水通道蛋白3在小鼠早期胚胎发育过程中的表达

目的 了解水通道蛋白3(aquaporin3,AQP3)在小鼠早期胚胎发育中的表达分布情况.方法 建立昆明小鼠控制性超排卵模型,收集小鼠四细胞期、八细胞期、桑葚期胚胎及早期囊胚,采用免疫荧光显微镜和激光共聚焦显微镜对AQP3在小鼠早期胚胎中的表达模式及亚细胞定位进行检测分析.结果 在小鼠早期胚胎的4个时期中均能检测到特异性AQP3荧光信号,激光共聚焦显微镜观察到AQP3的分布部位在各时期不完全相同,在四细胞期胚胎及八细胞期胚胎主要定位于卵裂球核膜,桑葚期胚胎主要定位于细胞膜,而早期囊胚主要定位于滋养层细胞膜及细胞质.结论 AQP3对小鼠早期胚胎的发育可能具有潜在的调控作用.     

Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos

OBJECTIVE: To evaluate the efficacy and efficiency of freezing cleaved human embryos through vitrification. DESIGN: Clinical study of vitrification of human embryos. SETTING: Assisted reproductive technology centers. PATIENT(S): Thirty-six patients undergoing IVF-ICSI treatment whose surplus embryos were frozen. INTERVENTION(S): Two hundred fifteen surplus embryos vitrified, subsequently thawed, and transferred in natural or controlled cycles. MAIN OUTCOME MEASURE(S): Embryo survival rate after thawing and resultant patient pregnancy rate. RESULT(S): From the 215 vitrified and thawed embryos, 106 survived, with an overall embryo survival rate of 49.3%. The survival rate was higher when embryos were vitrified at the eight-cell stage compared with at the six to seven-cell and six-cell stages (79.2%, 39.7%, and 21.1%, respectively). On average, 2.9 +/- 1.2 embryos per patient were transferred, resulting in 11 pregnancies (30.5%), with an implantation rate of 10.4% per embryo transferred. CONCLUSION(S): Ultrarapid embryo freezing by vitrification of eight-cell stage embryos is a reliable method, as evidenced by high rates of embryo survival and pregnancy, making it a superior alternative to the conventional slow-cooling method.     

Twin pregnancy after vitrification of 2-pronuclei human embryos

OBJECTIVE: To report an ongoing twin pregnancy after transfer of embryos that were vitrified at the 2-pronuclei stage in a new vitrification solution. DESIGN: Case report. SETTING: A tertiary-care infertility clinic. PATIENT(S): A 26-year-old infertile woman in whom two previous IVF implantations failed. INTERVENTION(S): Vitrification of 2-pronuclei embryos, in vitro culture for 48 hours, and transfer into the uterus. MAIN OUTCOME MEASURE(S): Survival and cleavage after vitrification and achievement of clinical pregnancy. RESULT(S): Six zygotes were vitrified by using a three-step protocol (4% ethylene glycol for 3 minutes, 20% ethylene glycol for 1 minute, and 38% ethylene glycol and 1.2 M trehalose for 0.5 minute). After 2 months of storage in a double-straw system in liquid nitrogen, two zygotes were warmed and cryoprotectants were removed by using a four-step protocol (1 M, 0.5 M, 0.25 M, and 0.125 M of trehalose). Two embryos were transferred after 48 hours of in vitro culture, cleaving to 5 and 6 cells. The resulting twin pregnancy was confirmed by ultrasonography at the sixth week. CONCLUSION(S): Vitrification by using ethylene glycol and trehalose appears to be a safe, promising method for cryopreservation of human zygotes. Storage of vitrified zygotes in a double-straw system does not compromise their subsequent potential for survival and development.     

Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologic studies

Cryopreservation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 100% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at 0 degree C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P less than 0.01) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 100% or 90% in HEPES buffered saline and 10 minutes' exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 70 and 80% solution and exposure for 5, 15, 20, or 30 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VS1 and cooling to 0 degree C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 101 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro.     

Closed-system solid surface vitrification versus slow programmable freezing of mouse 2-cell embryos

Purpose  To compare closed-system solid surface vitrification with slow freezing. Methods  Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. Results  Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). Conclusions  Closed-system vitrification was more effective than conventional slow freezing. Capsule   Closed system solid surface vitrification was more effective than conventional slow freezing in the cryopreservation of mouse 2-cell embryos.     

The effect of serum obtained before and after treatment for endometriosis on in vitro development of two-cell mouse embryos.

OBJECTIVE: To evaluate the effect of serum obtained before and after treatment for endometriosis on in vitro development of two-cell mouse embryos. DESIGN: Pretreatment and post-treatment comparison of mouse embryo development in serum supplements from patients with endometriosis; results were compared using chi 2 analysis. SETTING: Infertility Clinic, Shimane Medical University, Izumo, Japan. PATIENTS: Ten consecutive women with endometriosis who underwent treatment for the disease. Seven women who were managed expectantly served as controls. INTERVENTIONS: All patients underwent laparoscopic or conservative surgery. This was followed by a 6-month course of either danazol 400 mg/d (6 patients) or buserelin acetate 900 micrograms/d (4 patients). MAIN OUTCOME MEASURE(S): Total number of embryos that reached blastocyst stage after 72 hours of incubation were compared before and after treatment. RESULTS: Before treatment, 23% of the embryos reached blastocyst stage, whereas 8 of the 10 serum samples were embryo toxic. After treatment, significantly more embryos (59%) developed to blastocysts, and only one serum sample remained embryo toxic. In the control group, there was no significant difference in the number of embryos that reached blastocyst stage after expectant treatment. CONCLUSION: The embryo toxicity of serum samples from patients with endometriosis is lost after treatment.     

Sequential observation of mitochondrial distribution in mouse oocytes and embryos

Objective The purpose of this study was to elucidate changes in the distribution of mitochondria through the cell cycle.Materials and Methods Mouse oocytes and embryos were recovered sequentially from mice and stained with the vital fluorescent mitochondrial stain rhodamine 123. Mitochondrial staining pattern were classified into three types: aggregation (Ag), homogeneous (H), and perinuclear accumulation (PA).Results Sequential observations revealed that mitochondria of oocytes and embryos grown in vivo translocated in the cytoplasm during the cell cycle, showing the H pattern be fore human chorionic gonadotropin (hCG) administration, the PA pattern 8–9 hr post-hCG, the H pattern again 10–14 hr post-hCG, and the PA pattern again 24 and 31–32 hr post-hCG following fertilization. In the twocell stage, the Ag pattern was shown 35 hr post-hCG, the H pattern was observed 40 hr post-hCG, and the PA pattern was found 48 hr post-hCG. In the embryos cultured in vitro and showing developmental block, mitochondrial translocation was shown to be inhibited after they aggregated in the early two-cell stage (35 hr post-hCG). Moreover, the translocation of mitochondria was restored by the addition of superoxide dismutase or thioredoxin to the culture medium. Both of these enzymes have already been shown to have the ability to overcome developmental block.Conclusion The present study revealed that mitochondria translocated in the cell cycle and suggested that there is a close relationship between mitochondrial translocation and developmental arrest.     

Cryopreservation of human embryos by vitrification

Vitrification is a cryopreservation strategy where cells are converted into a glass-like amorphous solid which is free of any crystalline structure. Such process is achieved by a combination of high concentration of cryoprotectant and an extremely high cooling rate. In the last years, survival rates of up to 80% after thawing and pregnancy rates of almost 30% could be achieved after transfer of vitrified embryos at the zygote, cleavage, morula and blastocyst stages. Also deliveries of healthy babies have been reported numerous times. To this day, a limited interest in this technique can be noted. The explanation may lye in the apprehension of many ART units regarding exposure of embryos to high concentrations of cryoprotectants and storage in non sterile conditions. The aim of the first part of this article, is to analyse if such fears are justified on the basis that vitrification mimics conditions already in use for many years in slow-cooling procedures where cells are plunged into liquid nitrogen at around -30 degrees C and secondly since storage of embryos are now possible in high aseptic conditions. In the second part, results on survival after thawing, pregnancy rates and baby take home rates of vitrified embryos will be presented and the problems associated with vitrification of blastocysts will be discussed.     

A closed system supports the developmental competence of human embryos after vitrification

Purpose

Closed-system vitrification may enable the risk of contamination to be minimised. We performed three studies to compare the developmental competence of human embryos vitrified using either a closed vitrification system (CVS; Rapid-i®) or an open vitrification system (OVS; Cryo-top®).

Methods

The first study was performed in vitro using 66 zygotes previously vitrified at pronuclear stage. These were warmed and randomised 1:1 to revitrification using either the OVS or the CVS. After re-warming, embryo development and blastocyst cell number were assessed. For the second study, also performed in vitro, 60 vitrified–warmed blastocysts were randomised 1:1:1 into three groups (OVS or CVS revitrification, or no revitrification). The proportion of dead cells was assessed by staining. The third study was performed in vivo, using 263 high-grade blastocysts randomly assigned to vitrification using either the CVS (n = 100) or the OVS (n = 163). After warming, single blastocyst transfer was performed.

Results

There were no differences between the CVS and the OVS in survival rate (100 % vs. 97 %), blastulation rate (96 h: 50 % vs. 50 %; 120 h: 68 % vs. 56 %), proportion of good blastocysts (96 h: 32 % vs. 22 %, 120 h: 47 % vs. 41 %), or mean number of cells (137 vs. 138). The proportion of dead cells in blastocysts re-vitrified by CVS (31 %) was similar to that for OVS (38 %) and non-revitrification (32 %). In vivo, the implantation rate for blastocysts vitrified using the CVS (54 %) was similar to that with the OVS (53 %).

Conclusion

Our studies consistently indicate that human embryos may be vitrified using a CVS without impairment of developmental competence.     

The effects of vitrification on gene expression in mature mouse oocytes by nested quantitative PCR

Purpose  

This study was conducted on the effects of vitrification cryotop method on gene expression of mature oocytes in Mus musculus.     

The chromosome complement of first-cleavage mouse embryos after in vitro fertilization

A comparison of the first cleavage-stage chromosome complements of 1022 in vivo fertilized mouse embryos and 1033 in vitro fertilized mouse embryos is reported. The chromosome analysis of first-cleavage embryos allows us to study directly the chromosome complement of the sperm and oocyte that contribute to the embryo, since both chromosome clusters remain separate when an antimitotic agent is used to prevent syngamy. In this paper we show that the sex ratio and the incidence of aneuploidy are similar, irrespective of the fertilization system used. Male and female gametes have the same levels of aneuploidy. Triploidy is more frequent in the in vitro fertilized embryos and the difference can be ascribed to a higher incidence of polyspermy and diploid spermatozoa.     

Cryopreservation of animal and human embryos by vitrification

Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For embryo cryopreservation, the vitrification method has advantages over the slow freezing method. For example, injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short period without a programmed freezer. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulphoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and/or a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conventional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation.