patients' ovarian cyst wall, all of them accepted the laparoscopic surgery because of ovarian diseases, during2008.7～2008.12. The study used discarded ovarian organization came from of20patients, Signed consent form was obtained from each participant. The study protocols were approved by the Ethics Committee on Human Research. Cut the collected ovarian tissue into2x1x1mm3blocks.
2Contents
2.1Freezing and thawing of ovary tissue:using different concentration DMSO and EG to prepare two sets of vitrification agent(low concentration I group, high concentration II group). Steeping Ovarian tissue blocks separately into equilibrium liquid and two kinds of vitrification frozen agent for a period of time, then by means of straw method, microsphere method, SSV method to do vitrified frozen, rapid thawed method to recover human ovary tissue fragment.
2.2Human ovary tissue cultivation in vitro after recovery:regularly collecting tissue culture supernate and detecting E2level,14d later fished out ovarian tissue blocks and settled, coloured them then histology detection.
2.3Frozen recovery effect judgment of human ovary tissue:
2.3.1The morphological analysis of follicles of ovary tissue before and after frozen:before and after frozen and cultivation14d later in vitro, observing follicle density and distribution of tissue cortex and follicle morphological changes, count the ratio of Normal form and anomaly change, detect follicle density and distribution of tissue cortex of every group and proportion changes of level of follicles.
2.3.2Apoptosis situation of all kinds of cells in thawing ovarian tissue:using DNA in situ end labeling method, to detect apoptosis situation of all kinds cells in thawing ovarian tissue at DNA level.
2.3.3Estradiol detection of supernate culture in vitro:collect supernate of ovary tissue culture in vitro every48hours, keep it at-80℃after centrifugation. Detecting the level of E2in supernate by RIA method.
Results
1Follicles morphological change and proportion changes of each levels of follicle of human ovary tissue before and after frozen
The normal form rate (43.7%) of DMSO+EG high compatibility groups II group primordial follicles has a dramatic decline than fresh control group (75.5%) and low concentration group I group (69.4%). Among these three groups, the proportion of follicle of each levels has no significance difference, and the highest proportion is primordial follicles.
2Morphological changes of human ovary tissue structure in post-thaw
Interstitial cells of fresh ovary tissue arranged regularly and wrapped, follicles of each level. Two kinds of vitrification refrigerating fluid and use partly three carriers to cryopreserved human ovary tissue, there is no obvious influence on primordial follicles, but it is clear that part of the interstitial cells of ovary tissue arrange disorder, loose and even separate.
3Apoptosis of various cell of human ovary tissue after freezing and thawing
There are no brown or sepia apoptosis cells which are positive expressed in nucleus of interstitial cells. With one-way analysis of variance, there is no statistical significance on apoptotic rate of interstitial cells between vitrification and fresh control group.
4The changes of E2secretion of ovary tissue culture in vitro after frozen
In carriers groups of low concentration compatibility I group E2of ovary tissue secret higher than carriers groups of high concentration compatibility II groups, there is significant difference on SSV method between two carriers, while no significance among carriers of frozen groups.
Conclusions
1. The tolerance of primordial follicles to frozen damage is higher than primary follicles.
2. It is better for saving ovary tissue when use using low concentration frozen protective agent combined SSV vitrification frozen method.
Part IV:The experimental study of heterologous ectopic transplantation of human ovarian tissue
Purpose
On the basis of preliminary study, we used DMSO+EG with lower concentration as frozen protective agent, SSV method as vitrification freezing and thawing, heterotopia allograft to the Cervical subcutaneous of ovariectomized nude mice. To observe exfoliocytology of vagina, the secretion of E2in serum and morphology change of uterus to charge frozen effect.
Methods
1. Objects and groups
2009.11～2010.3,20cases of ovarian disease and line laparoscopic surgery stripping of ovarian cyst wall residual organization were collected to study. Through Ethics committee review and make patients informed consent. Cut the collected ovarian tissue into2×1×1mm3blocks. Transplantation host is healthy sexual maturity Nu/Nu nude mice, group them in accordance with whether to transplant into four: fresh transplantation group, cryopreservation group, ovariectomized group and common control group. The former three group cut Bilateral uterine.
2. Contents
2.1Freezing and thawing:use lower concentration DMSO+EG, vitrification frozen ovarian tissue by SSV method and reviving it by rapid thawing method for reviving.
2.2Transplantation method of ovarian tissue:fresh and frozen-thawed ovary tissue separately multipoint transplanted into the Cervical subcutaneous of ovariectomized nude mice in relevant group.
2.3Indicator for further observation:
2.3.1viginal exfoliocytology observation:observe estrous cycle from the fifth day after transplantation through viginal exfoliocytology.
2.3.2Estrogen level measurement:detecting blood serum E2level of transplanted group in estrus to charge the ovarian function; ovariectomized nude mice draw blood detection before death.
2.3.3Morphology observation and transplant collection of nude mice:settle removed nude ovary by methanal, stain sections and observe morphology of endometrial glands and mesenchyme in light microscope; cutting the transplanted site in neck of transplanted mice to find graft.
Results
1Recovery condition of oestrous cycle of transplanted nude mice
Control group keep regular estrogen periodic change,4/9of fresh transplanted group recovered oestrous cycle,3/9of frozen transplanted group recovered; in ovariectomized group epithelial cells with nuclear and keratinocyte fade away, white blood cells largely increased, cast-off cells did not seen cyclic variation. Frozen transplanted group Restore estrus cycle time for the first time [(22.33±1.20)d] later than fresh transplanted group [(19.00±1.58) d], but compare Estrus days of two groups [(5.67±1.15)d,(5.0±0.82) d] to common control group [(4.83±0.75) d], there was no significant difference(P>0.05).
2Uterine morphological changes of transplanted group
Transplanted group mice's uterine thickened and became ruddy, permanently resiliency, which was same to the common control group. There are no difference on uterine wet weight with transplanted group and control group, overweight than ovariectomized group obviously. By light microscopy transplanted group endometrial interstitial loose, endometrial glands bulky and abundance, gland cell is tall columnar, cytoplasm rich, false stratified epithelium cells, no significant difference with normal endometrial morphology.
Nude mice in ovariectomized control group uterine atrophy, become thin, colour etiolation, endometrial interstitial density. Endometrial atrophy thin, the quantity of glands is less, gland cavity smaller, gland cell is resting stage performance.
Though in transplanted group mice had similar E2secretion level with normal control group, no obvious grafts were found in neck graft sites.
3The average level of serum E2of nude mice
Fresh and frozen transplanted group have no significance difference in average level of serum E2, but both higher than ovariectomized control group.
Conclusions
1. Using low concentration ratio DMSO+EG cryogen, SSV vitrification freeze human ovarian tissue can got the same effect with fresh ovarian tissue transplantation.
2. It is proved that the transplantation of human ovarian tissue ectopic nude mouse cervical subcutaneous without vascular anastomosis can survive and recover nude mice oestrous cycle and endocrine function.
卵巢组织冷冻保存的优势在于其可冷冻卵巢皮质原位未成熟的始基卵泡,因其具有相对静止、体积小、缺乏透明带及皮质颗粒的特性,容易耐受冷冻及复苏造成的损伤,成功的卵巢组织冷冻和复苏,可保存卵巢组织中大量结构完整、具有生存活力及发育能力的原始卵泡,从而开辟了一条卵巢早衰和恶性肿瘤患者的治疗新途径。有效的卵巢冷冻保存及复苏技术是建立卵子库的基本前提,复苏后卵巢组织的成功移植对女性生育力的保存及恢复具有重要意义。为了拯救面临威胁和濒危动物,保存基因多样性,世界各地基因组储存中心正在收集和冷冻来自这些动物的卵巢组织。
卵巢冷冻保存及复苏还存在许多有待解决的问题,虽然在鼠类和大型灵长类动物中卵巢组织冷冻保存技术已有后代出生并获得了阶段性的研究成果,但用于人类卵巢组织的冷冻保存及复苏移植技术还存在许多问题,难以充分有效地的运用于临床,其关键难点包括确立最适宜的卵巢组织冷冻保存方案,因为冷冻过程中各个环节均有许多因素会影响卵巢组织的冻融效果,如组织切块的大小,所选择的冷冻方法、冷冻保护剂的种类及浓度、冷冻和复苏过程、操作人员的技术水平、硬件设备条件等,目前尚无定论的减少冷冻损伤的最佳冷冻方案。
卵巢组织复苏移植后的成活情况是决定生殖能力能否恢复的关键。影响移植成功的因素有很多,首先是移植后局部灌注损伤：Aubard等学者在羊卵巢组织自体移植模型实验中发现,只有5%的原始卵泡在移植后能够存活,分析原因可能是移植后的卵巢未行血管吻合,自身无血供,而移植局部的血管再生至少需要48小时,在此期间由于血供不足可能对卵巢造成了不可逆的损伤,如何保护移植组织不受缺血缺氧损害是卵巢组织冻融后移植亟待解决的一个问题；其次是最适宜移植部位的确立：迄今为止,国内外研究尚无最适宜卵巢组织移植的最佳部位定论,但能确认的是最适宜的移植部位必须具备能为组织的植入和成活发育提供良好条件、并且方便卵泡的检测和穿刺取卵的部位；最后是卵巢组织同种自体移植后的安全性问题的解决：卵巢组织的冷冻和移植的不断进步,为癌症患者恢复其自身卵巢功能和保存生育能力带来了新的希望。但卵巢组织移植仍然存在着一定的风险。冻融卵巢组织同种移植多数是为了对恶性肿瘤患者的生育能力进行保存,因此有学者质疑,移植恶性肿瘤患者的冷冻卵巢组织中是否存在微小癌灶,从而导致移植后肿瘤细胞发生播散或者再复发。MuellerA等实验研究发现大鼠卵巢经同种异位移植后,发生性索间质瘤的几率达100%。关于移植组织安全性的监测,Dror等研究表明采用RT-PCR(?)邕够敏感的检测到癌症患者卵巢组织内残存的微小病灶,从而提高移植的安全性。因此,在决定移植前,采用先进的检测手段如聚合酶链反应(PCR、FISH、基因芯片等技术,检测移植卵巢组织内残存的微小病灶,以降低卵巢移植所带来的肿瘤细胞转移风险,有利于提高卵巢组织移植的有效性和安全性。
关于卵巢组织冷冻、复苏、移植研究所采用的标本,人类卵巢组织冻存研究标本大多来自于因卵巢疾患而需要进行妇科手术的病人,由于她们年龄一般都比较大,卵巢中某些区域整群卵泡已经耗尽,同时合并有子宫内膜异位囊肿等卵巢病变,所以很难获得足够数量的卵泡标本,而且人类卵巢组织的冷冻、复苏、移植涉及诸多伦理问题,所以实验标本的获得非常珍贵而有限,实验数据不够充沛。有研究显示种属间卵巢原始卵泡没有明显差异,本实验前期选择家兔卵巢组织来进行研究,是由于其低廉的价格,广阔的来源,较短的繁殖周期,且人类与家兔卵巢始基卵泡中卵母细胞的大小及进入减数分裂的卵母细胞微细结构基本相似。在前期家兔卵巢冻存研究基础上,我们又对来自卵巢手术剥离的人类卵巢组织标本进行了冷冻、复苏和移植的研究。
本研究分四个部分,即家兔卵巢组织冷冻复苏的实验研究、家兔卵巢组织异种异位移植的实验研究、人卵巢组织冷冻复苏的实验研究、人卵巢组织异种异位移植的实验研究四个阶段。通过采用不同冷冻程序(慢速程序化法、快速玻璃化法)、不同冷冻载体(麦管法、微滴法、坚硬表面法)、不同冷冻试剂及配伍方法(二甲基亚砜DMSO、乙二醇EG、丙二醇PROH)等分别对家兔及人卵巢组织进行了冷冻、复苏、体外培养、异体移植的系统研究,以期寻找适宜的卵巢组织冷冻复苏方案及移植部位,为临床开展人类卵巢组织冷冻及移植提供实验数据参考,为建立卵巢组织冷冻库奠定基础。
第一部分：家兔卵巢组织冷冻复苏的实验研究
研究目的
采用不同冷冻保护剂(PROH、DMSO及EG)单独或联合应用于程序化或玻璃化法对家兔卵巢组织进行冷冻并复苏,观察复苏后卵巢组织中各级卵泡形态、卵巢组织Ⅱ型跨膜酪氨酸激酶受体(c-kit)和增殖细胞核抗原(ki67)以及主要组织相容性复合体类抗原(MHC-2)表达的变化,从而判断各种冷冻方案对家兔卵巢组织形态学、细胞增殖活性、卵巢组织抗原性的影响,以探讨相对适用于卵巢组织的冷冻复苏方案。
研究方法及结果
1四种不同冷冻复苏方法对家兔卵巢组织形态学的影响
1.1研究对象及分组：选择清洁级性成熟雌性日本大耳白兔12只,随机分为四组,麻醉后开腹取其卵巢组织并切成lxlxlmm3块,按随机方法分组将家兔卵巢组织块分别进行慢速程序化(分A2及B2两个亚组,冷冻剂分别采用PROH、 DMSO)及快速玻璃化(分C2及D2两个亚组,冷冻剂分别采用DMSO+PROH、 DMSO+EG)冷冻并复苏；冷冻前每组各取少量新鲜卵巢组织块做为对照组(A1、B1、C1、D1)。
1.2研究内容：通过卵巢组织学检测,观察新鲜对照组及各冷冻复苏组卵巢组织内各级卵泡比例、形态结构及正常形态率等变化。
1.3研究结果
1.3.1新鲜及冷冻复苏家兔卵巢组织形态学特点：来自各组家兔的新鲜卵巢组织切片中,显微镜下共计数各级卵泡1236枚,其中始基卵泡所占比率(88.2%)及其正常形态率(91.4%)均最高,初级卵泡及次级卵泡比率分别为8.4%和3.4%；计数来自三组冷冻复苏卵巢组织切片中的卵泡共954枚,其中始其卵泡857枚,占89.8%,初级卵泡占7.3%,次级卵泡仅占2.8%。
1.3.2采用不同冷冻程序和冷冻剂处理后家兔卵巢组织各级卵泡形态学的变化：冷冻组中,以PROH慢速程序化冷冻组始基卵泡的形态正常率最高,达80.1%,明显高于DMSO程序化组(70.5%)及两组玻璃化组(DMSO+PROH及DMSO+EG组分别为67.6%,69.7%),差异有统计学意义；将各冷冻组所观察到的各级卵泡合并计算,其中始基卵泡形态正常率(72.7%),明显高于初级卵泡的形态正常率(55.7%),因次级卵泡总数极少,所以未行统计学分析。
1.3.3冷冻复苏前后家兔卵巢组织结构变化：镜下观察冷冻前家兔卵巢组织内可见排裂规则的卵巢间质细胞紧密包裹的各级卵泡。所有冷冻复苏组卵巢组织均可见以初级卵泡和次级卵泡为主的受损情况,这些卵泡与周围间质细胞分离,但始基卵泡受影响较小；各冷冻组卵巢间质细胞排裂紊乱、连接疏松,形成裂隙。
2程序化及玻璃化冷冻对家兔卵巢抗原性及卵泡细胞增殖活性的影响
2.1研究对象及分组：选择9只4-5月龄清洁级性成熟雌性日本大耳白兔,随机分为3组(新鲜对照组、PROH慢速程序化冷冻组、DMSO+EG'决速玻璃化冷冻组),获取卵巢组织方法如前所述,切块待用。
2.2研究内容
2.2.1家兔卵巢组织相容性抗原(MHC-2)的表达：应用SP免疫组化法检测上述新鲜组及经两种冷冻方法处理后家兔卵巢组织MHC-2抗原表达的变化,通过观察MHC-2抗原的平均吸光度值,判断冷冻对卵巢组织抗原性的影响。
2.2.2冷冻复苏对各级卵泡细胞增殖活性的影响：分离新鲜对照组及冷冻复苏组卵巢组织内大、小卵泡(直径分别为100-150μm及300-500μm),对其进行体外培养后,采用3H标记的胸腺嘧啶核苷掺入试验(3H-TdR),移入液闪瓶中上机测其脉冲信号数(cpm),判断冷冻复苏对各级卵泡细胞增殖活性的影响。
2.3研究结果
2.3.1PROH程序化与DMSO+EG玻璃化冷冻法对家兔卵巢MHC-2表达的影响：免疫组化结果显示MHC-2主要表达于卵巢间质细胞和次级卵泡卵母细胞的胞膜,而始基卵泡及初级卵泡的卵母细胞和颗粒细胞均无表达；玻璃化冷冻组较程序化组和新鲜对照组卵巢组织MHC-2抗原的平均吸光度值明显降低；程序化组与新鲜对照组平均吸光度值无明显差异。
2.3.2PROH程序化与DMSO+EG玻璃化冷冻法对家兔卵泡细胞增殖活性的影响：通过3H-TdR掺入试验检测各组卵泡细胞增殖活性发现,程序化及玻璃化冷冻组与新鲜卵巢组小卵泡的cpm值差异无统计学意义,而冷冻组大卵泡的cpm值明显降低；两冷冻组间差异无统计学意义。
3.不同玻璃化冷冻载体对家兔卵巢组织c-kit和W67表达的影响
3.1研究对象及分组：选择12只4-5月龄清洁级性成熟雌性日本大耳白兔,按是否冷冻及玻璃化冷冻所选择的载体不同随机分为4组：新鲜对照组(A组)、麦管法玻璃化冷冻组(B组)、微滴法玻璃化冷冻组(C组)、坚硬表面法(SSV)玻璃化冷冻组(D组)。玻璃化冷冻试剂采用EG+DMSO;获取家兔卵巢组织方法如前所述,将卵巢组织切块待用。
3.2研究内容：通过对冻融卵巢组织的组织学观察,并采用免疫组化法检测冻融卵巢组织Ⅱ型跨膜酪氨酸激酶受体(c-kit)和增殖细胞核抗原(Ki67)表达,判断冷冻对卵巢组织增殖活性及发育潜能的影响。
3.3研究结果：采用免疫组化检测家兔新鲜及冻融卵巢组织,结果显示各级卵泡内卵母细胞的胞膜和/或胞浆中ki67蛋白均出现呈棕黄或棕褐色的阳性表达；与新鲜组相比较,经麦管法、微滴法、SSV法冷冻处理后Ki67和c-kit阳性率表达率差异无统计学意义。
结论
1. PROH慢速程序化冷冻方法用于家兔卵巢组织冷冻获得了较高的始基卵泡正常形态率及细胞增殖活性,可能是一种较好的冷冻方法。
2.玻璃化方法虽使始基卵泡形态正常率降低,但也能使始基及初级卵泡卵母细胞保持良好的增殖活性,并可降低家兔卵巢组织抗原性,可能会减轻复苏后卵巢组织移植的免疫排斥,加之该技术操作简便,费用低廉,可能会有良好的应用前景。
3.程序化冷冻及玻璃化冷冻对形态结构完整的小卵泡细胞增殖活力影响不明显,而大卵泡增殖活性明显下降。
第二部分：家兔卵巢组织异体异位移植的实验研究
研究目的
在前期研究基础上,选择低浓度配伍比的DMSO+EG做为冷冻保护剂,玻璃化冷冻家兔卵巢组织,将新鲜及冷冻复苏后卵巢组织块分别移植于去势裸鼠颈部皮下,观察接受移植后裸鼠动情周期、激素分泌、子宫形态变化及移植物变化,以评价冷冻效果。
研究方法
1研究对象及分组
选择清洁级性成熟雌性日本大耳白兔4只,取其卵巢组织处理后切块,分三组用于新鲜组织观察、新鲜移植、冷冻复苏移植；移植宿主采用8-10周龄的SPF级性成熟雌性Nu/Nu裸鼠36只,分4组(每组9只),除正常对照组外,其余三组均切除双侧卵巢去势,之后分别用于新鲜组织移植、冻融组织移植及去势对照组。
2研究方法
2.1卵巢组织冷冻复苏方法：采用低浓度比二甲基亚砜(DMSO)+乙二醇(EG)作为冷冻保护液,通过坚硬表面铜块(SSV)法对卵巢组织进行玻璃化冷冻后快速复苏。
2.2卵巢组织移植：麻醉裸鼠后,消毒并切开裸鼠左侧颈部皮肤,眼科镊分别夹取新鲜或冻融家兔卵巢组织多点移植于颈部皮下,缝合并标记移植部位。
2.3家兔卵巢组织冷冻复苏后移植的效果评价
2.3.1裸鼠阴道脱落细胞学观察：每天观察各组裸鼠阴道涂片上皮细胞变化以判断裸鼠动情周期恢复及持续时间。
2.3.2裸鼠血中雌激素测定：全部正常对照组及部分移植组裸鼠有动情周期出现；去势未移植组无动情周期。分别测定对照组、去势组及移植后出现动情周期的裸鼠血清雌激素(E2)水平,判定卵巢功能情况。
2.3.3裸鼠子宫形态学观察及移植物观察：移植35天后,切除各组裸鼠完整的子宫,分别于肉眼及镜下观察其形态学变化；切开新鲜及冷冻卵巢移植组裸鼠颈部皮肤,取出移植物,进行组织学观察。
研究结果
1裸鼠动情周期情况
正常对照组裸鼠全部有规律的动情周期；新鲜移植组9个裸鼠中7个出现动情周期,冷冻移植组9个中6个出现动情周期,两移植组裸鼠恢复动情周期天数无差异,正常及移植组动情周期持续天数无差异。
2裸鼠血中雌激素水平测定
测定全部出现动情周期的裸鼠和去势裸鼠血中E2水平,两移植组恢复动情周期的裸鼠血中E2水平与正常对照组无明显差异,均明显高于去势组。
3裸鼠子宫形态学观察
去势组裸鼠子宫明显有别于有动情周期的裸鼠子宫,呈萎缩、苍白状,内膜薄,腺体少,表面被覆立方或者单层扁平上皮细胞；而正常对照及移植组裸鼠子宫色泽红润而有弹性,形态饱满,内膜厚且腺体粗大、量多,腺细胞胞浆丰富,内膜表面被覆单层高柱状或假复层上皮细胞。
4卵巢组织移植物形态学观察
取出裸鼠颈部皮下移植物进行观察,新鲜移植组与冷冻移植组移植物肉眼可见大部分长成淡黄色圆形或椭圆体,体积增大明显,移植物柔软,部分移植组织块表面出现凹凸不平的串珠状突起。存活的移植物镜下可见毛细血管生成,皮质内可见到各级卵泡及闭锁卵泡。
结论
采用低浓度DMSO+EG玻璃化冷冻法能够较好地保存卵巢组织的结构和功能,新鲜及冻融后的家兔卵巢组织裸鼠异种异位移植后能够存活,并具有内分泌功能。
第三部分：人卵巢组织冷冻复苏的实验研究
研究目的
以不同浓度配伍的DMSO+EG做为玻璃化冷冻保护剂,分别以麦管法、微滴法及SSV法对人卵巢组织进行玻璃化冷冻,通过观察冷冻前后人卵巢组织中卵泡形态学变化、细胞凋亡率检测及复苏后卵巢组织体外培养上清液E2测定,评价不同冷冻复苏方案的效果。
研究方法
1研究对象
2008.7-12月共收集20例因卵巢疾病而行腹腔镜手术的患者剥离的卵巢囊肿壁上残留组织,利用患者手术切除的废弃组织进行研究,该研究通过伦理委员会审核,并经患者知情同意。将收集到的卵巢组织切成约lxlxlmm3的组织块待用。
2研究内容
2.1卵巢组织冷冻及复苏：通过采用不同浓度的DMSO和EG制备两组玻璃化试剂(低浓度Ⅰ组,高浓度Ⅱ组)。将卵巢组织块分别在平衡液中和两种玻璃化冷冻液中浸泡暴露一定的时间后,再分别将卵巢组织块通过微滴法、麦管法和SSV法进行玻璃化冷冻,快速复温后进行组织学检查及体外培养。
2.2复苏后人卵巢组织体外培养：定期收集组织培养上清液进行E2测定,14d后捞出卵巢组织块,固定染色行组织学检测。
2.3人卵巢组织冷冻复苏效果判断：
2.3.1冷冻前后卵巢组织中卵泡形态学分析：在卵巢组织块冷冻前后和体外培养14d后,观察各组卵巢组织结构及卵泡形态变化,计数正常形态和异常改变的卵泡比率,检测卵巢皮质内卵泡密度与分布以及各级卵泡的比例变化。
2.3.2冻融卵巢组织各种细胞凋亡情况：采用DNA断裂原位末端标记法检测(Tunel)试剂盒,从DNA水平上检测冻融后卵巢组织内各种细胞凋亡情况。
2.3.3卵巢组织体外培养上清液中雌二醇的检测：每48小时采集卵巢组织体外培养上清液,离心后保存在-80℃的冰箱中,以RIA法测定上清液中E2水平。
结果
1冷冻前后人卵巢组织中卵泡形态及各级卵泡比例变化
DMSO+EG高浓度配伍Ⅱ组原始卵泡正常形态率(43.7%)较新鲜对照组(75.5%)及低浓度Ⅰ组(69.4%)明显下降,三组间各级卵泡所占比例无明显差异,均以始基卵泡比例最高。
2冷冻复苏后人卵巢组织结构形态学变化
新鲜人卵巢组织间质细胞排裂致密而规则,卵巢皮质中间质细胞紧密包裹各级卵泡。经两种不同配伍比例的玻璃化冷冻液处理并分别采用三种载体冷冻保存的人卵巢组织复苏后对始基卵泡影响不明显,但可见卵巢组织内部分间质细胞排列紊乱,疏松甚至出现分离。
3人卵巢组织各类细胞冷冻复苏后凋亡情况
冻融后人卵巢组织中各级卵泡中都没有出现阳性表达的棕色或棕褐色凋亡细胞；而卵巢组织内间质细胞的细胞核内发现有凋亡细胞。经单因素方差分析,玻璃化冷冻各组卵巢组织内间质细胞凋亡率与新鲜对照组相比,差异无统计学意义。
4各冷冻组复苏后卵巢组织体外培养液中E2的变化
低浓度配伍Ⅰ组各载体组卵巢组织培养液中E2的分泌水平均高于高浓度配伍组Ⅱ各载体组,其中SSV法载体间差异更显著,而各冷冻组内载体间差异无统计学意义。
结论
1.人卵巢组织内始基卵泡对冷冻损伤的耐受性高于初级卵泡。
2.低浓度的冷冻保护剂配合SSV法玻璃化冷冻方案对保存人卵巢组织的效果较佳。
第四部分：人卵巢组织异种异位移植的实验研究
研究目的
在前期研究基础上,采用低浓度比DMSO+EG做为冷冻保护剂,通过SSV法对人卵巢组织进行玻璃化冷冻复苏,并将人卵巢复苏组织块多点移植至去势裸鼠颈部皮下,观察接受移植的裸鼠阴道脱落细胞学、血E2分泌及子宫形态学变化,以判断冷冻效果。
研究方法
1研究对象及分组
2009.11～2010.3,收集20例因卵巢疾病而行腹腔镜手术的患者剥离的卵巢囊肿壁上残留组织,利用患者手术切除的废弃组织进行冷冻复苏及异体移植研究,并在短期内处死移植裸鼠进行观察,对冷冻及移植效果进行评价,未对患者造成任何伤害,该研究通过伦理委员会审核,并经患者知情同意。将收集到的卵巢组织切成约1×1×1mm3的小块待用。移植宿主采用健康性成熟Nu/Nu裸鼠,按是否移植分为四组(正常对照组、新鲜移植组、冻存移植组、去势组),后三组裸鼠切除双侧卵巢去势。
2研究内容
2.1冷冻及复苏：采用低浓度比DMSO+EG通过SSV法对卵巢组织进行玻璃化冷冻,快速复温法进行复苏。
2.2卵巢组织移植方法：分别将新鲜及冻融复苏后人卵巢组织多点移植于相应组别裸鼠左侧颈部皮下。
2.3观察指标：
2.3.1裸鼠阴道脱落细胞学观察：移植术后第5天起每日对裸鼠阴道细胞学检测观察动情周期。
2.3.2裸鼠血雌激素水平测定：分别于动情期采集移植组裸鼠血测定血清E2水平,判定卵巢功能情况；去势裸鼠于处死前采血检测。
2.3.3裸鼠子宫形态观察及移植物收集：甲醛固定切除的各组裸鼠子宫,切片染色后于光镜下观察子宫内膜腺体和间质的形态；切开移植组裸鼠颈部移植部位寻找卵巢组织移植物。
结果
1移植后裸鼠动情周期恢复情况
正常对照组阴道细胞呈持续规律的动情周期性变化,新鲜移植组中4/9裸鼠恢复动情周期,冷冻移植组3/9裸鼠恢复动情周期；去势组阴道有核上皮细胞和角化细胞逐渐消失,白细胞大量增加,脱落细胞未见周期性变化。新鲜移植组裸鼠首次恢复动情周期时间[(19.0±1.6)d]稍早于冷冻移植裸鼠[(22.3±1.2)d],但两组动情周期持续天数[(5.0±0.8)、(5.8±1.2)d]与正常对照组[(4.8±0.7)d]比较,差异无显著性意义。
2移植组裸鼠子宫形态学变化
移植组小鼠子宫增粗红润,弹性好,与正常对照组相似。移植组与正常组裸鼠子宫湿重明显重于去势对照组。光镜下观察,正常对照组及移植组裸鼠子宫内膜间质疏松,内膜腺体粗大、量多,腺细胞呈高柱状,胞浆丰富,有假复层上皮细胞。与之相反,去势组裸鼠子宫变细变薄,外观苍白,内膜萎缩变薄,间质致密,腺体数量少,腺腔较小,腺细胞呈静止期表现。
恢复动情周期的移植组裸鼠虽有动情周期出现并有与正常对照组相近的E2分泌水平,但颈部移植部位却未寻找到明显移植物。
3裸鼠血清平均E2水平
新鲜与冷冻移植组有动情周期的裸鼠血清平均E2水平与正常对照组无明显差异,均显著高于去势裸鼠组。
结论
1.采用低浓度比DMSO+EG冷冻剂SSV法玻璃化冷冻人卵巢组织,复苏后行异体异位移植可获得与新鲜卵巢组织移植相同的效果。
2.人卵巢组织异体异位裸鼠颈部皮下无血管吻合移植能够存活并恢复裸鼠动情周期及内分泌功能。
With the development of society, the improvement of people's living standards and increasing concerning of their own health, much more critical demands on the preservation of women's reproductive capacity are proposed recently. This has greatly promotes the development and deeper of the study on the cryopreserved technology of human ovary tissue and clinical application. A study of World Health Organization (WHO) found that700thousand people of1.4million newly increased cancer patients were women in2008. And it will lead to the loss of young women's reproductive by large dose of radiotherapy, chemotherapy and ovarian surgery. However, cryopreserved ovarian tissue can provide a kind of compensation method for those patients, because of keeping large number of follicles. In early1950s, Parkes and other scholars using the method of slow freezing to make the rats ovarian tissue slice stored in-79℃glycerol mixture, and carried on the autologous transplantation after rapid thaw recovery. Then the growth and development of follicle in the transplantation ovarian tissue was observed. In1990s, along with the development and improvement of science and technology as well as a variety of antifreeze, the freezing technology of ovarian tissue has been developing rapidly, Many experimental study about ovarian tissue frozen of mice, sheep and non-human mammals was reported Constantly, as well as successful reports about freezing and thawing ovary tissue transplant. Sheep ovaries is similar with mankind no matter in the ovarian tissue structure or the ovulation cycle. Therefore the successful pregnancy after freezing thawing and transplantation of ovary tissue in sheep provide a meaningful model for technology development and application of human ovarian tissue freezing and thawing, and laid the solid experimental basis. Donnez J carried on autologous transplantation by laparoscopic surgery for a Hodgkin's lymphoma patient whose ovarian tissue has been frozen for six years,after six months, the patient restored the menstrual cycle, cyclical ovulation and corpus luteum generation.11months after surgery, HCG determination and ultrasonic confirmed that patients with successful pregnancy and a smooth delivery. Ovarian tissue freezing technology in our country is still in the exploratory stage, Zijiang Chen has gained some experience in frozening human ovarian cortex, studing at follicle morphology and ovarian cortex tissue in vitro culture.
The advantage of ovarian cryopreservation technology is freezing the immature cortical primordium follicles, which have the character of relatively quiet, small in size and lack oolemma and cortical granule, also tolerate the damage of freeze and recovery. Cryopreserved ovarian tissue has abundant primate follicles, complete structure, energy and viability. Successful cryopreservation and reviving will open up a new treatment way for the patiens with premature ovarian failure and malignancy. In order to survive threatened and endangered animals and to maintain gene diversity, gene group stock centers around the world are now collecting and freezing the ovarian tissue from these animals.
About human ovarian cryopreservation and recovery, there are still many problems to be resolved, although ovarian tissue cryopreservation technologies in rodents and large primates have already born offspring and achieved stage research. The application of this technology in clinical is difficult. The main difficulty is to establish the best frozen measure. Many factors will influence the effect of ovarian cryopreservation technology, including cryoprotectant choose, seeding temperature, thickness of tissue slice and process of freeze and thaw, the skill level of the operator, hardware conditions, etc. There are no best appropriate frozen measure for less injury now.
The key of recovery reproductive capacity is survival situation of ovarian tissue after transplantation. Firstly, partial reperfusion injury after transplantation. In the autologous transplantation model experiments of sheep ovarian tissue, Aubard and some scholars found there was only5%primordial follicle alive after transplantation in their experiment. It is likely because ovary lack vascular anastomosis and has no its own blood supply after transplantation. It needs at least48h to revascularization. During this time it has caused irreversible damage to ovarian, so how to protect the transplanted tissue out of hypoxic-ischemic damage after freezing and thawing is a problem which should be solved quickly. Secondly, to find the best transplantation site. So far, it is unknown where is the best site, however what should be clear is that the site should has fine live condition for tissue and be easy for follicle check and puncture oocyte retrieval. At last, solution of the problem of security after the transplantation of ovary tissue with autologous. With the development of frozen and transplant of ovary tissue, it brings new hope to the cancer patients who want to restore its own ovarian function and preservation of fertility, but it still has a certain risk about the transplantation of ovary tissue. Freezing and thawing ovarian tissue allograft mainly in order to save the fertility of cancer patients, some scholars have questioned that transplant the frozen ovarian tissue containing the tiny cancer lesions will lead the patiens to the occurrence of disseminated tumor cells after transplantation or recurrence. The experimental study by Mueller A,etc. found that the rat after heterotopic transplantation, the occurrence of a few of the sex cord-stromal tumors rate of100%. Dror and other studies have shown that using RT-PCR can detection the ovarian tissue remnants of small lesions of cancer patients sensitively, thereby enhancing the safety of the transplant. Before transplantation, using advanced detection methods, such as polymerase chain reaction (PCR), FISH technology, gene chip technology, detect the ovarian tissue remnants of small lesions to reduce tumor cell metastasis risk of ovarian transplant, benefit to improve the effectiveness and safety of ovarian tissue transplant.
The samples of ovarian tissue we used to study cry op reservation, recovery and transplantation are mostly obtained from the patients who need to carry on gynecological operation because of ovary disease. Because of their age are generally large, whole flock ovarian follicle in some region of ovary was exhausted and combined with ovary pathological changes such as endometrial implantation cyst, sufficient samples are hardly to obtain. Moreover ovarian cryopreservation, revive and transplantation all related to ethic problems, so the number of the samples were limited. It is proved that there are no obvious difference between species about primordial follicles. This article chose the ovary tissue of rabbit to study in the early stage, because of their popular prices, broad sources, short breeding cycle and the similar size and the minuteness structure of the meiosis ovary. Basic to the early study, this article did a research on the human ovarian cryopreservation, revive and transplantation which come off the ovary operation.
Our research study includes four stages:The experimental studies of the frozen and thawed of rabbit ovarian tissue; The experimental study of heterologous ectopic transplantation of rabbit ovarian tissue; The experimental study of frozen and thawed of human ovarian; The experimental study of heterologous ectopic transplantation of human ovarian tissue. We used different frozen procedure (including rapid vitrification, slow-freezing protocols), different frozen carrier (vasculature, microsphere and hard surface method), different frozen reagent and compatibility (DMSO, EG, PROH) to study ovary tissue of human and rabbit in frozen-thawed, in vitro culture, allotransplantation etc. In order to find appropriate ovary tissue frozen-thawed technology and transplantation site, to provide experimental data for developing human ovarian tissue frozen and transplantation, to make basement for founding ovarian tissue frozen bank.
Part I:The experimental studies of the frozen and thawed of rabbit ovarian tissue
Purpose
Application PROH, DMSO and EG alone or compatibility in slow programmed freezing or vitrification to frozen and recovery the rabbit ovarian tissue, then study the follicle shape of ovarian tissue, c-kit, ki67and MHC-Ⅱ expression, in order to determine a variety of frozen rabbit ovarian tissue morphology, cell proliferation activity, ovarian tissue antigenicity and immunogenicity to explore relatively suitable for freezing ovarian tissue freezing recovery program.
Methods and results
1The effect of4different frozen methods on rabbit ovarian morphology
1.1Objects and groups:Choose12healthy female Japanese white rabbits with big ear. The rabbits are randomly divided into4groups, laparotomy after anesthesia to take ovarian tissue and diced, according to group to take rabbits ovarian tissue blocks with slowly programmed freezing recovery (two subgroups, respectively using PROH and DMSO as frozen liquid, labeled A2and B2)), and rapid vitrification frozen recovery (two subgroups, respectively DMSO+PROH, labeled C2and DMSO+EG, labeled D2), frozen and thawed ovarian tissue; Prior to freezing, take a small amount blocks of fresh ovarian tissue from each group, as controls group.
1.2Contents:By histological examination of the ovarian tissue, observing ovarian tissue follicles proportion morphological structure and normal rate in the fresh control and recovery.
1.3Results
1.3.1The characteristics of fresh ovarian tissue of rabbits morphological:Slices of fresh ovarian tissue blocks from rabbits, the total number of follicles are1236under the microscope in the slices of fresh ovarian tissue blocks from rabbits, which accounted for88.2%of primordial follicles and the normal form rate of primordial follicles are all the highest91.4%,, the primary follicles8.4%, the secondary follicles accounted for only33.4%. Follicles are954which accounting from the three set of frozen-thawed ovarian tissue slice,857of them are primordial follicles account for89.8%, the primary follicles7.3%, the secondary follicles accounted for only2.8%.
1.3.2Follicles morphological changes in rabbit ovarian tissue after different freezing program and refrigerant:In frozen group, slowly programmed freezing PROH group of primordial follicles has the highest normal rate of80.1%, significantly higher than the programmed DMSO group (70.5%), rapid vitrification DMSO+PROH group (67.6%) and rapid vitrification DMSO+EG group (69.7%), the difference has statistically significant; make a calculation about all frozen groups at all levels of follicle, primordial follicles with normal morphology (72.7%) was significantly higher than the primary follicles morphologically normal rate (55.7%), due to the total number of secondary follicles is so exile, statistical analysis isn't be implemented.
1.3.3The effect of frozen to rabbit ovarian tissue structure:Observing the rabbit fresh ovarian tissue under the microscope to observe the rabbit fresh ovarian tissue,we can see the follicles packed by row close rules ovarian stromal cells tightly (Fig1-1). All the frozen resuscitation group ovarian tissue can visible the damage which mainly are primary follicles and secondary follicles. These follicles are isolated with surrounding stromal cells,but primordial follicles are less affected; interstitial cells in various frozen ovaries are disorders and the connection is loose and formation of cracks (Fig.1-2～1-6)
2Effects of slow-frozen method and vitrification of value-added activity of rabbit ovarian follicle cells
2.1Objects and groups:9healthy female Japanese white rabbits aged4-5month were chosed. The rabbits are randomly divided into3groups (fresh control group, PROH slow-freezing-thawing, DMSO+EG Rapid vitrification group). Get ovarian tissue as previously described, diced standby.
2.2Contents
2.2.1The expression of rabbit ovarian tissue MHC-2antigen:the variation of the two sets of frozen rabbit ovarian tissue MHC-2antigen expression was detected by SP immunohistochemical and judge the impact of frozen to the ovarian tissue antigen through observe the average absorbance value of the MHC-2antigen.
2.2.2Effect of freezing and thawing to Proliferation activity of all follicle cells: Separation the follicles in the fresh control and freezing/thawing ovarian tissue, cultured in vitro, then transferred to a liquid scintillation vial machine to measure cpm to observe the effect of freezing to all levels of follicle cells proliferative activity.
2.3Results
2.3.1The effect of procedures and vitrification to MHC-2expression of rabbit ovarian tissue:Immunohistochemistry results show that MHC-2mainly expressed in ovarian interstitial cells, secondary follicles oocytes, oocytes and granulosa cells which in primordial follicles and primary follicles had no expression; Vitrification DMSO+EG group compared with program PROH group and the control group-of ovarian tissue MHC-antigen,average absorbance values significantly lower; PROH group has no statistically significant in absorbance value compared to the control group.
2.3.1The effect to proliferative activity of rabbits follicle cells:After culture of follicle cells in vitro with program PROH and vitrification DMSO+EG frozen program, use thymidine marked by3H incorporation test to study the proliferative activity and found that DMSO+EG vitrification frozen program and program PROH has no statistically significant in cpm value differences with fresh group of small OGC, and then the cpm value of the big OGC significantly lower; there are no statistically significant between them.
3The effect of different vitrification carriers to c-kit and ki67expression of rabbit ovarian tissue
3.1Objects and Group:12healthy female Japanese white rabbits were chose,,according to different vitrification frozen carriers randomly divided into4groups:Control group(A), straw method group(B), microsphere group(C), SSV group(D). Taking ovary tissue as what mentioned before, make the ovarian tissue diced stand.
3.2Contents:According to observe the frozen-thawed ovarian tissue histology and test the expression of frozen-thawed ovary tissue c-kit and ki67which adopt immunohistochemical method, to judge the impact of frozen to ovarian tissue proliferation activity and development potential.
3.3Results:The effect of the expression of c-kit and Ki67in rabbit ovary tissue with different vitrification frozen carrier, the immunohistochemistry results demonstrated that the ki67protein in all levels of follicular oocytes membrane and/or cytoplasm ki67protein in rabbit ovarian tissue both showed positive expression in brown or tan,. Be frozen by straw, microsphere, SSV method,the expression rate of Ki67and c-kit has no statistically significant compared with the fresh group.
Conclusion:
1. The method of PROH slow programmed freezing of rabbit ovarian tissue achieved the higher primordial follicles normal morphology and cell proliferative activity, may be a better freezing methods
2. Vitrification method can reduce the primordial follicles morphologically normal rate,make primordial follicles good proliferative activity, and rabbit ovarian tissue immunogenicity is decreased significantly. And the vitrification method is simple, economical and practical, possible be a future research directions
3. There were no significant influence in the form of the structural integrity of small follicles cell viability about the freezing of PROH programmed and DMSO+EG vitrification.
Part Ⅱ:The experimental study of heterologous ectopic transplantation of rabbit ovarian tissue
Purpose
After heterotopic transplantation of the fresh and vitrification ovarian tissue into the neck of the castrated nude mice subcutaneously, to observe the nude mice estrous cycle hormone secretion, changes of mice uterine morphology and transplanted tissues, in order to evaluate the effect of freezing.
Methods
1Material and groups
We choose4cleanly female Japanese big ear white rabbits,and cut their ovarian tissue into block, then divided into3groups to observe fresh tissue,fresh transplants, frozen and thawed transplant; Transplant host using8to10weeks of age, of SPF grade mature female Nu/Nu nude mice36.The mouse were divided to4groups which have9mouse. Except the control,3other groups were used for fresh tissue transplantation, freeze-thaw transplantation and castration control after castrated.
2Methods
2.1The freezing recovery method of ovarian tissue:DG+DMSO in lower concentration as cryoprotectant solution was used in the rapid recovery after ovarian tissue vitrification, SSV method be used as a carrier.
2.2Ovarian tissue transplant:After anesthetizing nude mice, we disinfected and cut the skin of nude mice on the left side of the neck, and forceps gripping transplanted rabbit ovarian tissue which was fresh multi-point or freezing and thawing subcutaneously, then suture and mark transplantation part.
2.3The evaluation of transplant effect of freezing recovery rabbit ovarian tissue
2.3.1The observation of vaginal exfoliative cytology about Nude mice:observing the changes about the vaginal smear epithelial cells of nude mice to determine estrous cycle and its duration.
2.3.2The detection of estrogens in nude mice's blood:Both normal control group and the two transplant groups of nude mice have estrous cycle, the castration that not transplant group don't have estrous cycle. Testing serum estrogen (E2) level of each group respectively to determine the function of ovary.
2.3.3Uterine morphological observation and ovarian graft observation of Nude mouse:observing morphological changes of uterus removed from each group by the naked eye and microscope respectively; Cut the neck skin of the fresh and frozen ovary transplantation group to remove graft, then have histological observation.
Results
1Estrous cycle conditions of Nude mice:
Normal control group have regular estrous cycle;7/9of fresh transplant group have estrous cycle, while the frozen transplant group is6/9. There are no difference between the two transplanted group in recovering estrous cycle days, as well estrous duration between three groups.
2The detection of estrogens in nude mice blood:
Determining the E2levels in blood of all nude mice and ovariectomized which occur estrous cycle, there is no significant difference about the E2levels in blood between the two transplant group and the normal control group, but they were significantly higher than castrated group.
3The observation of uterus morphology about nude mice
Ovariectomized group'uterus displayed some special characters, such as atrophy, pale, thin endometrium, less glands, surface covered with cubic or simple squamous epithelial cells, which were significantly different with the other groups; then control group and transplanted group had a ruddy uterus with full shap thickness of coarse glands, the amount is more abundant cytoplasm of the glandular cells were high columnar or pseudostratified layer of the surface coated with a single layer of high columnar, endometrial epithelial cells.
4Morphological observation of the ovarian tissue graft
Removing subcutaneous graft from the nude mouse to be observed, it can be seen that most grafts grew into a pale yellow round or oval body, and its volume increased significantly. the surface of part transplanted tissue blocks were covered uneven beaded protrusions. Surviving transplants have rich capillaries by objective lens and follicles within the cortex
Conclusions
The low concentration of DMSO+EG vitrification method can save the structure and function of the ovarian tissue, fresh and freeze-thaw nude heterotopic ovarian tissue can live and maintain endocrine function after transplantation.
Part Ⅲ:The experimental study of frozen and thawed of human ovarian
Purpose
With different concentration of compatibility DMSO+EG as vitrification frozen protective agent, vitrified freeze ovarian tissue separately using straw method, microsphere method, SSV method, then observeing follicles'morphological change of human ovary tissue before and after frozen, apoptosis rate detection and E2determination in supernate of ovary tissue blocks cultivate in vitro after recovery, which help to evaluate the influence of different frozen-thawed methods.
Methods
1Objects
We collected20residual organization from patients' ovarian cyst wall, all of them accepted the laparoscopic surgery because of ovarian diseases, during2008.7～2008.12. The study used discarded ovarian organization came from of20patients, Signed consent form was obtained from each participant. The study protocols were approved by the Ethics Committee on Human Research. Cut the collected ovarian tissue into2x1x1mm3blocks.
2Contents
2.1Freezing and thawing of ovary tissue:using different concentration DMSO and EG to prepare two sets of vitrification agent(low concentration I group, high concentration II group). Steeping Ovarian tissue blocks separately into equilibrium liquid and two kinds of vitrification frozen agent for a period of time, then by means of straw method, microsphere method, SSV method to do vitrified frozen, rapid thawed method to recover human ovary tissue fragment.
2.2Human ovary tissue cultivation in vitro after recovery:regularly collecting tissue culture supernate and detecting E2level,14d later fished out ovarian tissue blocks and settled, coloured them then histology detection.
2.3Frozen recovery effect judgment of human ovary tissue:
2.3.1The morphological analysis of follicles of ovary tissue before and after frozen:before and after frozen and cultivation14d later in vitro, observing follicle density and distribution of tissue cortex and follicle morphological changes, count the ratio of Normal form and anomaly change, detect follicle density and distribution of tissue cortex of every group and proportion changes of level of follicles.
2.3.2Apoptosis situation of all kinds of cells in thawing ovarian tissue:using DNA in situ end labeling method, to detect apoptosis situation of all kinds cells in thawing ovarian tissue at DNA level.
2.3.3Estradiol detection of supernate culture in vitro:collect supernate of ovary tissue culture in vitro every48hours, keep it at-80℃after centrifugation. Detecting the level of E2in supernate by RIA method.
Results
1Follicles morphological change and proportion changes of each levels of follicle of human ovary tissue before and after frozen
The normal form rate (43.7%) of DMSO+EG high compatibility groups II group primordial follicles has a dramatic decline than fresh control group (75.5%) and low concentration group I group (69.4%). Among these three groups, the proportion of follicle of each levels has no significance difference, and the highest proportion is primordial follicles.
2Morphological changes of human ovary tissue structure in post-thaw
Interstitial cells of fresh ovary tissue arranged regularly and wrapped, follicles of each level. Two kinds of vitrification refrigerating fluid and use partly three carriers to cryopreserved human ovary tissue, there is no obvious influence on primordial follicles, but it is clear that part of the interstitial cells of ovary tissue arrange disorder, loose and even separate.
3Apoptosis of various cell of human ovary tissue after freezing and thawing
There are no brown or sepia apoptosis cells which are positive expressed in nucleus of interstitial cells. With one-way analysis of variance, there is no statistical significance on apoptotic rate of interstitial cells between vitrification and fresh control group.
4The changes of E2secretion of ovary tissue culture in vitro after frozen
In carriers groups of low concentration compatibility I group E2of ovary tissue secret higher than carriers groups of high concentration compatibility II groups, there is significant difference on SSV method between two carriers, while no significance among carriers of frozen groups.
Conclusions
1. The tolerance of primordial follicles to frozen damage is higher than primary follicles.
2. It is better for saving ovary tissue when use using low concentration frozen protective agent combined SSV vitrification frozen method.
Part IV:The experimental study of heterologous ectopic transplantation of human ovarian tissue
Purpose
On the basis of preliminary study, we used DMSO+EG with lower concentration as frozen protective agent, SSV method as vitrification freezing and thawing, heterotopia allograft to the Cervical subcutaneous of ovariectomized nude mice. To observe exfoliocytology of vagina, the secretion of E2in serum and morphology change of uterus to charge frozen effect.
Methods
1. Objects and groups
2009.11～2010.3,20cases of ovarian disease and line laparoscopic surgery stripping of ovarian cyst wall residual organization were collected to study. Through Ethics committee review and make patients informed consent. Cut the collected ovarian tissue into2×1×1mm3blocks. Transplantation host is healthy sexual maturity Nu/Nu nude mice, group them in accordance with whether to transplant into four: fresh transplantation group, cryopreservation group, ovariectomized group and common control group. The former three group cut Bilateral uterine.
2. Contents
2.1Freezing and thawing:use lower concentration DMSO+EG, vitrification frozen ovarian tissue by SSV method and reviving it by rapid thawing method for reviving.
2.2Transplantation method of ovarian tissue:fresh and frozen-thawed ovary tissue separately multipoint transplanted into the Cervical subcutaneous of ovariectomized nude mice in relevant group.
2.3Indicator for further observation:
2.3.1viginal exfoliocytology observation:observe estrous cycle from the fifth day after transplantation through viginal exfoliocytology.
2.3.2Estrogen level measurement:detecting blood serum E2level of transplanted group in estrus to charge the ovarian function; ovariectomized nude mice draw blood detection before death.
2.3.3Morphology observation and transplant collection of nude mice:settle removed nude ovary by methanal, stain sections and observe morphology of endometrial glands and mesenchyme in light microscope; cutting the transplanted site in neck of transplanted mice to find graft.
Results
1Recovery condition of oestrous cycle of transplanted nude mice
Control group keep regular estrogen periodic change,4/9of fresh transplanted group recovered oestrous cycle,3/9of frozen transplanted group recovered; in ovariectomized group epithelial cells with nuclear and keratinocyte fade away, white blood cells largely increased, cast-off cells did not seen cyclic variation. Frozen transplanted group Restore estrus cycle time for the first time [(22.33±1.20)d] later than fresh transplanted group [(19.00±1.58) d], but compare Estrus days of two groups [(5.67±1.15)d,(5.0±0.82) d] to common control group [(4.83±0.75) d], there was no significant difference(P>0.05).
2Uterine morphological changes of transplanted group
Transplanted group mice's uterine thickened and became ruddy, permanently resiliency, which was same to the common control group. There are no difference on uterine wet weight with transplanted group and control group, overweight than ovariectomized group obviously. By light microscopy transplanted group endometrial interstitial loose, endometrial glands bulky and abundance, gland cell is tall columnar, cytoplasm rich, false stratified epithelium cells, no significant difference with normal endometrial morphology.
Nude mice in ovariectomized control group uterine atrophy, become thin, colour etiolation, endometrial interstitial density. Endometrial atrophy thin, the quantity of glands is less, gland cavity smaller, gland cell is resting stage performance.
Though in transplanted group mice had similar E2secretion level with normal control group, no obvious grafts were found in neck graft sites.
3The average level of serum E2of nude mice
Fresh and frozen transplanted group have no significance difference in average level of serum E2, but both higher than ovariectomized control group.
Conclusions
1. Using low concentration ratio DMSO+EG cryogen, SSV vitrification freeze human ovarian tissue can got the same effect with fresh ovarian tissue transplantation.
2. It is proved that the transplantation of human ovarian tissue ectopic nude mouse cervical subcutaneous without vascular anastomosis can survive and recover nude mice oestrous cycle and endocrine function.
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