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INTRODUCTION: During the 25th symposium of assisted reproduction in Brno was lunch time organised as the lunch table discussion on the selected topics of assisted reproduction. More than 150 specialists reviewed themes related to gynecology and embryology.Discussed topics: Lunch table discussion covered the following topics: (1) Cross-border health care in assisted reproduction; (2) Indication for PGS (preimplantation genetic screening) in the context of actual information; (3) Does ovarian stimulation belong to the ambulance of registering gynecologists? (4) Therapy with clomifen - only for IVF specialists? (5) How and with whom should psychological support be directed during IVF? (6) Stimulation in women with low ovarian reserve; (7) Is basic semen analyses sufficient? (8) Time-lapse systems as relevant markers of embryonic development; (9) How to be oriented with choices of media and consumables in the IVF lab, and (10) "Freeze All" - is this new trend in cryopreservation suitable for all? CONCLUSIONS: Panel conclusions were presented during the afternoon session, which had great attendance, featured lively commentary, and produced some definitive consensus. Certain issues remained inconclusive, and these matters will be the subject of further discussion in the future. Specific summation of all deductions is presented in this paper.

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OBJECTIVE: Ovarian Hyperstimulation Syndrome (OHSS) is a severe health complication observed in some patients undergoing hormonal stimulation during IVF. Some authors present that OHSS is associated with Polycystic Ovary (PCO) and with a high count of growing follicles (more than 18) responding to FSH hyperstimulation, although none of them is sufficient to predict the onset of OHSS. The aim of this study is to analyze the association between PCOS and OHSS the Inhibin B-based TFF/SBM index. DESIGN: Retrospective analysis. SETTING: Assisted Reproduction Center, Department of Obstetrics and Gynecology, 1st Faculty of Medicine, Charles University and General Faculty Hospital in Prague, Sigma-Aldrich, Prague, Immunotech, a.s., Prague. METHODS: Serum and follicular fluid of 36 women (high responders with more than 18 growing follicles) in IVF program were collected at the day of oocyte pick up and used for analysis of Inhibin B. Age: mean 30.2 years (24-35 years, median 31.0 years), BMI: mean 22.34 (18.3-29.7, median 21.6). For every patient, the TFF/SBM index was calculated as follows: [concentration in FF] x [growing follicle count]/ [concentration in serum] x [body mass]. A distribution of the following parameters were compared: OHSS status, TFF/SBM index based on Inhibin B, growing follicle count and the incidence of PCOS. RESULTS: Values of the TFF/SBM index showed an association with the severe form of OHSS but not with the incidence of PCOS. CONCLUSION: These observations suggested that the incidence of PCOS is not associated with the development of severe form of OHSS, but may be still associated with a high count of growing follicles.

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OBJECTIVE: Compare the effectiveness of the IVF extended embryo culture day 4 versus day 2. DESIGN: Retrospective analysis. SETTING: Assisted Reproduction centre Apolinar, Department of Obstetrics and Gynecology, 1st Faculty of Medicine, Charles University and General Faculty Hospital in Prague. METHODS: We compared pregnancy rate (PR) and implantation rate (IR) of all IVF and IVF/ICSI embryo transfers (ET) performed between August 2004 and July 2008, in which 2 embryos were transfered. All transfers were done by the same physician. Maternal age was < or = 35 years (from 22 to 35 years, mean 30.4 years, median 31.1 years). The group of embryos evaluated and transfered on the day 2 was compared with the embryos evaluated and transfered on the day 4. Based on the evaluation, each embryo was assigned to one of the following categories: Grade A (top quality embryo) or grade B (medium quality embryo). RESULTS: On the day 2, ET of A+A embryos led to PR 69.0%; A+B 53.3% and B+B 36.4% (IR 50.0%, 40.0% and 22.7%), where number of transfers was 42, 30 and 11 in every group. On the day 4, ET of A+A embryos led to PR 54.3%, A+B 48.7%, B+B 26.6% (IR 45.6%, 34.6%, 13.3%), where number of transfers was 46.39 and 15 in every group. CONCLUSION: In our group, we found no statistical significant difference (p < 0.05) in PR between day 2 and day 4 embryotransfers. Results of the day 2 transfers have a trend to be better than results of day 4 transfers. Regardless of the transfer day, the prognosis of grade A embryos was significantly better (p < 0.05) than for grade B embryos.

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Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev, the causal agent of cucurbit downy mildew, was observed for the first time on Cucurbita moschata Duchesne in the Czech Republic (CR) in August 2009 and repeatedly in September 2010. Recently, C. moschata has not been an economically important crop in the CR; however, related crops C. pepo and C. maxima have increased in importance. Infected plants with P. cubensis were found in two locations: in a hobby garden in north Moravia (Nový Jicín - Kojetín [49°33'48.088″N, 17°59'16.632″E], 2009 and 2010) and in a commercial field in central Moravia (Olomouc-Holice [49°34'31.95″N, 17°17'35.462″E], 2010). The pathogen caused small, angular, yellowish or pale green lesions on the upper leaf surfaces and produced sporangiophores and sporangia on the lower leaf surfaces. The lesions were delimited by leaf veins and later turned necrotic. Sporangiophores were hyaline, branched, and emerged in groups from stomata. Olive brown-to-dark brown sporangia were ellipsoidal to oblong. Our morphological observations confirmed that the pathogen was P. cubensis (2). No previous reports are available of P. cubensis on C. moschata in CR or anywhere in Central Europe. However, P. cubensis is common on C. moschata in some parts of Asia and the United States (1,2). P. cubensis exhibiting clear host specialization has been reported in different countries and geographic areas (2). A C. moschata isolate (PC 88/2009) originating from the naturally infected plants was inoculated (1 × 105 spores per ml and incubation temperature of 18/15°C during light/dark cycles) according to the methodology described by Lebeda and Urban (3) onto the abaxial surface of leaf discs of all genotypes of a differential set of cucurbits for P. cubensis pathotype determination (4). C. moschata (line Novo5, Nohel-Garden, CR) was added to this set. The isolate PC 88/2009 was highly pathogenic to all screened Cucurbita spp. genotypes (C. pepo, C. maxima, and C. moschata). However, no infection was detected on most of the Cucumis accessions; only Cucumis melo subsp. agrestis var. conomon was susceptible. Also, no infection was observed on other differentials (Citrullus, Benincasa, Luffa, and Lagenaria). The pathotype was classified as Pc 4/15/0. This pathotype had not been previously detected in CR. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.arsgrin. gov/fungaldatabases/ . December 16, 2010. (2) A. Lebeda and Y. Cohen. Eur. J. Plant Pathol. 129:157, 2011. (3) A. Lebeda and J. Urban. Page 285 in: Mass Screening Techniques for Selecting Crops Resistant to Disease. M. M. Spencer and A. Lebeda, eds. International Atomic Energy Agency (IAEA), Vienna, Austria, 2010 (4) A. Lebeda and M. P. Widrlechner. J. Plant Dis. Protect. 110:337, 2003.

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OBJECTIVE: Ovarian Hyperstimulation Syndrome (OHSS) is a serious complication of In Vitro Fertilisation (IVF) treatment. It is estimated that 3-5% of treated women suffer of severe form of OHSS. OHSS pathogenesis remains unknown and its treatment is only symptomatic. It is difficult to predict the risk of OHSS of an individual woman, since a current criterion--the number of punctured follicles--is not very reliable. Our aim was to find, whether in patients with a high risk of OHSS (more than 18 follicles punctured for oocyte pick-up) is possible to predict the individual risk of OHSS. As a marker we tested concentrations of Pregnancy Associated Plasma Protein-A (PAPP-A) in follicular fluid (FF) and blood serum (S). STUDY DESIGN: Prospective study. SETTINGS: Assisted Reproduction Center, Clinic of Obstetrics and Gynecology, First Faculty of Medicine, Charles University in Prague and General Teaching Hospital in Prague. METHODOLOGY: Follicular fluid and serum of 118 female patients undergoing IVF treatment was collected at the day of oocyte pick-up and analysed for PAPP-A concentration. The resulting data were then correlated with the OHSS status of individual patient. RESULTS: Mean concentration of PAPP-A in FF was 0.81 +/- 0.29 IU/l, while in S it reached only 0.0017 +/- 0.0003 IU/l. Patients with subsequent OHSS grade 1, 2 and 3 reached FF levels of PAPP-A 0.81; 0.52; and 0.73 IU/l, respectively 0.0017; 0.0017 a 0.0017 IU/l in blood serum. No correlation was found between PAPP-A FF or PAPP-A serum concentrations and the degree of OHSS. No correlation was found between PAPP-A serum concentrations and the number of follicles. CONCLUSION: 1. Pathological response on hormonal stimulation leading to OHSS is not related to the concentrations of PAPP-A in either FF or in blood serum. 2. PAPP-A does not pass from follicles to blood in a significant amount.

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OBJECTIVE: To find the differences of polychlorinated biphenyls (PCBs) levels in follicular fluid in different indications to treatment with IVF + ET program. DESIGN: Pilot study. SETTING: Department of Obstetrics and Gynecology, 1st Faculty of Medicine, Charles University and General Faculty Hospital, Prague, AXYS Varilab, s.r.o., Vrané nad Vltavou, Prague, Euromise Centrum, Charles University and Academy of Art, Prague. METHODS: We examinated 100 infertile women undergoing IVF + ET program in our pilot study. After ovarian pick-up, frozen follicular fluid were transported to the laboratory. We detected the levels of polychlorinated biphenyls in follicular fluid of infertile women. The levels were compared according to the different indications of treatment. The control group consist of patients with male factor of sterility. RESULTS: We confirmed the higher levels of PCB 114, 153, 156, 157, 180 and 189 in indication endometriosis compared with the others and with control group. The levels of PCBs were in ng/gram of fat. CONCLUSION: It has become apparent that higher levels of PCB 114, 153, 156, 157, 180 and 189 in endometriosis patients may be one of many factors participating in the origin of the disease.

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OBJECTIVE: Summarize different ways of assisted reproduction performance evaluation. DESIGN: Review. SETTING: Centre for Assisted Reproduction, Department of Gynaecology and Obstetrics, 1st Faculty of Medicine, Charles University, Prague. METHODS: In databases of assisted reproduction centres, in national and international registers we evaluate data concerning in vitro fertilisation and related methods. Target of this evaluation is, first of all, to increase efficiency. RESULTS: Outputs of all the registers inform about absolute numbers of cycles, deliveries, born babies and about relative parameters (implantation rate, pregnancy rate), describing the efficiency of the treatment. CONCLUSION: The output evaluation must consider the influence of the age of female, number of transferred embryos and other factors.

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OBJECTIVE: The study of zona free oocytes fertilization using intracytoplasmic sperm injection (ICSI) with the aim to increase the pregnancy rate in the assisted reproduction. DESIGN: Pilot study. SETTING: Assisted Reproduction Centre, Gynecology Obstetrics Department of the 1st Medical Faculty and General Faculty Hospital, Charles University, Prague. METHODS: Zona free oocytes were fertilized using ICSI and cultured in standard conditions in vitro for 5 days until the blastocyst stage. Blastocysts were cryopreserved using 8% glycerol. RESULTS: The fertilization process of zona free oocyte was physiological, oocytes expulsed the polar body and in standard time, i.e. 18 hour post insemination, two distinct, morphologically normal pronuclei were apparent in cytoplasm. During early cleavage, from the first to the third day of in vitro culture, the dynamics of cell division was normal, the three-dimensional arrangement of cells was more flattened than in normal, zona intact embryo. The fourth and fifth day of culture, the morphological appearance of embryo corresponded to normal development. CONCLUSION: The fertilization of zona free oocytes using ICSI can give morphologically normal zygote and after 5 days culture in vitro these zygotes can develop into blastocyst stage. This method can increase the pregnancy rate in patients with repeatedly low number of oocytes or with defects of zona pellucida.

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Twitch and tetanic tension have been measured in single skeletal muscle fibres (m.ileofibularis, Rana esculenta and Rana temporaria). On the basis of resistance to fatigue produced by repetitive tetanic stable frequency stimulation with various numbers of stimulation trains, twitch fibers were subdivided in three groups (resembling those as described by Westerblad and Lännergren 1986 in Xenopus), that is fatigue-resistant (FR), moderately fatigued (MF) and easily fatiguing (EF). It was found further that the fibres differ in tetanic tension decline resistance i.e. fatiguability relating to some basic contractile parameters including the amplitude, the rates parameters of twitch and tetanus tension as well as the tetanus/twitch tension ratio. The main differences observed concern: 1) The inability to maintain the maximum tetanic tension plateau (IMT) during single tetanus. IMT was 18 times higher in EF fibres and 4 times higher in MF fibres, respectively, in comparison with FR fibres. IMT is the first parameter to change significantly during repetitive tetanic stimulation. 2) The different fibre types show pronounced differences in twitch contraction and tetanus tension during repetitive tetanic stimulation. There is a conspicuous facilitation of twitch tension during and after cessation of repetitive stimulation in FR fibres; the MF and EF fibres show, on the contrary, a depression of twitches. 3) Recovery to original (prefatigue) values is rapid in FR fibres, but slow, however, in EF fibres. 4) Removal of extracellular Ca2+ intensified the inability to maintain the maximum tetanic tension (IMT) and the tetanic tension decline, especially in fibres with an initial high fatigue-resistance. We assume that the results might be explained by a different refractoriness of transmission between the T-tubules and the sarcoplasmic reticulum in examined fibres and/or by a different dependency of the T-SR transmission on the extracellular calcium ions. A possible cause of the failure may be an intensification of the inactivation process.

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Muscle fibres of crustaceans represent an useful model for studying the mode of action of substances influencing calcium channels as the membrane generates the active responses on a pure calcium principle and the excitation-contraction link is dependent on external calcium. We followed the effect of Pb2+ ions (1-300 mumol/l) on contractile responses in single muscle fibres of the crayfish evoked by massive or intracellular electrical stimulation, by potassium depolarization and caffeine application, as well as on action potential in single intact muscle fibres and on calcium currents in voltage clamp conditions (vaseline gap) in internally perfused muscle fibre segments. All types of contractile responses, single twitches, tetanus, potassium and caffeine contractures were blocked. The strontium action potential was blocked very effectively by Pb2+ ions. The total calcium currents which can be split by means of Hodgkin-Huxley equations into two components (fast and slow respectively) differing in the rate of activation and inactivation were suppressed after addition of Pb(NO3)2 (50-300 mumol/l). The effect of Pb2+ was concentration and time dependent. At lower concentrations (100 mumol/l) the blocking effect was more pronounced on the fast inactivating Ca current component. The Pb2+ ions prolonged the time constant of inactivation tau h of the slow channel, while leaving that of the fast channel intact.

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The effect of ruthenium red (RR) on the electrical and contractile responses, membrane Ca currents, staining patterns of the external and internal membrane system were tested in intact and mechanically skinned muscle fibres of the crayfish Astacus fluviatilis. The following results were obtained: 1. Depression of the contractile responses following membrane depolarization (twitch, tetanus, potassium contractures). 2. Caffeine contractures were unaffected in intact (100 mumol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mumol/l RR). 3. Mechanical threshold and mechanical latency were increased and/or prolonged. 4. The rate of depolarization of the action potentials (AP) was decreased and decremental spread of AP was recorded. 5. Both fast and slowly inactivating Ca ionic currents were decreased and the time constants of activation (tau(m] and inactivation (tau(h] were prolonged after RR (100 mumol/l) pretreatment. 6. The penetration of RR into the T-system was inversely related to its binding to the sarcolemma. The depression of depolarization-induced contractions was most pronounced in fibres with unstained sarcolemma and stained T-tubules. In intact fibres, neither terminal cisternae nor other elements of SR were stained. On the contrary, all internal membrane structures were stained in skinned fibres. There was a gradient of staining intensity from surface toward the interior.

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Bilayer lipid membranes (BLM) of various composition were used to study the effects of local anesthetics (LA) carbisocaine and lidocaine on mechanical membrane characteristics and on the transport dynamics of ions across gramicidin D ionic channels. Carbisocaine concentrations of 20 mumols/l-0.1 mmol/l caused a considerable decrease (by 15-40%) in modulus of elasticity E1 in direction perpendicular to membrane surface. The effect of lidocaine was approx. one order of magnitude weaker. LA-induced changes in E1 were shown to depend on both the lipid composition of the membrane and the electrolyte pH. Neutral forms of LA induce marked changes in E1. An analysis of current-voltage (I-V) characteristics of BLM modified by the channel forming agent gramicidin D revealed that carbisocaine significantly affects the superlinear segment of the I-V relationship; this suggests a strong effect on the transport dynamics of ions through the internal channel region. The results of the study suggest that the action of both carbisocaine and lidocaine may be non-specific. The effectivity of the non-specific action of LA is determined by the hydrophobic moiety of the local anesthetic molecule.

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Kinetics of conductance and current-voltage characteristics (VACH) were studied on the bilayer lipid membranes from egg lecithin and cholesterol modified by gramicidin D and valinomycin. There were found significant changes of ionic channels conductance g, lifetime tau and non-linearity coefficient beta of VACH of modified membranes dependent on cholesterol content. In the region of high cholesterol concentrations, more than 20 mol%, there were observed abrupt changes of the ionic channels parameters: tau increased more than ten times, conductance decreased two times and beta greater than 0 in the whole region of electrolyte concentration.

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