Cardiotypic development in embryonic stem cellderived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase-like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H 2 O 2 or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N-acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3-kinase (PI-3-kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI-3-kinase in the regulation of the intracellular redox state. ß

The effects of electromagnetic fields (EMFs) on the differentiation of cardiomyocytes in embryoid bodies derived from pluripotent embryonic stem (ES) cells were investigated. A single direct current (DC) field pulse was applied to 4-day-old embryoid bodies. The electrical field induced a hyperpolarization of the anode-facing side of embryoid bodies and a depolarization at the cathode-facing side. Significant effects of a single electrical field pulse applied for 90 s on cardiomyocyte differentiation were achieved with field strengths of 250 and 500 V/m, which increased both the number of embryoid bodies differentiating beating foci of cardiomyocytes and the size of the beating foci. The 500-V/m electrical field increased intracellular reactive oxygen species (ROS), but not [Ca(2+)](i) and activated nuclear factor kappa B (NF-kappaB). A comparable increase in the number of beating embryoid bodies was achieved by an incubation for 1 h with H(2)O(2) (1-10 nM), indicating that the electrical field effect was transduced via the intracellular generation of ROS. Because the radical scavengers dehydroascorbate and pyrrolidinedithiocarbamate (APDC) and the NF-kappaB antagonist N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibited cardiac differentiation, we assume that ROS and NF-kappaB may play a role in early cardiac development.

As early as 1985, ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.

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The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-upprepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 mL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 mL of spermatozoa vitrified in 50-mL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P , .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P , .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P . .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

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The attitude toward initiating dialysis in neonates is changing and long-term outcome in the absence of severe comorbidity is promising. Prenatal prediction concerning respiratory and renal outcome in fetuses with ROH is difficult. Our data suggest that BOI (1d) and onset of ROH may be reliable predictors of respiratory prognosis in children born with ROH.