Fertilization and early embrology: Monozygotic twinning in the human is associated with the zona pellucida architecture

Six cases of identical twin pregnancies which occurred in 2163cycles of in-vitro fertilization during a 3 year period arereported. Monozygosity was confirmed when the number of fetusesexceeded the number of embryos replaced (n = 4) or when twoconcepti were observed in a single amniotic sac (n = 2). Eachof the reported pregnancies resulted from replacement of embryoseither with naturally thin zonae pellucida or embryos whosezonae had been breached during micromanipulation for assistedfertilization (subzonal sperminsertion) or assisted hatching.That such cases exclusively gave rise to monozygosity suggestsa link between the physical state of the zona pellucida, hatching,and generation of identical twins.     

Transfer of zona-free embryos improves outcome in poor prognosis patients: a prospective randomized controlled study

Assisted zona hatching (AZH) has been used in IVF programmes for several years. Recently one group has reported successful pregnancies after transfer of zona-free blastocysts. The aim of our study was to evaluate outcomes after transfer of zona-free day 3 embryos. Two groups of women undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Group A consisted of 52 women under the age of 40 years undergoing their first ICSI attempt. They were alternately randomized to receive zona-free embryos (27 women) and zona-intact embryos (25 women). The second group (group B) included 71 women with a poor prognosis, as defined by age 40 years or more, and/or at least two previous failed IVF/ICSI attempts. They were randomized in a 3:4 ratio (30 zona-free, 41 zona-intact). Acid Tyrode's solution was used to remove the zona pellucida before embryo transfer on day 3 after oocyte collection. The pregnancy rate in group A was not significantly improved when the zona pellucida was removed. However, in the poor prognosis group B, zona removal resulted in a significantly higher pregnancy rate when compared with controls (23 versus 7.3%). We conclude that complete removal of the zona pellucida can improve pregnancy rates in women with poor IVF/ICSI prognosis.     

Dual function of Langerhans cells in skin TSLP-promoted TFH differentiation in mouse atopic dermatitis

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Infertility: Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates?

In two separate prospectively randomized trials, intracytoplasmicsperm injection (ICSI) cycles were studied in a controlled mannerto monitor the effects of either bovine oviductal epithelialcell co-culture (n = 119) or assisted hatching by zona drilling(n = 100). In the first study, immediately following ICSI, alleggs were placed directly either onto partial monolayers ofbovine oviductal cells or into regular culture medium. Althoughthe embryo developmental rate was apparently compromised inpart by the presence of the co-culture cells, ultimately therewere no significant differences in either the viable pregnancyrate (31.6% co-culture versus 29.0% control) or the embryonicimplantation rate (11.4% co-culture versus 13.6% control). Assistedhatching also had no significant impact on ICSI cycle outcomein terms of either the viable pregnancy rate (30.0% assistedhatching versus 32.0% control) or the embryonic implantationrate (8.5% assisted hatching versus 13.5% control). However,in female patients aged 235 years, assisted hatching appearedto convey a marginally significant benefit in terms of boththe viable pregnancy rate (35.5% assisted hatching versus 11.1%control) and the embryonic implantation rate (103% assistedhatching versus 3.1 % control). It seems that the overall improvementof ICSI cycle outcome cannot be achieved by the general applicationof either co-culture or assisted hatching. Nevertheless, itis possible that there remain specific patient groups that mightbenefit from selected use of either of these modalities.     

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.     

Laser-assisted zona pellucida thinning prior to routine ICSI

BACKGROUND: In MII oocytes showing difficult oolemma breakage, ICSI can cause an increase in the degeneration rate. This may be overcome by laser-assisted ICSI using a 5-10 micro m opening in the zona pellucida for injection. However, such a small opening might impair the hatching process, especially if assisted hatching is applied in addition. In order to prevent this, the present study used routine injection through an area of zona pellucida in which laser zona thinning had been applied, providing for both a reduced mechanical stress to the oocyte and assisted hatching. METHODS: This prospective study involved 100 cycles with 1016 MII oocytes. Conventional ICSI (control group) was compared with a modified laser-assisted ICSI (study group) in sibling oocytes. In the latter group oocytes were injected through an extended area of zona thinning. RESULTS: Degeneration rate was significantly lower in the study group (P < 0.004). There were no differences in fertilization, or formation and quality of blastocysts. In the study group embryo quality on day 2 was significantly better (P = 0.004) and herniation of day 5 blastocysts was increased (P = 0.005). Rates of implantation and pregnancy were not affected. However, on day 3 laser-assisted ICSI proved beneficial (P = 0.038) in terms of clinical pregnancy rate. CONCLUSIONS: The new method combines a less invasive ICSI technique with assisted hatching. Our preliminary data indicate that in addition to an improved oocyte survival, this new approach increases the hatching rate in vitro, which may explain the increase in pregnancy rate, at least in day 3 transfers.     

Effects of thyroxine and temperature manipulations upon the hatching of chick embryos (Gallus domesticus)

Thyroxine levels within chick embryos (Gallus domesticus) and incubation temperature were manipulated late in development in order to determine those events most proximal to the time of hatching that are both necessary and sufficient for the appearance of the hatched state. Events recorded include the time of breathing initiation, changes in breathing rate, time of puncture of the chorioallantoic membrane and changes in rate of loss of yolk sac material. It was found that a certain level of thyroxine is necessary for an animal to hatch, that breathing and puncture of the chorioallantoic membrane are necessary but not sufficient to predict the occurrence of hatching, and that a change in rate of loss of yolk sac material during the last 18 hr prior to hatching is sufficient to predict the occurrence of hatching. The explanatory hypothesis offered is that the endogenous increase in thyroxine levels acting via increased metabolic rates is necessary and sufficient for the animals to hatch.     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

石灰水对埃及伊蚊不同发育阶段影响研究

目的通过石灰水对埃及伊蚊生活史幼期不同阶段生长发育影响的实验室研究,评价生石灰对防治埃及伊蚊的干预效果,为生石灰用于埃及伊蚊的防治提供实验数据。方法将一定数量的埃及伊蚊依据不同发育阶段(卵、Ⅰ~Ⅳ龄期蚊幼、蛹)分别饲养于石灰水中,观察蚊幼活动情况,记录不同时期相应孵化时间、孵化率和死亡率,同时将实验室过夜自来水(实验室脱氯水)饲养蚊虫作对照,分析比较不同饲养环境、不同发育阶段埃及伊蚊发育的变化差异。结果对照组埃及伊蚊卵的平均孵化时间为1.75 d,而试验组(1.3 g/L石灰水饲养)埃及伊蚊卵的平均孵化时间为3.84 d,是对照组的2.19倍,孵化时间的差异具有统计学意义(t=36.02,P<0.05)。试验组埃及伊蚊卵孵化率为59.3%,对照组为89.3%,两组之间孵化率的差异具有统计学意义(χ^(2)=35.38,P<0.05)。Ⅰ~Ⅳ龄试验组蚊幼死亡率分别为44.0%、92.0%、97.3%、98.7%,显著高于对照组蚊幼死亡率,两组之间死亡率的差异具有统计学意义(χ^(2)值为65.79~276.53,P<0.05)。试验组蛹的死亡率为98.7%,显著高于对照组的死亡率,两组之间死亡率的差异具有统计学意义(χ^(2)=280.35,P<0.05)。对照组埃及伊蚊卵、蚊幼和蛹的死亡率分别为10.7%、2.3%和2.0%,三组之间死亡率的差异具有统计学意义(χ^(2)=25.09,P<0.05)。对照组埃及伊蚊卵的死亡率明显高于蚊幼和蛹。试验组埃及伊蚊卵、蚊幼和蛹的死亡率分别为40.7%、83.0%和98.7%,三组之间死亡率差异具有统计学意义(χ^(2)=170.88,P<0.05)。结论石灰水对埃及伊蚊不同发育阶段均具有明显抑制作用,对蛹的杀灭作用最大,其次是蚊幼和卵。随着埃及伊蚊幼虫年龄的增长,石灰水对其抑制效果逐渐增强。但生石灰对埃及伊蚊成蚊的防治效果有待进一步的现场验证。     

A simplified technique for embryo biopsy: Use of the same micropipette for zona drilling and blastomere aspiration

Purpose: Using different micropipettes for zona drilling and blastomere aspiration for embryo biopsy is prevalent at centers of preimplantation genetic diagnosis. The purpose of our study was to simplify the technique by using only one micropipette. Methods: In this animal model, ICR mouse embryos at the four-cell stage (n=446) were randomly allocated into two groups: a biopsied group (n=224) for blastomere aspiration and a control group (n=222) without micromanipulation. We used a drilling/biopsy micropipette to drill a hole in the zona by expulsion of acidified Tyrode’s solution and to aspirate the blastomere by gentle suction with the same micropipette and pull it out of the zona. One blastomere was biopsied from each embryo. Results: In all, 222 (99.1%) intact blastomeres were successfully biopsied from 224 embryos. Only two blastomeres were damaged during aspiration. The capacity for blastocyst development (92.4 vs 93.7%) was not different between the two groups, but the percentages of embryos hatching (51.8 vs 18.0%) and hatched (29.9 vs 8.1%) were significantly higher in the biopsied group than in the control group. Conclusions: This simplified technique of embryo biopsy is safe and highly efficient for obtaining blastomeres for preimplantation genetic diagnosis and may also facilitate hatching of the blastocysts.