冷冻载体对扩张期囊胚解冻结局的影响

背景：囊胚的冷冻复苏对人类辅助生殖技术的发展具有重要意义,合适的冷冻载体可以降低冷冻毒性提高冷冻效率,是改善囊胚冷冻复苏结局的研究热点之一。 目的：观察不同冷冻载体对昆明小鼠扩张期囊胚玻璃化冷冻解冻结局的影响。 方法：以新鲜昆明小鼠扩张期囊胚作为对照组,随机选取同期小鼠胚进行玻璃化冷冻解冻,冷冻前使用激光仪行人工皱缩,使用玻璃化法进行冷冻和解冻,按实验载体不同分为冷冻环组、闭合拉细麦管组和自制麦管叶片组;冷冻环组和自制麦管叶片组投入液氮前与冷冻保护剂接触时间 <40 s,40-60 s和60-90 s;解冻后囊胚使用胚胎囊期培养基添加10%血清替代品进行微滴培养。比较解冻后组间囊胚丢失率、囊胚复苏率和培养24 h囊胚孵出率。 结果与结论：相比闭合拉细麦管组,冷冻环组和自制麦管叶片组的囊胚丢失率降低,胚胎复苏率增高(P < 0.05),各冷冻组复苏囊胚培养24 h孵出率均低于对照组(P < 0.05)。使用冷冻环载体和自制麦管叶片载体冷冻解冻囊胚,投入液氮前与冷冻保护剂接触时间90 s内不同时间胚胎复苏率和孵出率的比较差异无显著性意义。结果说明冷冻环载体和自制麦管叶片载体用于昆明小鼠扩张期囊胚玻璃化解冻复苏的效果较好。     

不同冷冻方案对人类卵巢组织形态学的影响

目的建立一种简便易行、冷冻保存效果稳定的人类卵巢组织冷冻保存技术。方法采用程序冷冻法,玻璃化冷冻法和液氮直投法冻存人卵巢组织,解冻复苏后,经HE染色,进行组织形态学分析计数各级形态正常和异常的卵泡。结果程序冷冻法,玻璃化冷冻法和液氮直投法始基卵泡正常率分别为80.1%、70.4%、71.6%,初级卵泡正常率分别为19%、15%、29%。在各冷冻复苏组的卵巢组织中均见到间质改变。结论各种冷冻方案均对人卵巢组织的各级卵泡和组织结构造成一定的损伤,对始基卵泡影响最小,程序冷冻法对始基卵泡的保存优于玻璃化冷冻法和液氮直投法,但液氮直投法操作简便,快捷,对生长卵泡的保存优于其它两种方法。     

玻璃化冷冻对小鼠卵母细胞和颗粒细胞的影响

目的探讨玻璃化冷冻法保存小鼠卵巢组织对卵泡内卵母细胞和颗粒细胞的影响。方法分别应用两种玻璃化冷冻方案(ED20和EE40)冷冻小鼠卵巢组织,常规组织学检测新鲜和复苏卵巢组织内卵泡形态,同时应用TUNEL方法观察冻融前后卵泡内卵母细胞和颗粒细胞凋亡情况。结果ED20组和EE40组卵泡存活率分别为78.99±5.99%和71.50±5.81%,明显低于新鲜卵巢组织。冻融组织卵泡凋亡率较冷冻前显著升高。ED20组、EE40组和新鲜对照组颗粒细胞凋亡的卵泡比例分别为8.30±2.14%、11.98±2.34%和4.95±1.62%,差异有显著性;而冷冻前后卵母细胞凋亡的卵泡比例无明显差异。结论玻璃化冻融过程对小鼠卵泡中颗粒细胞的损伤较大,而对卵母细胞的影响较小。     

不同冷冻方法对人卵巢组织血管内皮生长因子表达及血管生成的影响

背景:人卵巢组织冷冻已成为一种保存生育能力的手段,卵巢组织移植后必须经过血管重建过程才能恢复血供,冷冻保存和复苏技术是影响移植后卵巢组织血管重建的关键。目的:比较新型玻璃化冷冻和慢速冷冻后人卵巢组织血管内皮生长因子表达情况及微血管密度,探讨不同冷冻方法对人卵巢组织血管重建的影响。方法:8份卵巢组织标本取自子宫内膜癌患者手术切除的正常卵巢组织,将每份卵巢皮质切成1.5mm×1.5mm×1.0mm大小的组织块共12块后,随机数字表法分为3组:对照组(新鲜组)、新型玻璃化冷冻组和慢速冷冻组。将新型玻璃化冷冻组卵巢组织块依次在含体积分数7.5%乙二醇+体积分数7.5%二甲基亚砜+体积分数20%胎牛血清的TCM-199培养液和含体积分数15%乙二醇+体积分数15%二甲基亚砜+0.5mol/L蔗糖的TCM-199培养液中平衡脱水,然后直接浸入液氮并装入冷冻管保存,解冻时按浓度梯度1.0,0.5,0.25mol/L蔗糖和含体积分数20%牛血清的TCM-199培养液依次洗脱冷冻保护剂。将慢速冷冻组人卵巢组织块放入盛有1mL冷冻液(含1.5mol/L二甲基亚砜+体积分数20%牛血清+0.1mol/L蔗糖的TCM-199培养液)的1.8mL冷冻管内平衡,然后放入程序冷冻仪中按设定程序进行冷冻。解冻时按浓度梯度1.0mol/L二甲基亚砜+0.1mol/L蔗糖、0.5mol/L二甲基亚砜+0.1mol/L蔗糖、0.25mol/L二甲基亚砜+0.1mol/L蔗糖和0.1mol/L蔗糖依次洗脱冷冻保护剂。冻融组及对照组均进行体外培养。免疫组织化学观察培养0,2,4,6d各组人卵巢组织血管内皮生长因子和CD34表达情况,并进行微血管计数。结果与结论:3组人卵巢组织培养前后间质细胞中均见血管内皮生长因子成斑片状弱表达,培养2d血管内皮生长因子表达均增加并达到峰值,培养4d均开始减弱,6d时进一步减弱;与慢速冷冻组相比,新型玻璃化冷冻组血管内皮生长因子表达更接近对照组。3组人卵巢组织培养2d微血管密度均增加并达到峰值,对照组和新型玻璃化冷冻组明显高于慢速冷冻组(P0.05);慢速冷冻组培养4d时微血管密度较培养2d时显著下降(P0.05);3组培养6d时微血管密度较培养2d时显著下降(P0.05)。与慢速冷冻法相比,新型玻璃化冷冻法能更好地保存人卵巢组织间质细胞和细胞外基质,对卵巢组织血管内皮生长因子表达及微血管生成的影响更少。     

不同冷冻方案保存人类卵巢组织的活性比较

背景：卵巢组织冷冻保存被认为是保存女性生殖内分泌功能安全、有效的方法,但目前尚无统一确定的方案。目的：探讨3种冷冻方案对人类卵巢组织保存冷冻效果及卵泡活性的影响。方法：采用丙二醇慢速程序冷冻法、二甲基亚砜玻璃化冷冻法、液氮直投法对20例人类卵巢组织进行冷冻保存,复苏后采用细胞存活/死亡荧光分析法计数有活性的细胞,体外培养测定培养液雌二醇浓度及各级卵泡计数,判断3种不同冷冻方案对人类卵巢组织活性的影响。结果与结论：不同冷冻方案冻融后卵巢组织块的活性卵泡率均低于新鲜卵巢组(P < 0.05),二甲基亚砜组最低,液氮直投法组与慢速程序冷冻法组差异无显著性意义。体外培养各冷冻组分泌的雌二醇水平比较,第4天时二甲基亚砜组低于液氮直投法组和慢速程序冷冻法组(P < 0.05),至第8天时各冷冻组雌二醇水平与新鲜卵巢组一致。培养14 d组织学观察,各组卵巢组织内生长期卵泡比例增多,始基卵泡仍然占最主要的。新鲜卵巢组织正常卵泡的总数高于冷冻组(P < 0.05),二甲基亚砜组的正常卵泡数低于液氮直投法组和慢速程序冷冻法组(P < 0.05)。提示冷冻保存对卵巢组织卵泡有一定的损伤,但仍能保存大部分始基卵泡的活性,经体外培养后可进一步发育并具有分泌功能,在3种冷冻方案中,慢速程序冷冻法和液氮直投法的冷冻效果优于二甲基亚砜玻璃化冷冻法,液氮直投法操作简便,冷冻效果稳定。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

玻璃化保存肌腱的生物力学观察

目的探讨玻璃化保存法对兔肌腱力学性能的影响。方法玻璃化保存组，6条新鲜胫前肌肌腱，以18．64％二甲基亚砜＋13.37％乙酰胺＋9．17％1．2丙二醇＋浓度0．10mmol／L海藻糖＋10％小牛血清为玻璃化冷冻保护剂．将新西兰纯种大白兔肌腱采用三步法预处理．-196℃液氮保存14d；“两步法”深低温冷冻保存组，6条新鲜胫前肌肌腱，以15％二甲基亚砜＋10％小牛血清作为冷冻保护剂，“两步法”处理后-196℃液氮冷冻保存14d；对照组．6条新鲜新西兰纯种大白兔胫前肌肌腱。分别进行肌腱拉伸实验．检测肌腱破坏载荷峰值、最大载荷拉伸位移及杨氏弹性模量。结果肌腱破坏荷载峰值：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．002），玻璃化保存组与“两步法”深低温冷冻保存组无统计学意义（P=0．256）；最大载荷拉伸位移：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组3组间均无统计学意义（P=0．065）；杨氏弹性模量：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．006）．玻璃化保存组与“两步法”深低温冷冻保存组差异无统计学意义（P=0．577）。结论玻璃化保存法保存肌腱具有良好的发展前景。     

人卵巢组织冷冻及异种移植对始基卵泡凋亡的影响

目的:通过检测始基卵泡凋亡率的改变来探讨2种冷冻方法对人卵巢组织的冻融及异种移植后的效果。方法:共收集16例人卵巢组织标本,每例分成2份,其中1份应用程序化冷冻方法进行冷冻,另一份应用玻璃化冷冻方法进行冷冻。解冻后分别移植到去卵巢的雌性SCID裸鼠颈部皮下。32只裸鼠随机分为2组,程序化冷冻组移植以程序化方法冷冻复苏的人卵巢组织;玻璃化冷冻组移植以玻璃化方法冷冻复苏的人卵巢组织。解冻后及移植6周后观察2组卵巢组织中始基卵泡的形态学改变及凋亡情况。结果:解冻后,程序化冷冻组始基卵泡凋亡率明显低于玻璃化冷冻组,始基卵泡正常率2组相比无明显差别。移植后,始基卵泡正常率及凋亡率2组相比均无明显差别。无论是程序化冷冻组还是玻璃化冷冻组,解冻后和移植后相比,始基卵泡正常率及凋亡率均无明显差别。结论:与玻璃化冷冻相比,程序化冷冻更有利于人卵巢组织始基卵泡的存活。将解冻后的人卵巢组织进行异种移植不影响始基卵泡的凋亡。     

不同玻璃化冷冻方案对小鼠卵巢组织的影响

目的比较不同组合的冷冻保护剂对小鼠卵巢组织的影响,为人类卵巢组织玻璃化冷冻的保护剂选择提供实验依据。方法将20只小鼠的卵巢进行玻璃化冷冻,组织标本随机分为4组,新鲜的和冻融后的卵巢皮质分别进行光学显微镜观察、TUNEL实验和免疫组织化学分析。结果光镜观察发现各实验组的各级卵泡形态正常率均明显低于对照组,B组的始基卵泡形态正常率和初级卵泡形态正常率均高于A组和C组,差异有统计学意义（P〈0．05）。A组和C组冻融后卵泡凋亡率、卵母细胞凋亡率和颗粒细胞凋亡率均高于B组,有统计学差异（P〈0．05）。另外,B组卵泡Bax蛋白阳性表达率低于A组和C组,而B组Bcl-2蛋白阳性表达率高于A组和C组,差异具有统计学意义（P〈0．05）。结论实验结果提示：与其它组合相比,EG与PROH的组合更适合小鼠卵巢组织的玻璃化冷冻。     

不同冷冻方案对家兔卵巢组织卵泡细胞增殖活性的影响

目的探讨不同冷冻方案对家兔卵巢组织卵泡细胞增殖活性的影响。方法应用PROH慢速程序化冷冻及DMSO+EG玻璃化冷冻方案冻存家兔卵巢组织,复苏后机械性分离卵泡,采用3H标记的胸腺嘧啶核苷掺入试验测定2~5层颗粒细胞包裹的小次级卵泡(小OGC组)及5层以上颗粒细胞包裹的大次级卵泡(大OGC组)的cpm值,并以新鲜组为对照。结果PROH组及DMSO+EG玻璃化组复苏后形态正常的小OGC的cpm值分别为2554.67±93.59及2510.67±100.03,新鲜对照组为2507.00±25.94,两冷冻组与新鲜组比较,差异无显著性(P>0.05);而各大OGC组的cpm值分别为5784.00±188.95、5481.33±112.38及9629.00±265.64,两冷冻组与新鲜组比较,差异有显著性(P<0.05);两冷冻组间比较,大OGC及小OGC的cpm值均无显著性差异(P>0.05)。结论①两种冷冻方法对形态正常的小OGC的细胞增殖活性无明显影响,这些卵泡复苏后体外培养及成熟可能为今后卵巢组织冷冻复苏体外培养的研究方向。②两种冷冻方法使形态正常的大OGC细胞增殖活性受到明显抑制,冷冻复苏可能对颗粒细胞造成了损害。③两种冷冻方法对大小OGC细胞增殖活性的影响相似,其具体机制尚需进一步研究。     

卵巢组织玻璃化冷冻保存和移植的研究进展

背景：卵巢组织玻璃化冷冻技术作为一种快速、简便、经济的冷冻方式被逐渐应用于卵巢组织的保存。 目的：综述国内外关于卵巢组织玻璃化冷冻保存及移植的研究进展。 方法：由第一作者检索1995/2011 PubMed数据库及清华同方数据库有关卵巢组织玻璃化冷冻保存以及卵巢组织移植技术等方面的文献。 结果与结论：玻璃化冷冻是一个超高速的冷冻过程,形成高黏度的“玻璃样凝固状态”,可以避免由于冰晶形成所造成的细胞损伤。但至今玻璃化冷冻仍缺乏统一的标准化程序。影响卵巢组织玻璃化冷冻保存效果的主要因素有卵巢组织块的大小、冷冻保护剂的种类、渗透平衡的时间和温度、冷冻载体等。随着低温生物学的发展和卵巢组织冷冻保存效果的提高,卵巢组织的移植已经具备了一定的临床应用可行性。到目前为止,全世界已有一系列关于冻存卵巢组织移植后成功妊娠及分娩的报道,移植成功的关键在于减少缺血再灌注损伤和促进新生血管的形成。关键词：卵巢组织;玻璃化冷冻;移植;保存;综述 缩略语注释：SSV：solid-surface vitrification,固体表面;NIV：needle immersed vitrification,针浸润玻璃化冷冻法;DCV：direct cover vitrification,直接覆盖玻璃化方法 doi:10.3969/j.issn.1673-8225.2012.18.039     

Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes

BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.     

Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

BACKGROUND: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.     

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20,000 degrees C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200 degrees C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000 degrees C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.     

Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

Purpose

To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation.

Materials and Methods

In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay.

Results

A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups.

Conclusion

The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.     

The development of alternative vitrification solutions for microencapsulated islets

Bioartificial pancreas in which islets of Langerhans (islets) are enclosed in a semipermeable membrane is one of the approaches to treat insulin-dependent diabetic patients. Although there are advantages in this method, one of the issues that still remains is the long-term storage of tissue engineering devices before transplantation. One of the possible routes to address this is through cryopreservation. In this study, a freezing solution, 2 m DMSO in RPMI-1640, a conventional vitrification solution, VS55, and the newly developed vitrification solution KYO-1 were examined to cryopreserve microencapsulated islets in agarose hydrogel. The insulin release ability, morphology of islets, and physico-chemical properties of the agarose gel membrane were examined after a cryopreservation and thawing process. Frozen and vitrified (by KYO-1) groups showed a similar insulin secretion. Frozen groups by 2 m DMSO, however, showed destruction of agarose capsules and some islets were out of the capsule. When KYO-1 was used, islets still maintained the ability to release insulin in response to glucose stimulation, and agarose capsule showed morphological integrity, and mechanical properties. In conclusion, vitrification using KYO-1 which is composed of 5.38 m ethylene glycol, 2 m DMSO, 0.1 m PEG 1000 and 0.00175 m PVP K10 in EuroCollins, is a suitable method for cryopreservation of microencapsulated islets.     

乙二醇、丙二醇对冻融人卵巢卵泡凋亡的影响

目的:比较乙二醇和丙二醇对人卵巢组织中卵泡的凋亡及凋亡相关基因P53及Bcl-2表达的影响,为人卵巢组织的最佳冷冻保护剂的选择提供实验依据。方法：获取12例活检人卵巢组织每例标本分为3组：新鲜对照组、乙二醇（EG）组和丙二醇（PROH）组,冻融卵巢皮质块采用慢速冷冻法和快速解冻法,冻融后的卵巢皮质组织采用TUNEL实验检测卵泡凋亡,免疫组化法检测凋亡相关蛋白P53及Bcl-2表达变化。结果：新鲜对照组、PROH组、EG组的卵泡凋亡率分别为14.58%、23.08%、30.43%,EG组卵泡凋亡率与新鲜对照组相比差异显著（P<0.05）,但3组卵母细胞凋亡率之间差异无显著（P>0.05）。3组P53阳性卵泡率分别为13.48%、25.00%、33.93%,EG组卵泡凋亡率与新鲜对照组相比差异显著（P<0.05）,卵泡凋亡率与P53阳性率表达正相关（r=0.7791,P<0.01）。3组Bcl-2阳性卵泡率分别为5.29%、 3.65% 、 2.41%,其差异无显著（P>0.05）,凋亡与Bcl-2表达无相关性（P>0.05）。结论:结果提示对人卵巢组织进行冻存PROH优于EG;冻融过程对颗粒细胞的凋亡有明显影响,对卵母细胞凋亡无显著影响;P53蛋白在卵巢组织冻融过程中参与凋亡的发生,但不通过Bcl-2介导的途径。     

Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.     

Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles

Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent.     

微滴法及麦管法玻璃化冻存家兔卵巢组织的比较研究

目的观察微滴法及麦管法玻璃化冻存家兔卵巢组织对各级卵泡的形态学及卵母细胞增殖活性的影响,探讨适宜的卵巢组织玻璃化冻存方案及玻璃化冷冻容皿。方法将6只健康雌性日本大耳白家兔随机分为2组,分别采用无载体的微滴法及麦管法以DMSO+EG方案玻璃化冻存家兔卵巢组织,复苏后行HE组织切片染色及PCNA免疫组化染色,显微镜下观察各级卵泡组织形态学的改变及始基卵泡和初级卵泡卵母细胞PCNA表达的变化。结果1.微滴法及麦管法玻璃化冻存家兔卵巢组织复苏后始基卵泡、初级卵泡及次级卵泡的形态正常率分别下降为75.00%和78.80%、47.07%和41.18%及14.29%和29.49%,与新鲜对照组比较(86.92%、78.57%及86.67%),差异均有显著性(P0.05);而两组间比较,差异无显著性P0.05)。2.微滴法及麦管法玻璃化冻存家兔卵巢组织,始基卵泡及初级卵泡母细胞PCNA阳性表达率分别为73.77%及70.87%,与新鲜对照组比较(78.04%),差异无显著性(P0.05);两组间比较,差异也无显著性(P0.05)。结论1.微滴法及麦管法玻璃化冷冻方案均对家兔卵巢组织造成一定程度的损伤,各级卵泡的形态正常率明显下降。但始基卵泡仍能保持较高的形态正常率(75.00%和78.80%)。2.上述两种冷冻方法对家兔卵巢组织始基及初级卵泡卵母细胞的增殖活性未造成明显影响,PCNA的阳性表达率无明显变化。3.微滴法玻璃化冻存家兔卵巢组织对各级卵泡形态学的影响及对始基、初级卵泡卵母细胞PCNA的表达与麦管法比较无明显差异。4.麦管法因避免了组织与液氮的直接接触,故可避免微生物的污染,临床应用更安全可靠,所以目前玻璃化冷冻卵巢组织应以麦管法为宜。     

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts

BACKGROUND: The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). METHODS: Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. RESULTS: Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). CONCLUSIONS: These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.