两种玻璃化法冻存小鼠卵巢的研究

目的:探讨2种玻璃化法对小鼠卵巢组织、器官形态和功能保存作用的影响。方法:以改良的DMEM-F12为玻璃化液,分别采用常规玻璃化法(A组)和超速玻璃化法(B组)冻存小鼠卵巢组织及器官,解冻后通过组织学观察、卵巢组织异体、卵巢器官自体肾被膜下移植,观察动情周期恢复率、恢复时间、卵泡发育状况,并分别以新鲜卵巢组织异体移植、卵巢器官自体移植(C组)为对照,评价2种玻璃化法的冻存效果。结果:①A组、B组、C组卵巢组织异体移植小鼠动情周期出现率为100%,出现动情周期分别10.5±5.4d、8.0±2.2d、6.3±1.0d。A组与C组比,差异显著(P<0.05);B组与C组无差异,A组与B组间也无差异(P均>0.05)。②A组、B组、C组卵巢器官自体移植小鼠动情周期出现率为100%,出现动情周期分别为9.4±0.9d、6.9±1.1d、6.1±1.1d,A组与B、C组相比有统计学差异(P<0.05),而B组与C组间无差异(P>0.05)。移植存活的卵巢组织、器官内均可见不同发育阶段的卵泡,形态正常。结论:2种玻璃化法可有效地冻存卵巢组织及器官,但超速玻璃化法效果较优。     

兔卵巢组织玻璃化冷冻的实验研究

目的:探讨玻璃化冷冻法保存兔卵巢组织的效果。方法:随机将25只新西兰雌兔分为对照组(5只)、慢速冷冻组(10只)和玻璃化冷冻组(10只),比较各组冻融前后卵巢组织学、超微结构、卵泡凋亡(原位末端标记法,TUNEL)和子宫系膜内移植后卵巢功能的恢复情况。结果:新鲜组织、慢速冷冻复苏组织和玻璃化冷冻复苏组织中正常形态卵泡比例分别为87.36%、81.96%和82.72%,两冷冻组正常卵泡比例均低于对照组,差异有统计学意义?P(0.05),但玻璃化冷冻组与慢速冷冻组差异无统计学意义(P>0.05)。3组间卵泡凋亡比率分别为21.4%、13.5%和17.1%,差异无统计学意义(P>0.05);3组移植后兔动情周期出现率均为100%,动情周期出现天数差异无统计学意义?P>0.05);移植存活的卵巢组织内可见各级形态正常的卵泡发育。结论:玻璃化冷冻可有效保存卵巢组织的结构和功能,是一种简单、可行的兔卵巢组织冷冻保存法。     

人卵巢组织玻璃化冷冻技术研究进展

卵巢组织冷冻是女性生育力保存的重要方式,程序化慢速冷冻一直是卵巢组织冷冻的主流方法.随着玻璃化冷冻技术在辅助生殖领域的成功应用,玻璃化冷冻技术在卵巢组织冷冻领域中的应用也越来越广.本文概括性地对卵巢组织玻璃化冷冻的发展现状、实验室技术及临床应用效果做一综述,以期为临床和实验研究提供借鉴和参考.     

不同浓度保护剂对超速冻存小鼠卵巢组织的效果观察

目的:寻找适宜的小鼠卵巢组织超速冻存低温保护剂种类及浓度。方法:以不同浓度二甲亚砜(DMSO)和丙二醇(PROH)(1.5　mol/L、3　mol/L)作低温保护液时,观察超速冻存法对小鼠卵巢组织的冻存效果。解冻后行自体和异体卵巢的肾被膜下移植。通过对受体动情期的恢复及激素水平测定等方法对冻存效果进行观察。结果:低浓度组DMSO和PROH动情期的恢复率分别为75%和71%;血清E2分别为(10.5和16.8)×10-9　mmol/L,与作为对照的慢速程序法组无显著性差异(66.7%和100%;11.7×10-9　mmol/L和12.1×10-9　mmol/L)。高浓度动情期的恢复率分别为100%和62.5%;血清E2分别为14.1×10-9　mmol/L(DMSO)和7.4×10-9　mmol/L(PROH),与慢速组比也无显著性差异。结论:两种浓度的DMSO和PROH均适用于超速冻存法,尽管高浓度的DMSO在超速冻存时可取得较好的动情周期恢复率,但血清E2 水平的结果以低浓度的PROH为好。     

三种不同玻璃化冷冻方法对小鼠2-细胞期胚胎冷冻效果的比较

目的:比较三种玻璃化方法冷冻小鼠胚胎的存活率及继续发育能力的差异。方法:将573个小鼠2-细胞期胚胎随机分为四组:Straw冷冻组144个,OPS冷冻组142个,CPS冷冻组149个,比较胚胎复苏后存活率、囊胚形成率及孵化率,并以未冷冻为对照组(138个)。结果:Straw组及CPS组存活率均显著性高于OPS 组(P <0.05);囊胚形成率及孵化率OPS组和CPS组均显著性高于Straw组(P均<0.05),OPS和CPS两组之间无显著性差异(P均>0.05)。结论:OPS与CPS方法能够更好地保持胚胎复苏后的继续发育能力。且CPS方法存活率高于OPS方法,并降低了交叉感染的危险性,更具临床应用潜力。     

整体卵巢冷冻及移植的研究进展

卵巢组织冷冻是保存女性生育能力和内分泌功能的一种极具潜力的方法。卵巢组织移植后的缺血损伤导致卵泡大量丢失,同时由于小块卵巢组织所含的卵泡少,因此移植术后难以长久维持卵巢功能。整个卵巢行显微血管吻合移植术可避免移植后的缺血性损伤。适宜的整体卵巢的冷冻保存方法是施行整体卵巢移植的难点,目前未有统一的标准方法。综述国内外对整体卵巢冷冻及移植的研究进展,探讨如何选择合适的冷冻方法。     

自制冷冻环人卵巢组织玻璃化冷冻的可行性研究

目的探讨应用自制冷冻环进行人卵巢组织玻璃化冷冻的效果。方法2008年5月至9月在华中科技大学同济医学院附属同济医院收集5例人卵巢组织标本,以乙二醇、二甲基亚砜、蔗糖作为冷冻保护剂,自制冷冻环为载体进行玻璃化冷冻。并观察卵巢新鲜组织和冻融后组织始基卵泡和初级卵泡的形态学变化、凋亡情况、超微结构变化及体外培养内分泌功能。结果新鲜及冻融后组织形态正常卵泡比值分别是89.46±4.94、84.47±4.66,无统计学意义(P>0.05)。凋亡面积分别是(0.07±0.02)%、(0.10±0.05)%,无明显增加(P>0.05)。超微结构无明显改变。新鲜组织及冻融后组织体外培养上清液雌二醇含量分别(2549.73±711.87)pmol/L、(2514.87±714.66)pmol/L,无统计学意义(P>0.05)。结论自制冷冻环可作为一种便宜、方便、可行的人卵巢组织低温保存的可替代方法。     

人工皱缩后玻璃化冷冻小鼠扩张囊胚的效果观察

目的:观察冷冻前人工皱缩小鼠扩张期囊胚的囊胚腔,冻融后小鼠扩张期囊胚的存活率及体外和体内继续发育能力。方法:冻前先应用自制的玻璃微细管人工皱缩小鼠囊胚腔后,再应用冷冻环进行玻璃化冷冻小鼠扩张期囊胚150个。冻融后培养3h观察胚胎存活情况,以新鲜扩张囊胚为对照,并将存活胚胎及新鲜未冻囊胚移植受体孕鼠子宫内,观察胚胎的妊娠率和产仔率。结果:人工皱缩后小鼠扩张囊胚的存活率及孵出率均显著高于未皱缩组(P<0.05),皱缩组与对照组无显著差异(P>0.05)。移植后皱缩组的妊娠率及产仔率均显著高于未皱缩组(P<0.05),皱缩组与对照组无显著差异(P>0.05)。结论:冻前人工皱缩小鼠扩张期囊胚的囊胚腔后,再应用冷冻环进行玻璃化冷冻是个行之有效的方法,能明显提高囊胚的玻璃化冷冻效率。     

抗冷冻蛋白Ⅲ减少家兔卵巢组织冷冻损伤的效果观察

目的:评价抗冷冻蛋白Ⅲ(AFPⅢ)对玻璃化冷冻家兔卵巢组织的影响。方法:收集家兔卵巢30只,随机分为新鲜卵巢组、添加AFPⅢ玻璃化冷冻组(AFPⅢ终浓度为500 ng/ml)和常规玻璃化冷冻组,各组10只,解冻后分析各组卵巢的组织学结构、卵泡形态正常率、卵巢组织超微结构、卵母细胞凋亡率及卵泡存活率。结果:新鲜卵巢组的卵泡形态正常率(91.6%)、卵泡存活率(81.75%)显著高于两冷冻组(P0.01),卵母细胞凋亡率(12.0%)显著低于两冷冻组(P0.01);添加AFPⅢ玻璃化冷冻组卵泡形态正常率(77.5%)、卵泡存活率(45.31%)显著高于常规玻璃化冷冻组(分别为62.1%、37.25%)(P0.01),卵母细胞凋亡率(25.8%)显著低于常规玻璃化冷冻组(41.2%)(P0.01)。结论:家兔冷冻卵巢组织的卵泡形态正常率、卵泡存活率显著低于新鲜组织,冷冻保护剂中加入AFPⅢ可减少家兔卵巢组织冷冻损伤。     

4种冷冻-解冻方法对家兔卵巢组织形态学的影响

目的:探讨适宜的卵巢组织冻存方案。方法:采用PROH(A2组)及DMSO(B2组)慢速程序化冷冻和DMSO+PROH(C2组)及DMSO+EG(D2组)玻璃化冷冻方法,冻存家兔卵巢组织,解冻复苏后,以相应的4组新鲜组织为对照(A1-D1组),做HE染色,行组织形态学分析。结果:A1-D1组始基卵泡的形态正常率分别为90.1%、91.5%、91.8%、92.2%,其相对应的A2-D2组分别下降为67.6%、69.7%、70.5%、80.1%,差异均有统计学意义(P均<0.05)。冷冻组中A2组始基卵泡形态正常率最高,与B2、C2、D2组比,差异有显著性(P<0.05)。C2、D2组间比,差异无显著性(P>0.05)。各冷冻组合并后形态正常率始基卵泡为72.7%,初级卵泡为55.7%,两者比较有统计学差异(P<0.05)。4个冷冻组中均可见卵巢组织结构受损的表现。结论:4种冷冻解冻方法对卵巢皮质中各级卵泡及卵巢组织结构均造成一定程度的损害,使各级卵泡的形态正常率明显下降,卵巢间质细胞连接变得疏松;PROH慢速程序化冷冻法明显优于DMSO法及玻璃化法,较适合卵巢组织中始基卵泡的保存;冻存卵巢组织对初级卵泡的影响大于始基卵泡。     

两种玻璃化液冻存小鼠桑椹胚的研究

目的:研究EDS-40和EFS-40两种玻璃化溶液对小鼠桑椹胚冻存的影响。方法:随机选用NIH小鼠优质桑椹胚,先对两种玻璃化溶液EDS-40(40%乙二醇+18%葡聚糖+0.5mol蔗糖)和EFS-40(40%乙二醇+18%聚蔗糖+0.5mol蔗糖)行毒性测试:再在20℃±5℃室温下,将小鼠桑椹胚分别加入到两种玻璃化溶液,玻璃化后装管并迅速投入液氮中,冻存2-3个月。各组均在25℃的水浴中复温,胚胎用解冻液(S-PBS)和培养液反复洗涤后移入含Ham'sF12培养液培养48h,观察胚胎形态及发育。部分胚胎体外培养12-14h后行胚胎移植,观察妊娠及产仔情况。结果:两种玻璃化溶液的毒性有显著性差异,EDS-40较EFS-40毒性低(X2=6.415,P<0.05),透明带完好率两组间无显著性差异(X2=2.189,P>0.05)。胚胎优良率(X2=7.51,P<0.01)和发育率(X2=5.556,P<0.05)EDS-40较EFS-40更好,两组间妊娠率(X2=1.937,P>0.05)及产仔率(X2=0.245,P>0.05)无显著性差异。结论:EDS-40较EFS-40玻璃化溶液毒性低,冻存胚胎效果更好。     

Histological Evaluation of the Effects of Cryopreservation in Bovine Ovarian Tissue

The aim of this study was to show the effects of freezing and thawing in bovine ovarian tissue by histological analysis. Ten cortical slices (2-4 mm in diameter) were obtained from each ovary by tru-cut biopsy and randomly divided into two groups: five fragments were immediately processed as a fresh tissue control group, while the remaining 5 fragments were slowly frozen using DMSO plus sucrose as cryoprotectors, then stored for two weeks and quickly thawed. Histological examination of all cryopreserved ovarian fragments showed no damage in the structure of the organ. Furthermore, there was no difference in the average number of primordial and primary follicles between the two groups of ovarian tissue. These data suggest that the bovine ovary can be used as a suitable model to test new freezing and thawing procedures in search for a standard protocol of human ovary cryopreservation.     

Mouse Ovarian Tissue Cryopreservation Has Only a Minor Effect on In Vitro Follicular Maturation and Gene Expression

Purpose : To establish a protocol for ovarian tissue cryopreservation which can retain fertility potential after thawing and to evaluate the impact of cryopreservation on development and gene expression during folliculogenesis. Methods : A controlled randomized study in a clinical and academic research setting in a university medical center was conducted to study cryopreservation and in vitro maturation (IVM) of mouse ovarian follicles. Preantral follicles isolated from either fresh (Group A) or cryopreserved (Group B) murine ovarian tissues were used to test their fertility potential by in vitro culture–in vitro maturation (IVC-IVM). Expression of Graafian follicles derived from both groups were detected by DNA microarray techniques for comparison. Results : Although there were no significant differences in IVM outcomes and follicular gene expression between the two experimental groups, cryopreservation appears to induce the expression of heat shock proteins, DNA-damage-inducible protein 45 and death-related apoptosis genes (i.e., Fas and Fas-ligand). Conclusion : Cryopreservation may trigger biological events not amenable to normal cell function and follicular development. However, neither follicular development nor gene expression was dramatically changed after cryopreservation. These data suggest that although our current cryopreservation techniques yield competent follicles and mature oocytes, subtle changes observed in gene expression imply that the present cryopreservation techniques need to be further refined.     

Blastocyst Development After Cryopreservation and Subcutaneous Transplantation of Mouse Ovarian Tissue

Purpose To investigate follicle survival and developmental potential with IVF of cryopreserved, subcutaneously transplanted mouse ovarian tissue.Methods Fresh and frozen mouse ovarian tissue was autologously transplanted into subcutaneous tissue. Two weeks after the transplantation, the morphology and histology of the fresh and frozen grafts were compared. Superovulation and IVF was performed to evaluate the fertility potential of the frozen ovarian graft.Results Both fresh and frozen grafts of ovarian tissue survived in 14 of 16 mice (88%). Morphologically, both types of grafts resembled fresh ovarian tissue and contained follicles at all stages of folliculogenesis. A total of 73% of follicles in fresh grafts and 62% in frozen grafts survived after transplantation compared with fresh ovarian tissue. Sixteen ICR mice underwent superovulation. A total of 56 oocytes from antral follicles were recovered from the subcutaneously transplanted cryopreserved ovarian tissue. Fourteen (25%) oocytes were in metaphase II stage, 6 were fertilized by IVF, and 2 progressed to the blastocyst stage.Conclusions Cryopreservation and subcutaneous transplantation of ovarian tissue provides a possible means of fertility preservation. The main loss of follicles occurred during grafting rather than during freezing and thawing.     

Ovarian tissue and oocyte cryopreservation prior to iatrogenic premature ovarian insufficiency

Gonadotoxic treatments like chemotherapy or radiotherapy and ovarian surgery may result in an accelerated depletion of the ovarian reserve and subsequent premature ovarian insufficiency. Important determinants of this severe risk that require fertility preservation strategies are patient age, ovarian reserve, type of treatment, and administered dose. Oocytes and ovarian tissue can both be cryopreserved, with encouraging results in terms of pregnancy and live birth rates according to recent publications. Moreover, since ovarian tissue transplantation also results in long-term endocrine resumption, it represents a potential future therapeutic option for complete ovarian function restoration in patients with premature ovarian insufficiency.     

有无卵丘细胞对玻璃化冷冻后小鼠卵母细胞细胞骨架及体外发育能力的初步研究

目的:探讨卵丘细胞在玻璃化冷冻中对小鼠卵母细胞发育潜能及细胞骨架的影响。方法:采用玻璃化冷冻技术,保存带卵丘细胞或完全剥除卵丘细胞的小鼠MⅡ期和GV期卵丘复合体/裸卵(COC/DO),复苏且GV卵体外培养成熟后分别作体外受精或免疫荧光标记检查纺锤体和染色体的完整性。结果:MⅡ-COC和GV-COC的复苏率均显著高于MⅡ-DO和GV-DO(分别为86.49%vs60.92%和85.94%vs64.93%,P<0.01)。GV-COC组的受精率、囊胚率均高于GV-DO组,且MⅡ-COC组和GV-COC组的纺锤体和染色体均正常率均分别高于MⅡ-DO组和GV-DO组,但无显著性差异(P>0.05)。结论:卵丘细胞在玻璃化冷冻卵母细胞中能有效减少冷冻对细胞骨架的损伤,并改善卵子复苏及胚胎发育潜能。