维生素A酸对小鼠囊胚细胞凋亡的影响

目的:探讨全反式维生素A酸(ATRA)对体外培养小鼠囊胚中内细胞群(ICM)和滋养层(TE)细胞凋亡的影响。方法:获取妊娠3.5d小鼠囊胚,分别培养在含0μmol/l、1μmol/l和10μmol/l的ATRA的M199培养基中24h,用带有荧光(FITC)标记的原位末端标记(TUNEL)检测法,观察ATRA对小鼠囊胚细胞凋亡的效应。结果:ATRA可诱导囊胚细胞凋亡,并增强小鼠囊胚Fas蛋白表达。结论:维生素A酸对小鼠胚胎的发育具有细胞毒性作用。     

囊胚期冷冻胚胎移植的临床应用探讨

目的探讨囊胚期冷冻胚胎移植的临床应用价值.方法对2002年10月～2003年12月在湘雅医院生殖医学中心接受囊胚培养并行囊胚期冷冻胚胎移植的病例进行分析,了解囊胚期与卵裂期冷冻胚胎解冻后的胚胎成活率、植入率及妊娠率.结果冷冻囊胚解冻后胚胎数、胚胎成活率、植入率和妊娠率与卵裂期冷冻胚胎无显著差别(P＞0.05),囊胚解冻后经15 h过夜培养,42枚中有22枚成活,其中8枚Ⅰ,Ⅱ级继续发育或囊胚腔扩张,3例获得妊娠,妊娠率为37.5%.结论囊胚期冷冻胚胎移植同样能达到卵裂期冷冻胚胎移植的临床效果,同时增加解冻与移植间隔时间可更好地选择冻融囊胚移植,对提高妊娠率可能有一定意义.     

Comparison of clinical outcomes of frozen-thawed blastocysts transfer in natural and hormonally controlled cycles

Objectives:To assess the clinical outcomes of frozen-thawed blastocysts transfer in natural and hormonally controlled cycles.Methods:A retrospective analysis of natural and hormonally controlled cycle for 246 frozen-thawed blastocyst transfer cycles,the clinical pregnancy rate,implantation rate,early abortion rate were compared.Results:Of the 192 hormonally controlled cycles,the cancel rate,clinical pregnancy rate per ET,implantation rate and abortion rate were 7.3%(14/192),53.9%(96/178),38.8%(131/338)and 11.5%(11/96)respectively,whereas in 54 natural cycles,these rates were 16.7%(9/54),68.9%(31/45),52.9%(45/85)and 16.1%(5/31)respectively.There was no significant difference between the two groups with regard to the clinical pregnancy and abortion rate per ET,but the cancel rate and implantation rate were higher in natural cycles.However,the pregnancy and implantation rates of patients without PCOS in hormonal control cycles(57.2%,40.9%)were similar with those in natural cycles(P>0.05).Conclusion:These findings suggested that both hormonally controlled and natural cycles had similar pregnancy outcomes in frozen-thawed blastocysts transfer.     

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.     

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.     

Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst

During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.