玻璃化冷冻技术临床应用的安全性

随着体外受精一胚胎移植（IVF—ET）及其衍生技术的不断发展,低温冷冻保存配子、胚胎以及卵巢或睾丸组织已经成为人类辅助生殖技术（ART）中一个重要的环节。近年来,玻璃化冷冻技术成功用于冻存人类卵母细胞和胚胎。然而,玻璃化冷冻要达到普及性应用之前仍有许多安全性问题有待解决,包括高浓冷冻保护剂毒性、开放式冷冻载体交叉污染、液氮病原微生物潜在污染以及复苏后胚胎发育潜能及损伤鉴定等。度     

保存女性生育力技术研究进展

保存女性生育力技术是低温生物学与生殖医学紧密结合的学科。本文介绍了保存女性生育力的方法、适用范围、临床应用、发展趋势以及研究进展，并就卵母细胞玻璃化冷冻和卵巢组织冷冻的发展方向作一概述。     

卵母细胞玻璃化冷冻技术在体外受精-胚胎移植中的应用

目的探讨卵母细胞玻璃化冷冻技术在体外受精-胚胎移植中的应用价值。方法选取取卵日取精失败进行卵母细胞冷冻的174枚卵母细胞,按冷冻方法分为两组,A组共101枚卵母细胞,行慢速程序化冷冻;B组共73枚卵母细胞行玻璃化冷冻。比较两组卵母细胞的复苏率、受精率、2PN受精率、可利用胚胎率、优质胚胎率、临床妊娠率。结果 B组(玻璃化冷冻组)的复苏率、受精率、2PN率、可利用胚胎率、优质胚胎率,均显著高于A组(慢速冷冻组),两组患者平均年龄、平均移植胚胎数、妊娠率均无显著性差异。结论与慢速冷冻相比,卵母细胞玻璃化冷冻的冷冻效率更高,卵母细胞玻璃化冷冻是体外受精治疗中取精失败后保存女性生育力最佳的补救措施,避免了女性生殖细胞的浪费。     

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。     

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     

人成熟卵母细胞玻璃化冷冻技术的临床应用

目的探讨玻璃化冷冻技术应用于人成熟卵母细胞冷冻保存的临床效果。方法使用开放式载体cryotop进行人成熟卵母细胞的玻璃化冻存,对10例患者的冷冻卵子复苏后进行卵母细胞浆内单精子（ICSI）注射后行胚胎移植,观察复苏卵母细胞受精、卵裂、胚胎发育及临床妊娠情况。结果 10例患者共86枚玻璃化冻存的成熟卵母细胞复苏后77枚存活,69枚正常受精,获得65枚胚胎,共移植17枚胚胎,3例患者获临床妊娠,其中2例双胎妊娠。结论玻璃化冷冻技术是有效地保存人成熟卵母细胞的方法,有一定的临床应用价值。     

卵母细胞深低温保存的策略分析

背景：卵母细胞具有特殊而复杂的低温生物学性质,致使卵母细胞在深低温保存中易受到多因素的损伤而影响复温后的存活率、受精率和受精后发育潜能。 目的：综述卵母细胞深低温保存技术的研究进展,阐明存在的技术缺陷以及解决问题的思路与研究路线。 方法：以“卵母细胞,深低温保存/慢速冷冻,玻璃化”为中文捡索词,以“oocyte, cryopreservation/slow freezing, vitrification”为英文检索词,在中国知网(CNKI)期刊全文数据库和PubMed数据库检索2004 年1月至2014年10月有关卵母细胞深低温保存文献,排除陈旧性、重复性研究,最终纳入41篇文献进行综述。 结果与结论：慢速冷冻、玻璃化方法深低温保存卵母细胞得到较好的临床应用,但复温存活率、受精能力、发育能力损伤等仍困扰医生们。卵母细胞深低温保存技术的进一步完善或突破在于对深低温保存技术环节的细化、卵母细胞低温生物学特性的研究以及相关技术的创新,如深低温保护剂选择、冷冻载体的改进、卵母细胞时相选择、卵母细胞体外成熟技术、卵巢组织保存与移植等。     

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。     

GMP玻璃化法冻存人早期胚胎的临床应用

目的探讨GMP玻璃化法冷冻技术的临床应用效果。方法应用GMP玻璃化法冷冻技术保存了解12例患者的33个优质胚胎并复苏后移植。结果复苏了12例患者的33枚胚胎,32枚存活并移植,胚胎存活率为96.97%（32/33）,6枚胚胎种植,种植率为18.75%（6/32）,5例获得妊娠,妊娠率为41.67%（5/12）。结论GMP玻璃化法是一种可应用于临床的冷冻保存技术。     

卵巢组织的冷冻及其应用的研究进展

卵巢不仅是女性的内生殖器官也是重要的内分泌器官，对于女性生育力和第二性征的维持具有重要的意义。女性肿瘤患者特别是年轻女性由于接受各种抗肿瘤治疗如手术、放疗、化疗，有可能会导致卵巢早衰甚至会切除卵巢从而导致终身不孕，丧失生育力。目前保存女性生育力的方法主要有胚胎冷冻保存，卵母细胞冷冻保存和卵巢组织冷冻保存。相对于前两种方法卵巢组织冷冻保存有其优越性。对于那些因疾病必须切除卵巢，或必须行放疗、     

Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report

We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.     

Ovarian tissue banking for cancer patients: fertility preservation, not just ovarian cryopreservation

While ovarian tissue cryopreservation has commonly been equated with fertility preservation in cancer patients, there is a range of alternative options to preserve fertility. Based on the type and timing of chemotherapy, the type of cancer, the patient's age and the partner status, a different strategy of fertility preservation may be needed. If the patient has a partner or accepts donor sperm, embryo cryopreservation should be considered first, since this is a clinically well established procedure. Despite relatively low pregnancy rates, when there is time for ovarian stimulation and the patient is single, oocyte cryopreservation may also be preferred to ovarian tissue banking. In breast cancer patients, tamoxifen or aromatase inhibitors can be used for ovarian stimulation prior to oocyte or embryo cryopreservation. In endometrial cancer patients, aromatase inhibitors may be the only choice for ovarian stimulation. When only pelvic radiotherapy is used, ovarian transposition can be performed, but the success rates vary because of scatter radiation and vascular compromise. Lack of FSH and GnRH receptors on primordial follicles and oocytes does not make gonadal suppression an effective strategy of gonadal protection. Fertility preservation should be an integral part of improving the quality of life in cancer survivors; however, it is neither possible nor ethical to recommend the same recipe for every cancer patient.     

Human oocyte cryopreservation

The clinical role of oocyte cryopreservation in assisted reproduction, as an adjunct to sperm and embryo cryopreservation, has been comparatively slow to evolve as a consequence of theoretical concerns related to efficacy and safety. Basic biological studies in the 1990's alleviated many of these concerns leading to more widespread adoption of the technology. While a number of babies were born from the approach validated in the 1990's, its perceived clinical inefficiency led to the search for improved methods. Introduction of elevated dehydrating sucrose concentrations during cryopreservation increased survival and fertilization rates, but there is no well-controlled evidence of improved clinical outcome. Similarly, the use of sodium-depleted cryopreservation media has not been demonstrated to increase clinical efficiency. More recently, and in the absence of basic biological studies addressing safety issues, the application of vitrification techniques to human oocytes has resulted in reports of a number of live births. The small number of babies born from clinical oocyte cryopreservation and the paucity of well-controlled studies currently preclude valid comparisons between approaches. Legal restrictions on the ability to select embryos from cryopreserved oocytes in Italy, where many of the available reports originate, also obscure attempts to assess oocyte cryopreservation objectively.     

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。     

Ovary cryopreservation and transplantation for fertility preservation

The aim of this review is to summarize the state-of-the-art of ovarian transplantation and cryopreservation. This field has progressed over the last half century from simple animal experiments to sophisticated application in humans. The initial poor results in humans began to improve when a series of nine monozygotic (MZ) twin pairs discordant for premature ovarian failure (POF) underwent ovary transplantation at one center. All of these fresh ovary transplants were successful, resulting in 11 healthy babies in 7 of the 9 recipients. The same surgical techniques were then applied to 3 frozen ovary tissue transplants, up to 14 years after the ovary had been frozen, resulting in 3 more healthy babies. Around the world, the number of healthy babies has now risen to 28. Even ovary allotransplantation is being attempted in the not so uncommon situation where a previous bone marrow donor is now willing to donate ovarian tissue to the same recipient. Recipients routinely reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 4.5 months. Most conceived naturally (three of them twice or three times from the same graft). The duration of function of fresh ovarian grafts, contrary to initial expectations, indicated minimal oocyte loss from ischemia time. Grafts of just modest portions of ovarian tissue have lasted >7 years. In vitro studies suggest that vitrification of ovarian tissue may be an improvement over the 70% oocyte viability loss from slow freeze.     

卵子冷冻技术在IVF-ET中的应用

介绍作为辅助生殖技术一项新技术，卵子冷冻技术，方法 包括卵子冷冻的选取。卵子的预处理，卵子的冷冻，复苏，复温，复苏后的培养与受精，以及影响卵子冻存率和受精率的因素。文中还介绍了该技术的临床应用和发展前景。     

Ovarian tissue cryopreservation and transplantation: a review

The review covers current options for ovarian tissue cryopreservation and transplantation and provides a systematic review of the existing literature from the last 10 years, taking into account all previously published reviews on the subject. The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The choice depends on various parameters: the type and timing of chemotherapy, the type of cancer, the patient's age and the partner status. The different options and their results are discussed, as well as their putative indications and efficacy. The review concludes that advances in reproductive technology have made fertility preservation techniques a real possibility for patients whose gonadal function is threatened by premature menopause, or by treatments such as radiotherapy, chemotherapy or surgical castration.     

Fertility after breast cancer

Breast cancer is the most common tumor in childbearing women. In the last decades, considerable improvement in breast cancer-related death has been achieved with adjuvant therapies (chemotherapy, endocrine and targeted therapies, radiotherapy) but at cost of significant long-term sequels, including infertility. Reproductive issues are of great importance to young women, in particular for those who did not complete their families before breast cancer diagnosis: patients should be adequately informed at the time of diagnosis about the risk of infertility and the available methods for fertility preservation. This review will focus on incidence and impact of infertility secondary to breast cancer treatment, the available options for ovarian function preservation, including embryo and oocyte cryopreservation, ovarian tissue cryopreservation, and ovarian suppression with gonadotropin-releasing hormone agonists. We will also discuss the optimal time of subsequent pregnancy, the potential risks for the mother and the fetus, and the impact of therapies on breastfeeding.