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。     

小鼠卵巢组织玻璃化冷冻的初步研究

目的建立一种有效的卵巢组织玻璃化冷冻方法。方法采用乙二醇和蔗糖两步法、小鼠卵巢组织经5m in、10m in、15m in 3种不同的预平衡时间处理,在玻璃化溶液中暴露相同的时间后直接投入液氮进行玻璃化冷冻。复苏后与新鲜的卵巢组织均进行HE染色、提取DNA进行琼脂糖凝胶电泳。结果玻璃化冷冻复苏后,预平衡时间为10m in的实验组小鼠卵巢组织中的卵泡保持了较好的形态结构。提取DNA进行琼脂糖凝胶电泳,各实验组与新鲜对照组的结果相似。结论采用乙二醇和蔗糖两步法、预平衡为10m in、直接投入液氮的玻璃化冷冻方法可能是一种有效的卵巢组织片冷冻保存方法。该玻璃化冷冻方法没有造成细胞DNA的损伤。     

卵巢组织的冷冻和移植研究进展

卵巢组织冷冻技术可以为患有良、恶性肿瘤的女性提供生育保险 ,已经成为生殖医学领域的新型技术和研究的热点之一。冷冻卵巢组织的移植途径有异体移植、异位自体移植、原位移植和卵泡的体外成熟培养 ,其应用前景广阔。     

卵巢组织冷冻移植在女性生育力保存中的研究进展

医疗技术的发展使得癌症患者的存活率越来越高,但放化疗治疗会导致女性卵巢功能早衰,出现过早绝经甚至丧失生育能力。卵巢组织冷冻保存是保存女性生育力的一种方式,甚至是有些患者唯一的生育力保存的选择。目前在世界范围已有17例婴儿是来自冷冻/解冻-移植后的卵巢组织,这给我们带来了希望,同时也带来了挑战,因为这项技术还存在一定的局限性。目前使用的冷冻方法可以保存卵巢内大量的始基卵泡,但是在移植的初期,因组织缺血缺氧会导致大量的卵泡丢失,影响了移植物的存活,缩减了卵巢组织在体内发挥功能的时间。卵巢组织移植存活程度的影响因素很多,包括移植前个体因素和基础疾病及治疗对卵巢的损伤,冷冻的损伤和移植组织块的大小,移植后血管再生情况等。这篇文章将对卵巢组织冷冻作为女性生育力保存方式的研究进展做一综述。     

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。     

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。     

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     

两种冷冻方法冻融人卵巢组织异种移植后血管生成素-2表达变化的比较

目的 通过检测血管生成素-2（Ang-2）的表达,探讨两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1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裸鼠颈部皮下。32只SCID裸鼠随机分为两组：A组16只,移植以程序化方法冷冻复苏的人卵巢组织;B组16只,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周后观察两组卵巢组织中始基卵泡的存活及颗粒细胞Ang-2的表达情况。 结果 解冻后,A组Ang-2的阳性率明显高于B组（P<0.05）,始基卵泡正常率两组相比差别不明显（P＞0.05）。移植后,始基卵泡正常率及Ang-2的阳性率两组相比均无明显差别（P＞0.05）。解冻后和移植后相比,两组的Ang-2阳性率及A组的始基卵泡正常率差别均不明显（P＞0.05）,B组移植后始基卵泡正常率明显低于解冻后（P<0.05）。 结论 与玻璃化冷冻相比,程序化冷冻对人卵巢组织始基卵泡颗粒细胞Ang-2的表达影响更小。将解冻后的人卵巢组织进行异种移植不影响Ang-2的表达。     

两种冷冻方法对人大块卵巢组织卵泡活性的影响

目的 探讨两种冷冻方法冻融人类大块卵巢组织的冷冻效果及对卵泡活性的影响。方法 收集15例人卵巢组织,每例修剪成3块卵巢组织
（约15 mm×15 mm×2mm）,分别随机分配到新鲜对照组（A组）、玻璃化冷冻组（B组）、两步法冷冻组（C组）,组织学分析3组卵泡形态学变
化,并进行卵巢组织体外培养后测定培养液上清中雌激素、孕激素浓度,免疫组织化学染色测定培养后的卵巢组织细胞核增殖相关抗原Ki67 及
B细胞淋巴瘤/白血病-2基因（Bcl-2）表达水平,评估卵泡增殖及凋亡活性。结果 A组正常形态卵泡比率（91.9%）显著高于B组（71.3%）和C组
（82.9%）,差异有统计学意义(P<0.05),C组高于B组,差异有统计学意义(P<0.05)。C组的始基卵泡和初级卵泡的正常形态率分别为86.8%、
54.4%,均分别高于B组73.8%、44.4%,差异有统计学意义(P<0.05)。同一时期3组激素测定结果差异不显著(P>0.05)。C组Ki67的阳性率为73%,
分别显著高于A组50%和B组55%(P<0.05),A组低于B组(P<0.05)。Bcl-2阳性率,A组为57%和C组61%相比无差异(P>0.05),但B组42%阳性率分别低
于A组和C组(P<0.05)。结论 人体大块卵巢组织冻融后仍具有活性,并且两步法冻存效果优于玻璃化冷冻法。     

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。     

Human ovarian tissue cryopreservation: indications and feasibility

BACKGROUND: The cryopreservation of ovarian tissue may enable women exposed to gonadotoxic treatments to have children at a later date. METHODS: Between April 1998 and October 2000, we evaluated the feasibility of long-term ovarian tissue cryopreservation in 51 women who were all at risk of becoming sterile following treatment. RESULTS: Ovarian tissue was not cryopreserved in 20 cases because of the woman's age or premature ovarian failure. In 31 patients, ovarian tissue was frozen by a slow cooling technique using DMSO and sucrose as cryoprotectants. The patients were aged 2.7-34 years and 16 of them were <18 years old. Cryopreservation could be performed in all cases. Ovarian cortex histology was performed for all patients to evaluate the concentration of follicles. The mean number of primordial and primary follicles per mm(2) was 20.36 +/- 19.03 before 10 years of age, 4.13 +/- 2.9 between 10 and 15 years of age and 1.63 +/- 3.35 after 15 years of age. An average mean number of 26 +/- 8.2 ovarian fragments (range 13-50) were cryopreserved per patient for future autografts or for in-vitro growth of follicles. CONCLUSION: Cryopreservation of ovarian tissue may be systematically proposed to young women and girls at risk of becoming sterile as a result of gonadotoxic treatment.     

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.     

快速深低温冻存对新生大鼠卵巢组织免疫原性的影响

目的:探讨快速冻存法冻存新生大鼠卵巢器官的效果,以及冻存对卵巢抗原性的影响。方法:采用快速冻存法冻存出生1 d的鼠(新生鼠)卵巢组织,移植入同系去卵巢成年雌性大鼠的肾被膜下,利用免疫组织化学方法及Western blot检测观察移植物内CD8 和CD4 T淋巴细胞的浸润情况。结果:新生鼠卵巢组织移植后动情周期恢复率(66.67%)低于新鲜移植组(90.51%),但无显著性差异。各移植物内毛细血管丰富;有正常发育阶段的各级卵泡、间质腺及闭锁卵泡;CD8 和CD4 阳性细胞计数值及其蛋白表达明显低于新鲜移植组。结论:快速冻存法可有效地冻存新生鼠的卵巢;冻存降低新生鼠卵巢组织抗原性。     

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix

BACKGROUND: Ovarian tissue cryopreservation is a promising technique tosafeguard fertility in cancer patients. However, in some typesof cancer, there is a risk of transmitting malignant cells presentin the cryopreserved tissue. To avoid such a risk, pre-antralfollicles could be isolated from ovarian tissue and grown invitro. On the basis of this assumption, the aim of our studywas to investigate in vitro survival and growth of pre-antralfollicles after cryopreservation of ovarian tissue and follicularisolation, followed by encapsulation in alginate beads. METHODS: Ovarian biopsies from four patients were frozen and thawed.Pre-antral follicles were then isolated and embedded in an alginatematrix before in vitro culture for 7 days. RESULTS: Small pre-antral follicles (42.98 ± 9.06 µm) fromfrozen–thawed tissue can survive and develop after enzymaticisolation and in vitro culture. A total of 159 follicles wereincubated in a three-dimensional system (alginate hydrogel)and, after 7 days, all of them showed an increase in size (finalsize 56.73 ± 13.10 µm). The survival rate of thefollicles was 90% (oocyte and all granulosa cells viable). CONCLUSION: Our preliminary results indicate that alginate hydrogels maybe a suitable system for in vitro culture of isolated humanpre-antral follicles. However, more studies are required toestablish whether follicular morphology and functionality canbe maintained using this matrix.     

Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation

The freezing of ovarian tissue and the growth of immature oocytes from primordial follicles is an interesting concept in ovarian tissue transplantation and in-vitro fertilization. In this study, the morphology and distribution of primordial follicles were studied in ovarian tissue from 24 women before and after cryopreservation. Cryopreservation did not significantly change either the morphology or number per unit volume of morphologically normal follicles in frozen ovarian tissue. Primordial follicles were predominant, accounting for 78.6% and 82.6% of total follicles in fresh and frozen ovarian tissues respectively. The distribution of follicles was extremely uneven in ovarian tissue. A large variation in follicle numbers was observed in ovarian tissue samples from patient to patient, and even in the same patient, indicating that the number of follicles counted in one sample of ovarian tissue may not represent the number of follicles in other tissue samples. Ovarian tissue could be frozen in the form of strips instead of fragments for fast processing and better viability of ovarian tissue in cryopreservation. The number of follicles in ovarian tissue declined with the increasing age of the patients. An immunohistochemical study showed that immunoreactivity for the epidermal growth factor (EGF) receptor was detected in primordial follicles of adult ovarian tissue. EGF receptor staining was most intense in the oocytes of primordial follicles. Weak staining for EGF receptor was observed in some surrounding pregranulosa cells. Immunohistochemical staining for EGF receptor was also present in the stromal cells of ovarian tissue, but to a much lesser degree. There was no significant difference in the immunohistochemical staining for EGF receptor in ovarian tissue before and after cryopreservation.     

Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation

BACKGROUND: Cryopreservation of ovarian tissue for fertility preservation is becoming increasingly common. Treatment of diseases that may deprive the ovaries of follicles is often performed at local hospitals that are without the necessary facilities and expertise to cryopreserve ovarian tissue. The aim of the present study was to evaluate whether primordial follicles of ovarian cortex survive transport for up to 4 h prior to cryopreservation. METHODS: Immediately after recovery of one ovary from each of four patients, the cortex was roughly isolated, placed in IVF culture medium, kept on ice and transported for 3-4 h to the centre where final dissection and cryopreservation took place. Transplantation of pieces of thawed ovarian cortex under the skin of ovariectomized immunodeficient mice for a period of 4 weeks was used to assess the survival of primordial follicles. RESULTS: After transplantation, ovarian tissue from each of the four patients contained surviving follicles. CONCLUSIONS: Transport of roughly isolated ovarian cortex cooled on ice for a period of up to 4 h allows survival of primordial follicles following cryopreservation and transplantation to immunodeficient mice.     

Development of antral follicles in human cryopreserved ovarian tissue following xenografting

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.     

Indications for cryopreservation of ovarian tissue

For patients who are planning to have chemotherapy, radiotherapyor to undergo bilateral oophorectomy, loss of ovarian functionwill result in premature ovarian menopause and loss of fertility.For these women, although there is no successful method forthe cryopreservation of human oocytes, ovarian tissue cryobankingis proposed with a view to its autotransplantation at a laterdate or the isolation and in-vitro maturation of oocytes. Embryopreservation is indeed not an option for single women and evenfor married women because delaying treatment for at least 2months of in-vitro fertilization cycles is inappropriate andlife-threatening. Following the success of animal experiments,there have been reports of ovarian cryopreservation for womenhaving to receive chemotherapy and/or radiotherapy. We presentfour case reports of ovarian tissue cryobanking and review theconsequences of chemotherapy and radiotherapy on gonadal function,as well as the indications for freezing ovarian tissue.     

Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol.

Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.     

Two novel techniques to detect follicles in human ovarian cortical tissue

BACKGROUND: Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. METHODS: The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The first method used the fluorescent mitochondrial stain rhodamine 123 (R123) in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottom dishes for detecting primordial follicles in ovarian cortical tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. RESULTS: Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glass-bottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. DISCUSSION: Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottom dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.