The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.

The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.     

异丙酚麻醉不同阶段对卵细胞受精和对早期胚胎质量的影响

目的探讨异丙酚静脉麻醉下取卵手术中卵泡抽吸液中异丙酚的浓度变化及各阶段对卵细胞受精和早期胚胎质量的影响.方法 17例实施超促排卵下体外受精-胚胎移植术的妇女预测卵泡至少有15个,按取卵的先后分3组(早、中、晚)分别培养.记录从静脉给异丙酚到每个卵泡取出时间.检测卵泡抽取液中异丙酚的浓度及观察卵细胞的受精率及卵裂率等.结果取出第G一个卵泡的平均时间是200秒,取出卵泡的平均速率是17.6s/ 个.所有患者的最后一个卵泡抽吸液中异丙酚的浓度明显高于第一个卵泡抽吸液中异丙酚的浓度(P<0.01).除未成熟的卵细胞占总卵泡百分比在3组间有明显的区别外,受精率和卵裂率无明显区别.结论随着麻醉的进展,卵泡抽吸液中异丙酚的浓度逐渐增加,但与所给剂量或持续的时间之间无必然的关联.我们不能显示卵泡抽吸液中升高的异丙酚浓度对卵细胞质量的不利影响.     

In normal development pulmonary veins are connected to the sinus venosus segment in the left atrium

Background: Classic theories descibe that the common pulmonary vein develops as an outgrowth from either the sinus venosus or atrial segment. Recent studies show that the pulmonary veins are connected to the sinu-atrial region before its differentiation into a sinus venosus and atrial segment. Methods: The development of the sinu-atrial region with regard to the developing common pulmonary vein and the growth of the atrial septum was investigated in avian embryos, using both scanning electron microscopy and immunohistochemistry. Embryos ranging between stage HH12 and HH28 were incubated with QH-1 that recognizes quail endothelial cells and precursors, HNK-1, that appears in this study to detect the myocardium of the sinus venosus, or with HHF-35, being specific for muscle actins. Also vascular casts of the heart were produced by injecting prepolymerized Mercox into the vascular system. Results: In preseptation stages the common pulmonary vein drains into the left part of the sinus venosus, that is clearly demarcated by the sinuatrial fold and HNK-1 expression. During atrial septation the left part of the sinus venosus, in contrast to the right part, loses its HNK-1 antigen from stage HH23 onwards, while at the same time the sinu-atrial fold in the left atrial dorsal wall flattens and disappears. From stage HH25 onwards HNK-1 expression is restricted to the right part of the sinus venosus, which contributes to the right atrium. The myocardial atrial septum never expresses the HNK-1 antigen, suggesting that the septum is of atrial origin. Discussion: It appeared that the sinus venosus does not only contribute to the sinus venarum of the right atrium, but also to the left atrium. © 1995 Wiley-Liss, Inc.     

促进胚胎干细胞来源造血干/祖细胞移植后细胞免疫功能恢复

目的:体外诱导胚胎干细胞分化为造血干/祖细胞过程中, 增加成熟T淋巴细胞的含量, 以促进其重建致死量照射小鼠的造血功能后免疫功能的早期重建。方法:胚胎干细胞在含甲基纤维素的培养基中自由分化形成胚胎体, 分化第6d添加造血生长因子, 同时添加胸腺肽, 流式细胞仪检测分化细胞中CD₃₄⁺的造血干/祖细胞和CD⁺₃的成熟T淋巴细胞含量, 最后将分化细胞注射入致死量照射小鼠体内, 观察60d, 以移植物抗宿主病(GVHD)发病率作为T淋巴细胞免疫功能的指标, 用PCR检测Sry反映移植细胞在宿主体内的存活。结果:分化第13d, 未加胸腺肽, CD⁺₃的成熟T淋巴细胞含量仅10.52%, 重建造血后无GVHD发生;添加胸腺肽, CD⁺₃的成熟T淋巴细胞含量升高达22.93%, 重建造血后GVHD发病率100%。结论:胚胎干细胞体外分化为造血干/祖细胞过程中, 添加胸腺肽, 能增加CD⁺₃的成熟T淋巴细胞含量, 体内重建造血后细胞免疫功能恢复较快。     

Cryopreservation of human oocytes: a review of current problems and perspectives

It is well recognized that the ability to cryopreserve unfertilizedhuman oocytes would make a significant contribution to infertilitytreatment. However, despite considerable interest, very fewsuccessful pregnancies have arisen from cryopreserved oocytesafter thawing, insemination and transfer of the subsequent embryo.The reasons for this lack of progress may well result from adearth of information on how the various biophysical changesduring a cryopreservation regimen affect human oocyte function.Recently, fundamental studies on the effects of cooling, membranepermeability, cryoprotectant addition and ice formation havebeen performed on human oocytes by a number of groups, and theseform the basis of the current review. It is likely that successfulhuman oocyte cryopreservation will only follow once these factorsare fully understood, but the existing base of knowledge shouldprovide a platform for further improvements in the techniquescurrently employed.     

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.     

光照对胎兔视觉器官发育影响的研究

近的来众多学者对振声等刺激范围的胎教研究已广泛开展，有些已在临床实际应用中取得了良好的成效。而光照机制的研究，特别是有关胎体在光照条件下视觉器官所发生的变化研究报道尚属罕见。本研究应用组织学技术对胎兔在光照强度不同的条件下视觉器官所发生改变的实验结果，进行了分析，并证实在一定的光照强度及限定时间内胎兔视神经、视网膜等组织发育状况良好。本研究结果为进一步开展恍照胎教的理论学研究及早日应用于临床提供了     

Embryo donation: attitudes toward donation procedures and factors predicting willingness to donate

BACKGROUND: The aim of the study was to assess infertile couples' attitudes toward the procedures of embryo donation (ED) and to identify factors predicting interest in donation. METHODS: Fifty-one couples who had received IVF treatment and had subsequently had embryos cryopreserved for >3 years were located and sent written information about the procedures for ED and possible implications of donation. A total of 49 couples agreed to participate in the study with 36 women and 31 men subsequently returning questionnaires describing their reasons for not claiming unused embryos and attitudes towards ED. RESULTS: Patients were supportive of donor screening procedures, but less comfortable sharing non-identifying information. Comfort levels declined as information became increasingly personal. Support for unconditional (i.e. the donation of embryos without conditions attached) and conditional (i.e. where couples could limit the donation of their embryos to persons/couples according to their preferences) models of donation was highly polarized and a substantial minority expressed strong opposition to each model. Willingness to donate was associated with greater comfort about disclosing personal information, a desire to know the outcome of donation and willingness to have future contact with a child, but not with current family size. CONCLUSIONS: Comfort in sharing information with a recipient couple is more important than acceptance of screening procedures, or attainment of family size goals in predicting willingness to donate embryos. Offering the option of conditional donation could increase the acceptability of ED for some patients.     

Fertilization and early embryology: The effect of equilibration temperature and time on the outcome of ultrarapid freezing of 1-cell mouse embryos

Mouse 1-cell embryos were frozen ultrarapidly at a rate of 2500°C/minin solutions containing 0.25 M sucrose, 0.5% (w/v) bovine serumalbumin (BSA) and 3 or 4.5 M dimethyl sulphoxide (DMSO) or 3or 4.5 M 1, 2-propanediol (PROH) in HEPES-buffered modifiedEarle's medium. We investigated the effect of pre-freeze equilibrationfor 1, 3, 5 or 10 min at 22°C and for 1, 3, 5, 10, 15 or20 min at 4°C. After thawing in a 22°C water bath ata rate of 2500°C/min and dilution in 1 M sucrose in HEPES-bufferedmodified Earle's medium, embryos were cultured in vitro in bicarbonate-bufferedmodified Earle‘s medium with 0.5% (w/v) crystalline BSA.Embryo viability was expressed as the percentage of hatchingor hatched blastocysts resulting from the initial number offrozen-thawed 1-cell embryos. To determine the toxicity of thefreezing solutions, embryo viability was evaluated after equilibrationwithout freezing. Our results demonstrated that the concentration,the equilibration temperature and time are very important factorsin ultrarapid freezing of mouse 1-cell embryos. Optimal viabilitywas found when equilibration was done in 4.5 M DMSO for 3–5min at 22°C and in 4.5 M PROH for 3–5 min at 4°C.The results with regard to exposure to the freezing solutionsindicated that the loss of viability beyond an optimum is notdue solely to cryoprotectant toxicity, in particular not at4°C and not for DMSO. It is suggested that the temperatureand time of equilibration influence the degree of cryoprotectantpermeation and subsequent rehydration, which play a role indetermining freezing susceptibility in terms of ice formation.We conclude that both DMSO and, in contradiction to previousreports, PROH can be used perfectly adequately for ultrarapidfreezing on condition that concentration, and the temperatureand time of equilibration are controlled.     

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.