激光辅助孵化对快速玻璃化冻融妊娠结局的影响

目的:探讨激光辅助孵化对移植玻璃化冻融胚胎妊娠结局的影响。方法:分析292个玻璃化冻融胚胎移植周期,其中激光辅助孵化组(171个周期)胚胎解冻后行激光辅助孵化,对照组(121个周期)胚胎解冻后不作辅助孵化,比较两组胚胎种植率和临床妊娠率,同时比较透明带削薄法与透明带打孔法对妊娠结局的影响。结果:激光辅助孵化组临床妊娠率32.35%,胚胎种植率20.70%;对照组临床妊娠率35.65%,胚胎种植率21.05%,两组间差异均无统计学意义(P>0.05)。透明带削薄法胚胎种植率及临床妊娠率均高于激光打孔法,差异有统计学意义(P<0.05)。结论:激光辅助孵化并不能提高玻璃化冻融周期的胚胎种植率和临床妊娠率,故不应常规对玻璃化冻融胚胎行激光辅助孵化;需要作激光辅助孵化时,应选择透明带削薄法。     

Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: A golgi study

Nucleus magnocellularis in the chicken consists predominantly of a population of mediu-msized cells which receive large, axosomatic endings from the auditory nerve. The morphological development of these cells and their auditory input were studied with the Golgi methods. At 7 1/2–9 days of incubation (embryonic days 7 1/2–9, staged according to the Hamburger-Hamilton series), cells in nucleus magnocellularis have several long, branched dendrites, which often end in bulbous swellings. By embryonic day 10, efferent axons have already grown out from the cells and characteristic terminal plexuses of these axons are seen in nucleus laminaris bilaterally. The dendrites of cells in nucleus magnocellularis have been replaced by a multitude of long somatic processes, giving the cell body a shaggy appearance. This arrangement is maintained up to embryonic day 15, when a remarkable second transformation occurs. The cells lose their somatic processes and present bald, round profiles. Around embryonic days 17–18 a primitive-looking process with a tip like a growth cone emerges from the cell body and somatic spines are evident. By days 19–20, one or two thin, frail dendritic processes can be seen.Correlated with this dramatic series of changes in the cells is a fixed sequence of transformations of the incoming axons. Around embryonic day 10, primary sensory axons in nucleus magnocellularis end in swellings resembling growth cones. Between days 11 and 13, following the explosive growth of somatic processes there is a corresponding expansion and ramification of the auditory nerve endings. On embryonic day 14, there is a condensation of the terminal axon branches, which now form a compact, highly branched plexus. Between days 16 and 17, the plexus coalesces into a calycine structure, now approaching its final form, the end-bulb of Held, which is achieved by embryonic days 19–20. The transformation of the plexus to the calycine form occurs around the same time that the cell loses its somatic processes.The parallel sequence in the morphogenetic stages of the assembly of the end-bulbs and their target cells evinces a correlation, if not a causal relationship between the sensory axons and the developing neurons. The arrangement of the somatic processes and axonal branches during the early, multipolar stage would provide an opportunity for optimum interactions between the synaptogenetic processes of the afferent axons and the target cells. The later morphological transformations could orchestrate the specific, cell-to-cell interactions which accompany, or even depend on the activity of the definitive end-bulb synapse.     

In situ detection of apoptotic cells by TUNEL in the gill epithelium of the developing brown trout (Salmo trutta)

Apoptosis is a form of naturally occurring cell death during development and it is characterised by extensive DNA fragmentation. Apoptosis is easily detected in the gill epithelium of brown trout embryos in ultrathin sections (Rojo et al. 1997). Here we provide the first biochemical evidence for apoptosis in the gill epithelium of brown trout embryos, using in situ end-labelling of DNA breaks (Gavrieli et al. 1992). Embryos at d 57 of development as well as those at hatching, were processed to analyse the distribution of apoptotic cells in the gills. The extent of apoptosis revealed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling method technique is considerably greater than apoptosis detected by nuclear morphology. This method revealed that apoptosis was frequent at hatching, although it was also present during embryonic development. The presence and distribution of stained nuclei were different depending on the developmental stage. In embryos of 57 d, apoptotic flattened nuclei were dispersed in the gill epithelium, whereas at hatching, they were mainly grouped in the tips of the filaments and in the gill arches. TUNEL also revealed a distinct pattern of nuclear staining: at hatching, the intense staining covered the entire cell, but in embryos it was restricted to the nucleus. These results show the functional relevance of apoptosis at hatching, when apoptosis seems to be the unique process by which cell numbers in the gill epithelium are adjusted, in order to prepare for the new extrinsic conditions affecting the free-living life of alevins.     

Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing

BACKGROUND: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage. METHODS: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles. RESULTS: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development. CONCLUSIONS: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.     

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching

Immature oocyte recovery followed by in-vitro oocyte maturationand in-vitro fertilization is a promising new technology forthe treatment of human infertility. The technology is attractiveto potential oocyte donors and infertile couples because ofits reduced treatment intervention. Immature oocytes were recoveredby ultrasoundguided transvaginal follicular aspiration. Oocyteswere matured in vitro for 36–48 h followed by intracytoplasmicsperm injection (ICSI). Embryos were cultured in vitro for 3or 5 days before replacement. Assisted hatching was performedon a day 5 blastocyst stage embryo. Embryo and uterine synchronywere potentially enhanced by luteinization of the dominant follicleat the time of immature oocyte recovery. Mature oocyte and embryoproduction from immature oocyte recovery were similar to theprevious IVF results of the patients. A blastocyst stage embryo,produced as a result of in-vitro maturation, ICSI, in-vitroculture and assisted hatching, resulted in the birth of a healthybaby girl at 39 weeks of gestation.     

Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system

Purpose A noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-sec pulses at a wave-length of 2.1 m and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two-to four-cell FVB mouse embryos.Results In the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO₂ humidified air incubator at 37°C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group.Conclusions These preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.     

Fertilization and early embryology: Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon

Of 48 spare human pre-embryos achieving the expanded blastocyststage, 22 (45.6%) secreted significant amounts of human chorionicgonadotrophin (HCG) (>5 IU/l/day). Of these, nine remainedintrazonal, seven partially hatched and six fully hatched. Embryonicproduction of HCG in vitro appeared to be time-dependent, startingafter a certain minimum time (160 h post-insemination) and risingexponentially, with maximal HCG production around day 10. Hatchingwas not a prerequisite for HCG secretion, since similar amountswere produced by intrazonal blastocysts. Blastocysts derivedfrom abnormally fertilized oocytes also began secreting HCGexponentially but secretion was delayed and the upper limitof maximum HCG secretion rate was comparatively low. The actualamount of HCG is thought to reflect the number of viable trophectodermcells producing the hormone. HCG doubling times for blastocystsin vitro were rapid when compared to implanting blastocystsof a similar age in vivo, with 19/22 (86.4%) blastocysts havinga doubling time of < 10 h. Provided a pre-embryo can secreteHCG and maintain an adequate doubling time, sufficient HCG shouldbe produced for initial stages of embryonic recognition in vivo.Since intrazonal blastocysts are capable of fulfilling bothof these criteria, the limiting factor in realizing their fullpotential may be escaping from the zona pellucida.     

Continuous pressure measurements in the evaluation of patients for laser assisted uvulopalatoplasty

The aim of the present study was to evaluate the effect of laser-assisted uvulopalatoplasty (LUPP) performed under local anesthesia in an outpatient setting. No procedure included tonsillectomy. Obstruction related mainly to the velopharyngeal segment of the airway was defined in 16 consecutive patients by clinical examination, nocturnal polysomnography and pressure measurements. The mean follow-up period was 7 months, range 3–16 months. Using five pressure sensors, four separate upper airway segments could be defined. The preoperative location of obstructive segments in obstructive apneas was proximal and was identified in the velopharyngeal segments in 90% of the patients. This was the case in 92% of the patients with hypopneas. After LUPP, there were statistically significant improvements in the duration of respiratory events (P < 0.02), incidence of sleep with snoring (P < 0.001), apnea-hypopnea index (P < 0.01), microarousal index (P < 0.02) and the mean duration of non-rapid eye movement sleep with oxygen saturation < 80% (P < 0.0001). Four patients still had proximal obstructions after LUPP, but these were clinically tolerable.     

胚胎透明带显微操作与单卵双胎妊娠

目的探讨体外受精-胚胎移植(IVF-ET)后单卵双胎妊娠与胚胎透明带显微操作的关系。方法回顾分析在我院行IVF-ET后获得临床妊娠的1,099个周期,比较行胚胎透明带显微操作(ZPM)和未经显微操作的两组患者单卵双胎(MZT)妊娠的发生率。结果IVF-ET后临床妊娠1,099周期中,MZT16周期,发生率为1.46%,占多胎的7.6%。显微操作组临床妊娠462周期,MZT6周期,发生率为1.30%;无显微操作组临床妊娠637周期,MZT10周期,发生率为1.60%,两组无显著差异(P>0.05)。结论单卵双胎妊娠与胚胎透明带显微操作之间无密切联系,积极控制IVF-ET的多胎妊娠可能是减少单卵双胎的有效途径。