Promastigote secretory gel from natural and unnatural sand fly vectors exacerbate Leishmania major and Leishmania tropica cutaneous leishmaniasis in mice.

Leishmania rely heavily on glycans to complete their digenetic life cycle in both mammalian and phlebotomine sand fly hosts. Leishmania promastigotes secrete a proteophosphoglycan-rich gel (Promastigote Secretory Gel, PSG) that is regurgitated during transmission and can exacerbate infection in the skin. Here we explored the role of PSG from natural Leishmania-sand fly vector combinations by obtaining PSG from Leishmania (L.) major-infected Phlebotomus (P.) papatasi and P. duboscqi and L. tropica-infected P. arabicus. We found that, in addition to the vector's saliva, the PSG from L. major and L. tropica potently exacerbated cutaneous infection in BALB/c mice, improved the probability of developing a patent cutaneous lesion, parasite growth and the evolution of the lesion. Of note, the presence of PSG in the inoculum more than halved the prepatent period of cutaneous L. tropica infection from an average of 32 weeks to 13 weeks. In addition, L. major and L. tropica PSG extracted from the permissive experimental vector, Lutzomyia (Lu.) longipalpis, also exacerbated infections in mice. These results reinforce and extend the hypothesis that PSG is an important and evolutionarily conserved component of Leishmania infection that can be used to facilitate experimental infection for drug and vaccine screening.

Induction of lipogenesis in white fat during cold exposure in mice: link to lean phenotype.

This corrects the article DOI: 10.1038/ijo.2016.228.

Induction of lipogenesis in white fat during cold exposure in mice: link to lean phenotype.

BACKGROUND/OBJECTIVE: Futile substrate cycling based on lipolytic release of fatty acids (FA) from intracellular triacylglycerols (TAG) and their re-esterification (TAG/FA cycling), as well as de novo FA synthesis (de novo lipogenesis (DNL)), represent the core energy-consuming biochemical activities of white adipose tissue (WAT). We aimed to characterize their roles in cold-induced thermogenesis and energy homeostasis. METHODS: Male obesity-resistant A/J and obesity-prone C57BL/6J mice maintained at 30 °C were exposed to 6 °C for 2 or 7 days. In epididymal WAT (eWAT), TAG synthesis and DNL were determined using in vivo 2H incorporation from 2H2O into tissue TAG and nuclear magnetic resonance spectroscopy. Quantitative real-time-PCR and/or immunohistochemistry and western blotting were used to determine the expression of selected genes and proteins in WAT and liver. RESULTS: The mass of WAT depots declined during cold exposure (CE). Plasma levels of TAG and non-esterified FA were decreased by day 2 but tended to normalize by day 7 of CE. TAG synthesis (reflecting TAG/FA cycle activity) gradually increased during CE. DNL decreased by day 2 of CE but increased several fold over the control values by day 7. Expression of genes involved in lipolysis, glyceroneogenesis, FA re-esterification, FA oxidation and mitochondrial biogenesis in eWAT was induced during CE. All these changes were more pronounced in obesity-resistant A/J than in B6 mice and occurred in the absence of uncoupling protein 1 in eWAT. Expression of markers of glyceroneogenesis in eWAT correlated negatively with hepatic FA synthesis by day 7 in both strains. Leptin and fibroblast growth factor 21 plasma levels were differentially affected by CE in the two mouse strains. CONCLUSIONS: Our results indicate integrated involvement of (i) TAG/FA cycling and DNL in WAT, and (ii) hepatic very-low-density lipoprotein-TAG synthesis in the control of blood lipid levels and provision of FA fuels for thermogenesis in cold. They suggest that lipogenesis in WAT contributes to a lean phenotype.

Trypanosomatids in ornithophilic bloodsucking Diptera.

Trypanosomes are known as widespread blood parasites of birds; however, knowledge of their prevalences in vectors and their overall biodiversity is rather limited. To assess the prevalences in potential vectors, we have microscopically examined ornithophilic bloodsucking Diptera (Culicidae, Simuliidae and Hippoboscidae) for the presence of trypanosomatids in their guts. In total, 3270 specimens were dissected, namely Culex pipiens Linnaeus, 1758 (n = 898), C. modestus Ficalbi, 1890 (136), Simulium vernum (Macquart, 1838) (1455), S. angustipes Edwards, 1915 (221) and Ornithomyia avicularia (Linnaeus, 1758) (560). All insect species were found to be infected with trypanosomatids, and the prevalence ranged from 4 to 8% but reached 60% in S. vernum. Blackflies and hippoboscids exclusively harboured trypanosomes (both T. cf. avium s.s. Danilewsky, 1885; T. corvi/culicavium group in hippoboscids). Mosquitoes were infected with T. culicavium Votypka, 2012 and T. avium s. l. but also with monoxenous parasites, namely Crithidia brevicula Frolov and Malysheva, 1989, and Paratrypanosoma confusum Votypka and Lukes, 2013. Only 4% of the isolated parasite strains were monoxenous whereas the majority were avian trypanosomes, confirming the vectorial status of the studied insects.

[Analysis of complications and clinical and pathologic factors in relation to the laparoscopic cholecystectomy]. / Analýza komplikací a klinicko-patologických faktoru ve vztahu k laparoskopické cholecystektomii.

INTRODUCTION: Laparoscopic cholecystectomy (LCHCE) is connected with a different spectrum of postoperative complications than classic cholecystectomy. The aim of this study was to analyse the complications and define their relation to some chosen clinical and pathological factors and their incidence. MATERIAL AND METHODS: We included 1014 patients that were operated (laparoscopic or converted cholecystectomy) at the Department of Surgery, University Hospital in Pilsen between January 1st 2008 and December 31st 2011. All included patients were at least one year after cholecystectomy in the time of the realization of this study therefore we could analyse late complications. The group of patients included 351 men (34.6%) and 663 women (65.4%). The median age was 57 years (range: 8-87 years). We evaluated the following clinical and pathological factors: a histopathological diagnosis, early surgical and internal complications (with manifestation within 30 days after cholecystectomy) classified by the Clavien-Dindo score, incisional hernia as a late complication, conversion, perforation of the gallbladder wall, the duration of hospital stay, the age of patients and the use of drain. The results of the study were calculated with use of statistical tests: the test of population probability, the two-sample t-test, the ODDS ratio) and then were compared with published literature data. RESULTS: Surgical and internal complications occurred in 138 patients (13.6%) within 30 postoperative days. The most common complication was connected with the wound - 92 patients (9.1%), then a hematoma in the gallbladder bed - 16 patients (1.6%) and a biliary leak - 16 patients (1,6%). The bile duct injury which needed a reoperation affected 3 patients (0.3%). The laparoscopic operation had to be converted in 77 cases (7.6%). Incisional hernia after the placing of trocars or in the scar after the laparotomy (in the case of conversion) occurred in 32 patients (3.16%) during one year after the cholecystectomy. The influence of the age of patients on the incidence and the spectrum of postoperative complications were proved as statistically significant. Postoperative complications were more common in the case of male gender. We found a correlation between the diagnosis and postoperative complications. Men with solitary cholecystolithiasis had more common incidence of hematoma in the gallbladder bed. A biliary leak occurred three times more often after the procedures with a gallbladder wall perforation. Three of four bile duct injuries occurred during the non-converted laparoscopic cholecystectomies. CONCLUSION: Our study proved the influence of the age of patients, the histopathological diagnosis, conversion and the perforation of gallbladder wall on the incidence of postoperative complications after laparoscopic cholecystectomy. The incidence of bile duct injury, which is the most serious complication, in our group of patients is comparable with published literature.

Probing high-affinity 11-mer DNA aptamer against Lup an 1 (ß-conglutin).

Aptamers are synthetic nucleic acids with great potential as analytical tools. However, the length of selected aptamers (typically 60-100 bases) can affect affinity, due to the presence of bases not required for interaction with the target, and therefore, the truncation of these selected sequences and identification of binding domains is a critical step to produce potent aptamers with higher affinities and specificities and lowered production costs. In this paper we report the truncation of an aptamer that specifically binds to ß-conglutin (Lup an 1), an anaphylactic allergen. Through comparing the predicted secondary structures of the aptamers, a hairpin structure with a G-rich loop was determined to be the binding motif. The highest affinity was observed with a truncation resulting in an 11-mer sequence that had an apparent equilibrium dissociation constant (K D) of 1.7 × 10(-9) M. This 11-mer sequence was demonstrated to have high specificity for ß-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin. Finally, the structure of the truncated 11-mer aptamer was preliminarily elucidated, and the GQRS Mapper strongly predicted the presence of a G-quadruplex, which was subsequently corroborated using one-dimensional NMR, thus highlighting the stability of the truncated structure.

Selection of 2'F-modified RNA aptamers against prostate-specific antigen and their evaluation for diagnostic and therapeutic applications.

Currently, prostate-specific antigen (PSA) is considered to be the most sensitive marker available for prostate cancer detection and for monitoring of disease progression. In addition to its importance as a tumor marker, PSA has a role in the biological activity of cancer growth and proliferation. Therefore, the inhibition or activation of its biological activity may be used in prostate cancer therapy. Here, we describe the isolation and characterization of new 2'F-modified RNA aptamers directed against PSA. Binding studies demonstrate the ability of these new aptamers to specifically recognize their target with dissociation constants in the nanomolar range. In order to demonstrate the functionality of the selected aptamers, an apta-PCR approach was used for the quantitative detection of PSA, achieving a limit of detection of 11 nM. Furthermore, the potential use of the selected aptamers in therapeutics was demonstrated with the 2'F-modified aptamers being highly stable in human serum and having the ability to moderate the activity of PSA, which will be explored for the treatment of prostate cancer.

Comparison of different methods for generation of single-stranded DNA for SELEX processes.

Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).

Synergistic induction of lipid catabolism and anti-inflammatory lipids in white fat of dietary obese mice in response to calorie restriction and n-3 fatty acids.

AIMS/HYPOTHESIS: Calorie restriction is an essential component in the treatment of obesity and associated diseases. Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) act as natural hypolipidaemics, reduce the risk of cardiovascular disease and could prevent the development of obesity and insulin resistance. We aimed to characterise the effectiveness and underlying mechanisms of the combination treatment with LC n-3 PUFA and 10% calorie restriction in the prevention of obesity and associated disorders in mice. METHODS: Male mice (C57BL/6J) were habituated to a corn-oil-based high-fat diet (cHF) for 2 weeks and then randomly assigned to various dietary treatments for 5 weeks or 15 weeks: (1) cHF, ad libitum; (2) cHF with LC n-3 PUFA concentrate replacing 15% (wt/wt) of dietary lipids (cHF + F), ad libitum; (3) cHF with calorie restriction (CR; cHF + CR); and (4) cHF + F + CR. Mice fed a chow diet were also studied. RESULTS: We show that white adipose tissue plays an active role in the amelioration of obesity and the improvement of glucose homeostasis by combining LC n-3 PUFA intake and calorie restriction in cHF-fed mice. Specifically in the epididymal fat in the abdomen, but not in other fat depots, synergistic induction of mitochondrial oxidative capacity and lipid catabolism was observed, resulting in increased oxidation of metabolic fuels in the absence of mitochondrial uncoupling, while low-grade inflammation was suppressed, reflecting changes in tissue levels of anti-inflammatory lipid mediators, namely 15-deoxy-Δ(12,15)-prostaglandin J(2) and protectin D1. CONCLUSIONS/INTERPRETATION: White adipose tissue metabolism linked to its inflammatory status in obesity could be modulated by combination treatment using calorie restriction and dietary LC n-3 PUFA to improve therapeutic strategies for metabolic syndrome.

Spatial feeding preferences of ornithophilic mosquitoes, blackflies and biting midges.

The section of habitat used by particular bloodsucking insects when seeking bloodmeals may influence the spectrum of hosts to which they have access and consequently the diseases they transmit. The vertical distribution of ornithophilic bloodsucking Diptera (Culicidae, Simuliidae and Ceratopogonidae) was studied using bird-baited traps set at both ground and tree canopy levels. In total, 1240 mosquito females of eight species, 1201 biting midge females of 11 species, and 218 blackfly females of two species were captured during 2003-2005. Culex pipiens (L.) (Diptera: Culicidae) was found to prefer ground-level habitats, whereas Anopheles plumbeus (Stephens) (Diptera: Culicidae), biting midges [Culicoides spp. (Diptera: Ceratopogonidae)] and Eusimulium angustipes (Edwards) (Diptera: Simuliidae) preferred the canopy. The results of this study with regard to Cx. pipiens behaviour differ from those of most previous studies and may indicate different spatial feeding preferences in geographically separate populations. The occurrence of E. angustipes in the canopy is concordant with its role in the transmission of avian trypanosomes. These findings may be important for surveillance programmes focusing on ornithophilic Diptera which transmit various pathogenic agents.

[The use of IgG antibody avidity assays in the diagnosis of cytomegalovirus infection]. / Vyuzití testu avidity IgG protilátek v diagnostice cytomegalovirové infekce.

Human cytomegalovirus (CMV) is the most common cause of congenital infection. Primary CMV infection can lead to severe disease and complications in patients immunocompromised as a result of disease or therapy. IgG antibody avidity assays make it possible to differentiate between primary infection and reactivation of latent infection or reinfection. The study objective was to determine CMV IgG avidity by enzyme-linked immunosorbent assay (ELISA) with denaturation of IgG antibody binding to the antigen and by chemiluminiscent microparticle immunoassay (CMIA) on an Abbott Architect analyzer. Both methods yielded comparable CMV IgG avidity results. In some cases, the Abbott test was superior in reflecting IgG antibody maturation during primary infection to microplate ELISA using antigen-antibody complex dissociation by a denaturing agent.

Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples.

BACKGROUND: Reactive oxygen species (ROS) are essential for sperm function. However, excessive ROS production can impair sperm function and might be a factor contributing to male infertility. METHODS: We investigated the levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) as well as lipid peroxidation, as represented by thiobarbituric acid reactive species (TBARS), in blood and seminal plasma of 38 normozoospermic males from infertile couples (NSI-males), compared with that of 17 fertile volunteers (FV-males). RESULTS: TBARS levels in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.0002, P < 0.0003, respectively), as were AA levels (P < 0.0003, P < 0.00004, respectively). On the contrary, the blood and seminal plasma levels of DHA were lower in NSI-males than in FV-males (P < 0.02 and P < 0.05, respectively). The AA/DHA ratios in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.003, P < 0.0007, respectively). Significant correlations between seminal and blood plasma levels of TBARS (P < 0.0001, r = 0.548), AA (P < 0.0001, r = 0.571) and DHA (P < 0.0001, r = 0.506) were found. CONCLUSIONS: Our data provide new insight into lipid metabolism in male infertility and indicate that systemic oxidative stress resulting in increased lipid peroxidation and an altered fatty acid profile may be, at least in part, responsible for infertility even in normozoospermic males.

[Tick-borne encephalitis in the East Bohemia Region and its microbiological diagnostic pitfalls]. / Klíst'ová encefalitida ve vychodoceském regionu a úskalí její mikrobiologické diagnostiky.

AIM OF THE STUDY: The analysis of cases of tick-borne encephalitis (TBE) laboratory diagnosed at the Department of Virology of the Institute of Clinical Microbiology of the University Hospital in Hradec Králové in 2003-2006 and comparison of the data from 2003-2006, 1999-2002 and 1995-1998. Another objective was to compare two routinely used ELISA (enzyme-linked immunosorbent assay) diagnostic kits for TBE. MATERIAL AND METHODS: The samples from 3284 patients tested by the Department of Virology of the University Hospital in Hradec Králové in 2003-2006 were analyzed. The detection of the specific IgM and IgG antibodies in serum and cerebrospinal fluid specimens was performed using two ELISA diagnostic kits (Euroimmun, Germany, and Test-Line Ltd, Clinical Diagnostics, Czech Republic). Non-specific reactivity of IgM antibodies was only analyzed in 2004-2006. RESULTS: In 2003-2006, TBE was diagnosed in 163 patients of the University Hospital in Hradec Králové aged from 3.5 months to 80 years. The number was by 48 cases (29%) higher than that in 1995-1998 and by 36 cases (22%) higher than that in 1999-2002. TBE was confirmed in 94 males (57.7%) and 69 females (42.3%). More than half of the patients were aged above 41 years (94/163, i.e., 57.6 %) and 60 patients (37 %) were older than 51 years. A biphasic course of infection was observed in 93/136 cases (68.4%). As many as 101/136 patients (74 %) had a history of a tick bite, one patient reported the consumption of non-pasteurized milk. Thirty-eight (1.4 %) specimens showed non-specific reactivity of IgM antibodies. None of the cases showed cerebrospinal fluid reactivity in the absence of serum reactivity. CONCLUSIONS: Increase in the diagnosed TBE cases was reported in 2003-2006 in the East Bohemia Region. It was due mainly to more severe clinical forms of TBE that were more frequent than the milder ones. The distribution of the confirmed TBE cases remained unchanged in terms of age and sex, but the seasonal peak moved from summer to autumn. ELISA proved suitable for use in the routine diagnosis of TBE. Both of the tested commercial kits are of good quality. Nevertheless, in view of possible non-specific reactivity of IgM antibodies, IgG antibodies need to be tested in both the first acute serum specimen and paired sera. The detection of antibodies in cerebrospinal fluid is unlikely to be of diagnostic benefit for TBE. Close cooperation between the attending physician and clinical microbiologist is crucial.

Endophagy of biting midges attacking cavity-nesting birds.

Feeding behaviour, host preferences and the spectrum of available hosts determine the role of vectors in pathogen transmission. Feeding preferences of blood-feeding Diptera depend on, among others factors, the willingness of flies to attack their hosts either in the open (exophagy) or in enclosed places (endophagy). As far as ornithophilic blood-feeding Diptera are concerned, the biting midges (Diptera: Ceratopogonidae) and blackflies (Diptera: Simuliidae) are generally considered to be strictly exophagous. We determined which blood-sucking Diptera enter nest cavities and feed on birds by placing sticky foil traps inside artificial nest boxes. A total of 667 females of eight species of biting midges of the genus Culicoides (Latreille, 1809) were captured on traps during 2006-2007, with Culicoides truncorum (Edwards, 1939) being the dominant species. DNA blood analyses of blood-engorged females proved that midges actually fed on birds nesting in the boxes. Three species were identified as endophagous: Culicoides truncorum, Culicoides pictipennis (Staeger, 1839), and Culicoides minutissimus (Zetterstedt, 1855). Our study represents the first evidence that ornithophilic biting midges are endophagous. The fact that we caught no blackflies in the bird boxes supports the exophagy of blackflies. We believe that our findings are important for surveillance programmes focusing on Diptera that transmit various bird pathogens.

[Comparison of reactive oxygen species production in neat semen and washed spermatozoa]. / Porovnání produkce reaktivních kyslíkových cástic v nativním ejakulátu a v suspenzi spermií.

OBJECTIVE: To determine Reactive Oxygen Species (ROS) production in neat semen and spermatozoa suspension using chemiluminescence and to examine correlation between both methods. SUBJECT: Prospective laboratory study. SETTING: Department of Obstetric and Gynecology, University Hospital, Olomouc. METHODS: The study included fertile volunteers (FV, n = 17), men from infertile couples (NM, n = 19) and men with idiopathic infertility (NMI, n = 15). ROS levels were determined by the same method in neat and washed semen samples. RESULTS: The ROS production in neat semen was lower than that in spermatozoa suspension. There was no significant diference in ROS production between volunteers and males from infertile couples. There was a significant correlation between log ROS in neat semen and in spermatozoa suspension in studied groups (FV r = 0.85, p = 1.5 x 10(-5); NM r = 0.76, p < 2 x 10(-4); NMI r = 0.75, p < 1.5 x 10(-3)). CONCLUSIONS: Measurement of ROS in neat semen is simpler, faster and better reflecting the actual level of oxidative stress than the same measurement in spermatozoa suspension. The implementation of this method can complement the algorithm of diagnostics and treatment of male infertility and be helpful in selection of patients for antioxidant or antibiotic treatment.

[Oxidative stress in diagnosis and treatment of idiopathic male infertility]. / Oxidacní stres v diagnostice a lécbe idiopatické muzské neplodnosti.

OBJECTIVE: To summarize available data concerning reactive oxygen species in sperm physiology and male infertility. DESIGN: Review article. SETTING: Dept. of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc. METHODS: Compilation of published data from scientific literature. CONCLUSIONS: The article describes the importance of reactive oxygen species and their role in male reproductive health.

[EmbryoGlue the transfer medium with hyaluronan in the IVF+ET program]. / Transferové médium s hyaluronanem embryoGlue v programu IVF+ET.

OBJECTIVE: To assess, if the replacement of serum albumin by recombinant human albumin and hyaluronan in EmbryoGlue transfer medium effects embryo implantation within IVF+ET programme. DESIGN: A prospective study. SETTING: Department of Obstetric and Gynecology, University Hospital, Olomouc. METHODS: 297 IVF cycles with the embryotransfer (ET) were evaluated. The patients were divided into 2 groups according to the type of transfer medium (culture medium; G2.2, n = 112 and the transfer medium EmbyoGlue (EG) n = 185) and to three subgroups according to the age of the patients (< 30, 30-38, > 38). In both groups the following entry parameters were compared: average age, number of oocytes obtained, fertilization rate (FR), number of frozen embryos, number of transferred embryos. The outcome parameters compared were: pregnancy rate (PR), implantation rate (IR), abortions (AB) and the number of multiple pregnancies. RESULTS: In the group of patients up to 30 (EG = 72 vs. G2.2 = 51) and over 38 years of age EG = 18 vs. G2.2 = 18) no differences in input and output parameters have been discovered, while in the group of patients between 30 and 38 years of age (EG = 94 vs. G2.2 = 44) the results of EG group embryo implantation have been improved significantly (IR 27.7% vs. 153%, p < 0.01). No differences in any other output parameters have been encountered (PR 43.6% vs. 39.5%, AB 7.3% vs. 11.7%). CONCLUSION: Human serum albumin can be replaced by hyaluronan as a sole macromolecule in a human embryo transfer medium and by recombinant human albumin, without reduction of pregnancy rate (PR) and implantation rate (IR). Not only similar results were achieved in EG medium compared to currently used G2.2 medium, but in the group of 30-38 year old patients a statistically significant positive influence of EG on the implantation of embryos has been observed.

[Lycopene therapy in male infertility]. / Lykopen v terapii muzské neplodnosti.

OBJECTIVE: To summarize available data concerning lycopene and male infertility treatment. DESIGN: Review article. SETTING: Dept. of Obstetrics and Gynecology, University Hospital Olomouc. METHODS: Compilation of published data from scientific literature. CONCLUSIONS: The article describes occurrence, biochemistry, metabolism of lycopene and its role in male reproductive health.

[Embryo quality evaluation according to the speed of the first cleavage after IntraCytoplasmic Sperm Injection (ICSI)]. / Kvalita embryí po ICSI v závislosti na rychlosti nástupu prvního bunecného delení.

OBJECTIVE: To evaluate morphological parameters of embryos obtained in the process of ICSI. DESIGN: A prospective study. SETTING: Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc. METHODS: In the present study 1116 embryos developing after ICSI (IntraCytoplasmic Sperm Injection) procedure in the period of 2001-2004 were evaluated. The beginning of the mitotic cleavage was assessed within the interval of 22-27 hours after insemination. The embryos were divided into three groups according to the speed of their division as Early Cleavage (EC) embryos, where two blastomeres were present at the time of assessment, Break Down ProNuclei stage (BDPN) where the pronuclei had already disappeared, and ProNuclei (PN) embryos, where both pronuclei were still present. In these groups the degree of fragmentation was evaluated on day two of cultivation and embryos were divided into four categories as: A--regular blastomeres, without fragmentation, B--irregular blastomeres or fragmentation below 30%, C--fragmentation 30-50%, D--fragmentation above 50%. The speed of further cleavage and average number of blastomeres were evaluated on day two and three of cultivation. Statistical analysis was preformed at the Palacky University Computer Centre. The chi2 test and t-test for independent samples were used. RESULTS: EC embryos were found in 37.4%, BDPN in 33.1% and PN in 29.5%. The degree of fragmentation between evaluated groups of embryos were statistically significant (p = 0.000). EC embryos were less fragmented (p = 0.000), had more blastomeres at the time of evaluation (p = 0.000) and their speed of cleavage was faster (p = 0.000). The cleavage of EC embryos was faster in comparison with the PN group (p = 0.000), but there were no significant differences between the EC and BDPN groups on day two of cultivation. On day three significant differences were found also between the EC and BDPN groups (p = 0.000). The embryonic developmental arrest was found only in PN embryos. CONCLUSION: The speed of the first cell cleavage is a useful additional criterion for the embryo selection for embryotransfer. EC embryos usually have better morphology and more blastomeres than the BPDN and PN ones.

[Embryo quality evaluation according to the speed of the first cleavage after conventional IVF]. / Kvalita embryí po klasickém IVF v závislosti na rychlosti nástupu prvního bunecného delení.

OBJECTIVE: To evaluate morphological parametres of embryos obtained in the process of conventional IVF. DESIGN: A prospective study. SETTING: Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc. METHODS: In the present study 549 embryos developing after conventional IVF in the period of 2001-2004 were evaluated. The beginning of the mitotic cleavage was assessed within the interval of 24 - 27 hours after insemination. The embryos were divided into three groups according to the speed of their division as early cleavage (EC) embryos, where two blastomeres were present at the time of assessment, break down pronuclei stage (BDPN) where the pronuclei had already disappeared, and pronuclei (PN) embryos, where both pronuclei were still present. In these groups the degree of fragmentation was evaluated on day two of cultivation and embryos were divided into four categories as: A - with regular blastomeres, without fragmentation, B - with irregular blastomeres or with fragmentation below 30%, C - with fragmentation 30-50%, D - with fragmentation above 50%. The speed of further cleavage and average number of blastomeres were evaluated on days two and three of cultivation. Statistical analysis was preformed in the Palacký University Computer Centre. The chi-square test and t test for independent samples were used. RESULTS: EC embryos were found in 45.5%, BDPN in 33.5% and PN in 21.0%. EC embryos were less fragmented (p = 0.000), had more blastomeres at the time of evaluation (p = 0.000) and their speed of cleavage was faster (p = 0.000). The cleavage of EC embryos was faster in comparisson with the PN group (p = 0.000), but there were no significant differences between the EC and BDPN groups on day two of cultivation. On day three significant difference was found also between the EC and BDPN groups (p = 0.000). CONCLUSION: The speed of the first cell cleavage is a useful additional criterion for the embryo selection for ET. EC embryos usually have better morphology and more blastomeres that the BPDN and PN ones.