胚胎玻璃化冷冻技术的临床应用(附成功分娩1例报道)

目的 探讨胚胎玻璃化冷冻技术的临床应用效果。方法 运用玻璃化冷冻技术保存75例患者的397个胚胎，复苏13例69个胚胎，并根据患者的情况进行胚胎移植。结果 69个胚胎中，丢失3个，存活26个，存活率为39，4％。复苏后8例患者因胚胎质量差未移植，另外5例患者共移植了14个胚胎，其中1例获得单胎妊娠，分娩1名健康女要。结论 玻璃化冷冻技术简便、经济，并能够有效地冻存人类早期胚胎，可成为胚胎冷冻方法的另一个选择。     

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.     

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.     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.     

Study on the developing potentiality of mouse morula produced in vitro or in vivo after vitrification

Great horovement haS been made in the cryopreservation Of inalnlnalian elnbmp since wnttingham['] reported his Pioneering wold about successful cryOPreservationof mouse embryo. It results in the availability of cryOPreserVation of embryos of all stages[']. HOwever, cryopreserved embryos can not avoid the damages by ice crystal,which is a major cause of cen death in the freezing andthawing ProceduresL',']. SO it is necesseq to seareh for anew and more efficient method for embryO cryOPrese…     

Measurement of Glass Transition Temperatures of Freeze-Concentrated Solutes by Differential Scanning Calorimetry

Thermal analysis of aqueous solutions in which the solute does not crystallize immediately upon freezing was carried out to define the effects of experimental parameters on thermograms in the glass transition region. The intensity of enthalpy relaxations in the glass transition region is related to both the rate of cooling and the rate of heating through the glass transition region—slow cooling or slow heating increases the extent of structural relaxation in the glassy state and increases the intensity of the endotherm. Plots of the logarithm of heating rate versus l /Tg are linear, and activation enthalpies for structural relaxation are in the range of 210–350 kJ/mol. For polymeric solutes, both the activation enthalpies for structural relaxation and the heat capacity change accompanying the glass transition increase with increasing molecular weight of the solute. Molecular weight dependence of the observed midpoint of the glass transition agrees with the Fox–Flory relationship. Results are compared and contrasted with glass transitions in solid polymers and with the glass transition of hyperquenched water. Practical implications for characterization of formulations intended for freeze-drying are discussed.     

两种冷冻保护剂玻璃化冷冻小鼠卵巢的比较研究

目的:探讨两种玻璃化冷冻保护剂对小鼠卵巢组织学和功能的影响。方法:将23只4周龄ICR雌鼠随机分为新鲜卵巢移植组(6只)、去势组(5只)、EG40组(6只)和ED20组(6只)。EG40组和ED20组分别应用乙二醇(EG)和联合应用EG与二甲基亚砜(DMSO)玻璃化冷冻小鼠卵巢组织,一周后解冻,将一部分复苏卵巢组织自体移植入小鼠肾被膜下,另一部分组织行组织学观察。移植术后5d开始观察所有小鼠的动情周期,一个月后处死小鼠,对存活的卵巢组织行组织学观察。结果:EG40组、ED20组和新鲜卵巢移植组小鼠动情周期出现率均为100%,出现动情周期的天数分别为10.2±1.2d、8.0±0.9d和6.8±1.0d,EG40组动情周期出现天数明显多于新鲜卵巢移植组(P<0.01);ED20组与新鲜卵巢移植组差异无显著性(P>0.05)。移植存活的卵巢组织内可见不同发育阶段的卵泡,形态正常,但冻融卵巢组织内卵泡数量比新鲜组织少。结论:联合应用玻璃化冷冻保护剂EG和DMSO对小鼠卵巢组织学和功能的影响较小。     

Effects of different open cryo-carriers on embryo survival and clinical outcome in frozen embryo transfer cycle patients

The purpose of this study was to compare the efficacy of different open cryo-carriers: the CryoloopTM, CryotopTM, and CryoleafTM, in embryo survival and clinical outcome in patients with frozen embryo transfer (FET) cycle. We analyzed the embryo survival rate and clinical outcome in 325 patients of 348 FET cycles vitrified with the CryoloopTM (160 cycles), CryotopTM (105 cycles), or CryoleafTM (83 cycles). No significant differences were observed in embryo survival rate (98.8% vs. 100% vs. 97.7%, p > 0.05), HCG positive rate (58.8% vs. 63.8% vs. 57.8%, p > 0.05), biochemical pregnancy rate (6.9% vs. 11.4% vs. 9.6%, p > 0.05), or implantation rate (33.2% vs. 37.4% vs. 34.1%, p > 0.05) in the three groups respectively. The early abortion rate of the CryoloopTM group was significantly higher than that of the CryotopTM and CryoleafTM group (27.1% vs. 3.6% and 7.5%, p < 0.05). At the same time, the average female age of the CryoloopTM group was significantly older by 1 year than that of the CryotopTM and CryoleafTM group (33.29 ± 4.71 years vs. 31.96 ± 4.27 years and 31.1 ± 4.28 years, p < 0.05). There was no significant difference in take home baby rate (38.1% vs. 46.7% vs. 43.4, p > 0.05) or birth weight among the groups (2893.5 ± 780.8 g vs. 2778.4 ± 710.0 g vs. 2724.5 ± 838.8 g, p > 0.05). No case of neonatal malformation was observed in the present study. Overall, CryotopTM and CryoleafTM were effective for embryo vitrification at both the cleavage and blastocyst stage according to the results of clinical outcome and infant characteristics. However, CryoloopTM led to a decreased positive HCG rate and increased early abortion rate, heightened at the cleavage stage.

Abbreviations: LN₂: liquid nitrogen; CPA: cryoprotectant; ART: assisted reproductive technology; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; BMI: body mass index; FSH: follicular stimulation hormone; COH: controlled ovarian hyperstimulation; FET: frozen embryo transfer; mm: millimeter; HCG: human chorionic gonadotropin; RCT: randomized clinical trial; NC: natural cycle; AC: artificial cycle; EM: equilibration medium; DMSO: dimethyl sulphoxide; EG: ethylene glycol; VM: vitrification medium; WM: warming medium     

Potential use of melatonin supplementation to protect vitrified testicular grafts from hypoxic–ischaemic damage

This study aimed to assess the morphological changes in neonate vitrified testicular grafts after host treatment with melatonin. Neonate vitrified testes, candidates for transplantation to treated and nontreated groups receiving melatonin, were thawed in media containing or not containing 100 μm melatonin. Following transplantation, melatonin (20 mg kg^?1 per day) and saline were given to the treated and nontreated groups for up to 9 weeks. The testicular status was assayed by terminal deoxynucleotidyl transferase (TdT)‐mediated deoxyuridine triphosphate (dUTP)‐biotin nick‐end labelling TUNEL, semi‐thin section and ultra‐structure studies. Chi‐squared test was used to compare categorical variables between the groups. Overall, the degenerative and apoptosis changes in the vitrified testis parenchyma were not significant. However, atrophic seminiferous cords and jumbled appearance of the interstitial space were more often observed in the nontreated group than in the treated ones. Semi‐thin sections showed that the germinal epithelium was taken in a normal arrangement on the testicular grafts of both groups. Nevertheless, ultrastructural analysis revealed that the characteristics of interstitial space cells, basement membrane BM and epithelial cells of seminiferous tubules in the treated group were better than those in the nontreated group. The study revealed a beneficial effect of melatonin on vitrified neonatal testes and after that, on restoring testicular grafts.     

组织玻璃化冻存技术：优势与尚未完全解决的问题

文题释义：玻璃化冷冻保存：是利用这些高浓度的低温保护剂组合成玻璃化冻存液,通过与水分子发生强烈的水合作用,增加溶液黏性,降低冰晶形成速度,从而使细胞在快速降温或复温过程中得以保护。 玻璃化：对于非晶高分子,当高分子通过降温从高弹态转变为玻璃态,或者通过升温从玻璃态转变为高弹态的过程称之为玻璃化转变,发生玻璃化转变的温度叫玻璃化转变温度。对于结晶高分子,玻璃化转变是指其非晶部分所发生的由高弹态向玻璃态(或者玻璃态向高弹态)的转变。因此,玻璃化转变是高分子中普遍存在的现象。但是玻璃化转变现象并不局限于高分子,一些小分子化合物也存在玻璃化转变。 背景：玻璃化冷冻保存是一种应用前途广阔的低温冷冻方法,通过使用高浓度的玻璃化冻存试剂将生物材料进行玻璃态转变,从而实现活性保存。 目的：就玻璃化冻存的生物学原理及玻璃化冻存试剂的分类,卵巢、皮肤与角膜等医学组织标本的玻璃化冻存进行综述。 方法：以“tissue;vitrification;cryopreservation”为英文检索词;“组织;玻璃化;冷冻保存”为中文检索词,检索1994年1月至2019年10月 PubMed 数据库及万方医学网相关文献。按照纳入与排除标准筛选后,对最终纳入的45篇文献进行归纳总结。 结果与结论：玻璃化冻存可以防止细胞内外冰晶形成,避免了冰晶给细胞带来的多种损伤,有效保留了细胞的生物活性与基本功能。玻璃化冻存试剂主要分为渗透性和非渗透性2种,其操作简便、高效,唯一的缺点是高浓度的冻存试剂对细胞具有一定的毒性损伤。为了降低对组织整体损伤风险,可以混合使用多种低毒冻存试剂。目前玻璃化冻存技术已经成功应用于多种细胞,但组织冻存的技术难题尚未完全解决。 ORCID: 0000-0003-0140-9935(张源) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程