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PURPOSE: We sought to determine whether pregnancies conceived in those with male factor infertility have unique placental pathology profiles compared to those undergoing infertility treatments for other indications. METHODS: This was a retrospective cohort study of placental pathology from 464 live births conceived from autologous fresh IVF cycles at an academic fertility center from 2004 to 2017. Placental pathology was compared between live births arising from patients with male factor infertility alone and those with another infertility diagnosis. Placental outcomes were compared with parametric or non-parametric tests; logistic regression was performed to account for potential confounders. RESULTS: Compared to cycles performed for a non-male factor diagnosis, male factor infertility cycles had a higher mean paternal age (38.2 years vs. 36.5 years, p < 0.001), a higher female mean BMI (24.3 vs. 23.3 kg/m2, p = 0.01), and a lower day 3 follicle stimulating hormone (FSH) level (6.8 vs. 7.3 IU/mL, p = 0.02). The mean numbers of embryos transferred, and day of transfer were similar between groups, and more cycles used ICSI in the male factor infertility group (90.6% vs. 22.5%, p < 0.001). Placental pathology in our adjusted model was similar between the male factor and non-male factor groups. In our unadjusted subgroup analysis, cycles for male factor using ICSI appeared to lead to more small placentas by weight compared to cycles performed with conventional insemination (45.8% < 10th percentile vs. 18.8%, p = 0.04). CONCLUSION: Male factor infertility is not associated with significantly different placental pathology compared to other infertility diagnoses.
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Infertilidad Masculina/patología , Enfermedades Placentarias/patología , Placenta/patología , Adulto , Peso al Nacer/fisiología , Transferencia de Embrión/métodos , Femenino , Fertilización/fisiología , Fertilización In Vitro/métodos , Humanos , Nacimiento Vivo , Masculino , Hombres , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
PURPOSE: A deep learning artificial intelligence (AI) algorithm has been demonstrated to outperform embryologists in identifying euploid embryos destined to implant with an accuracy of 75.3% (1). Our aim was to evaluate the performance of highly trained embryologists in selecting top quality day 5 euploid blastocysts with and without the aid of a deep learning algorithm. MATERIALS AND METHODS: A non-overlapping series of 200 sets of day 5 euploid embryo images with known implantation outcomes was distributed to 17 highly trained embryologists. One embryo in each set was known to have implanted and one failed implantation. They were asked to select which embryo to transfer from each set. The same 200 sets of embryos, with indication of which embryo in each set had been identified by the algorithm as more likely to implant was then distributed. Chi-squared, t-test, and receiver operating curves were performed to compare the embryologist performeance with and without AI. RESULTS: Fourteen embryologists completed both assessments. Embryologists provided with AI results selected successfully implanted embryos in 73.6% of cases compared to 65.5% for those selected using visual assessments alone (p < 0.001). All embryologists improved in their ability to select embryos with the aid of the AI algorithm with a mean percent improvement of 11.1% (range 1.4% to 15.5%). There were no differences in degree of improvement by embryologist level of experience (junior, intermediate, senior). CONCLUSIONS: The incorporation of an AI framework for blastocyst selection enhanced the performance of trained embryologists in identifying PGT-A euploid embryos destined to implant.
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Algoritmos , Inteligencia Artificial , Blastocisto/clasificación , Blastocisto/citología , Técnicas de Apoyo para la Decisión , Implantación del Embrión , Fertilización In Vitro/métodos , Adulto , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
STUDY QUESTION: Is there an effect of male factor infertility (MFI) on either early or late morphokinetic parameters obtained during embryonic culture to blastocyst stage in a time-lapse imaging (TLI) incubator? SUMMARY ANSWER: Neither mild nor severe MFI had an impact on overall time to blastocyst or duration of individual cleavage stages in the total embryo population. WHAT IS KNOWN ALREADY: Prior studies have suggested that paternal DNA and sperm quality affect embryo morphokinetic parameters, but the impact of MFI is not fully understood. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study, at a major academic fertility centre, included 536 couples (women, ≤44 years of age) undergoing IVF between September 2013 and September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 4126 embryos cultured to the blastocyst stage in a TLI-monitored incubator were retrospectively reviewed. Embryos derived from the sperm of men with MFI were compared with those derived from patients with other infertility diagnoses. Generalized fixed and random effects models, t-test and χ2 were used as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE: Couples with MFI had a higher rate of ICSI utilization and fewer usable embryos on average, and the men were older compared with couples with other diagnoses. Additionally, the women in MFI couples were younger and had higher antral follicle counts (AFCs) and higher anti-Müllerian hormone (AMH) levels compared with the other women undergoing IVF. When controlling for maternal and paternal ages, AMH and fertilization method (conventional IVF versus ICSI), neither mild nor severe MFI affected duration of individual cleavage stages or overall time to the blastocyst stage, when all or only usable embryos were examined (coefficient 0.44 hours in all embryos, P = 0.57; coefficient 0.39 hours in usable embryos, P = 0.60). Whether the sperm was surgically extracted similarly had no significant effect on embryo morphokinetic parameters. When the fertilization method was assessed independently, ICSI lengthened the overall time to blastocyst stage by 1.66 hours (P = 0.03) on average, primarily due to an increase in duration of the time from 5-cell embryo stage to early blastulation (P5SB). LIMITATIONS, REASONS FOR CAUTION: This large cohort study avoided embryo selection bias due to random assignment of embryos to the TLI incubators. However, our findings may not be generalizable to groups under-represented in our clinic population. Future studies should also evaluate the impact of male hormonal status and detailed sperm morphology, such as head versus flagellum defects, on embryo morphokinetic development. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that the fertilization method rather than MFI per se impacts time to early blastulation. The clinical implications of this effect on embryo development warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S): There were no sources of funding for this study. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.
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Blastocisto , Técnicas de Cultivo de Embriones , Estudios de Cohortes , Femenino , Fertilización In Vitro , Humanos , Masculino , Estudios Retrospectivos , Imagen de Lapso de TiempoRESUMEN
PURPOSE: To evaluate the differences in implantation and pregnancy rates when embryo transfer occurs on D2 versus D3 in women with a low yield of fertilized oocytes. METHODS: A total of 156 IVF/ICSI cycles from 141 women at an academic fertility center were analyzed in a retrospective fashion. Women with a low number of fertilized oocytes (≤ 2 two pronuclei (2PN) stage zygotes) who had their fresh embryo transfer on D2 or D3 were included in the study. Positive pregnancy test per IVF cycle (PPT), clinical pregnancy rate (CPR), spontaneous abortion rate (SABR), and implantation rate (IMPR) were the main outcome measures assessed. Mann-Whitney U test and χ2 test were used as appropriate. A generalized linear mixed effect model adjusted for relevant covariates was conducted. P < 0.05 was considered significant. RESULTS: Patients having their embryo transfer on D2, when compared to those who had a D3 embryo transfer, experienced similar PPT [30.8 vs. 28.2%, respectively; adjusted OR (95%CI): 0.49 (0.16, 1.52)], CPR [26.9 vs. 25.6%, respectively; adjusted OR (95%CI): 0.44 (0.12, 1.67)], and IMPR [17.3 vs. 16.7%, respectively; adjusted ß (95%CI) - 5.6% (- 15.0, 3.9)]. CONCLUSION: Our findings suggest that transferring embryos on D2 versus D3 in women with a limited number of 2PN stage zygotes does not affect early pregnancy outcomes. These results indicate that there can be some flexibility in scheduling the day of transfer at the convenience of both the patient and the center.
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Transferencia de Embrión/métodos , Fertilización In Vitro/métodos , Aborto Espontáneo , Adulto , Implantación del Embrión , Femenino , Humanos , Recuperación del Oocito , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios Retrospectivos , Factores de TiempoRESUMEN
The corpus luteum (CL) is a transient endocrine organ that is essential for maintenance of pregnancy in both ruminants and primates. The cellular and endocrine mechanisms that regulate the CL in these species have commonalities and some distinct and intriguing differences. Both species have similar cellular content with large luteal cells derived from the granulosa cells of the follicle, small luteal cells from follicular thecal cells, and large numbers of capillary endothelial cells that form the vasculature that has an essential role in optimal CL function. Intriguingly, the large luteal cells in ruminants grow larger than in primates and acquire a capacity for high constitutive progesterone (P4) production that is independent of stimulation from LH. In contrast, the primate CL and the granulosa lutein cells from primates continue to require stimulation by LH/CG throughout the luteal phase. Although the preovulatory follicle of women and cows had similar size and steroidogenic output (10 to 20 mg/h), the bovine CL had about ten-fold greater P4 output compared to the human CL (17.4 vs. 1.4 mg/h), possibly due to the development of high constitutive P4 output by the bovine large luteal cells. The continued dependence of the primate CL on LH/CG/cAMP also seems to underlie luteolysis, as there seems to be a requirement for greater luteotropic support in the older primate CL than is provided by the endogenous LH pulses. Conversely, regression of the ruminant CL is initiated by PGF from the nonpregnant uterus. Consequently, the short luteal phase in ruminants is primarily due to premature secretion of PGF by the nonpregnant uterus and early CL regression, whereas CL insufficiency in primates is related to inadequate luteotropic support and premature CL regression. Thus, the key functions of the CL, pregnancy maintenance and CL regression in the absence of pregnancy, are produced by common cellular and enzymatic pathways regulated by very distinct luteotropic and luteolytic mechanisms in the CL of primates and ruminants.
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Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.
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Blastocisto , Células Madre Embrionarias , Microfluídica , Oocitos , Técnicas Reproductivas Asistidas , Espermatozoides , Técnicas de Cultivo de Célula , Separación Celular/instrumentación , Separación Celular/métodos , Técnicas de Cultivo de Embriones , Femenino , Fertilización In Vitro/métodos , Humanos , Masculino , Microfluídica/instrumentación , Microfluídica/métodos , Nanotecnología , Técnicas Reproductivas Asistidas/tendencias , Espermatozoides/citologíaRESUMEN
BACKGROUND: Despite advances in in vitro manipulation of preimplantation embryos, there is still a reduction in the quality of embryos produced leading to lower pregnancy rates compared with embryos produced in vivo. We hypothesized that a dynamic microfunnel embryo culture system would enhance outcomes by better mimicking the fluid-mechanical and biochemical stimulation embryos experience in vivo from ciliary currents and oviductal contractions. METHODS AND RESULTS: Mouse embryos were cultured in microdrop-static control, microfunnel-static control or microfunnel-dynamic conditions with microfluidics. All groups tested had greater than 90% total blastocyst development from zygotes after 96 h culture. Blastocyst developmental stage was significantly enhanced (P < 0.01) under dynamic microfunnel culture conditions as evidenced by an increased percentage of hatching or hatched blastocysts (Microdrop-control 31%; Microfunnel-control 23%; Microfunnel-pulsatile 71%) and significantly higher (P < 0.01) average number of cells per blastocyst (Microdrop-control 67 +/- 3; Microfunnel-control 60 +/- 3; Microfunnel-pulsatile 109 +/- 5). Blastocyst cell numbers in dynamic microfunnel cultures (109 +/- 5) more closely matched numbers obtained from in vivo grown blastocysts (144 +/- 9). Importantly, dynamic microfunnel culture significantly improved embryo implantation and ongoing pregnancy rates over static culture to levels approaching that of in utero derived preimplantation embryos. CONCLUSIONS: The improved pregnancy outcomes along with the simple and user-friendly design of the microfluidic/microfunnel system has potential to alleviate many inefficiencies in embryo production for biomedical research, genetic gain in domestic species and assisted reproductive technologies in humans.
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Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/fisiología , Microfluídica , Animales , Femenino , Ratones , Embarazo , Índice de EmbarazoRESUMEN
BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.
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Coagulación Sanguínea/genética , Factor VIII/genética , Hemartrosis/genética , Hemofilia A/genética , Factores de Edad , Envejecimiento , Animales , Secuencia de Bases , Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Codón sin Sentido , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exones , Factor VIII/metabolismo , Factor VIII/farmacología , Femenino , Predisposición Genética a la Enfermedad , Hemartrosis/sangre , Hemartrosis/tratamiento farmacológico , Hemartrosis/patología , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Hemofilia A/patología , Humanos , Masculino , Datos de Secuencia Molecular , Tiempo de Tromboplastina Parcial , Fenotipo , ARN Mensajero/sangre , Técnicas Reproductivas Asistidas , Ovinos , Especificidad de la EspecieRESUMEN
In almost every European country health surveys are conducted in order to describe the health status of the population and to use these data for planning health services and preventive activities. In the last 10-15 years the availability and quality of data from population-based health surveys in Europe have greatly improved, particularly in the countries of Central and Eastern Europe. Nevertheless the design, quality of data and the comparability across countries, nations and cultures remain a challenge. Therefore WHO and EU have started various activities to harmonize the methods and instruments in health surveys. These activities are described in this contribution.
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Bases de Datos Factuales , Unión Europea/organización & administración , Encuestas Epidemiológicas , Almacenamiento y Recuperación de la Información/métodos , Cooperación Internacional , Sistemas de Registros Médicos Computarizados/organización & administración , Informática en Salud Pública , Sistemas de Administración de Bases de Datos/organización & administración , Difusión de la Información/métodos , Gestión de la Información/organización & administraciónRESUMEN
Individual longitudinal data are indispensable for studying the differential incidence of diseases and of possible chronic conditions, health care interventions and outcomes in the course of time, the effects of changing population structure on morbidity, the demand and use of health services, as well as the efficacy and efficiency of health care guidelines. Here, we present an up to date survey of panel studies in Germany, in so far as these are either still active or were completed only recently, and in so far as they are accessible via public use files or equivalent solutions for the scientific community. There are some health related items on existing general purpose national panel studies, but no health related representative national panel study. In addition to the well known unique potential of panel data, we demonstrate, for special populations (e. g. foreigners and people living in institutionalised settings), as well as for special items (salutogenetic potentials and preventive resources), the particular need for health related panel data in the German context.
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Recolección de Datos/estadística & datos numéricos , Directrices para la Planificación en Salud , Encuestas Epidemiológicas , Morbilidad/tendencias , Mortalidad/tendencias , Programas Nacionales de Salud/estadística & datos numéricos , Vigilancia de la Población/métodos , Predicción , Alemania , Necesidades y Demandas de Servicios de Salud/estadística & datos numéricos , Humanos , Dinámica PoblacionalRESUMEN
Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.
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Adenosina Trifosfato/metabolismo , Glutatión/metabolismo , Oocitos/metabolismo , Porcinos/metabolismo , Animales , Femenino , Técnicas In VitroRESUMEN
BACKGROUND AND OBJECTIVE: Malignant melanomas show a higher heterogeneity in their architecture and a higher vessel density because of neovascularization compared to benign melanocytic skin tumours. In this study the validity of the newly developed Laser Doppler Perfusion Imager (LDPI) with a lateral resolution of 100 microns in the differential diagnosis of pigmented skin tumours was investigated. PATIENTS/METHODS: The perfusion of 116 pigmented skin tumours was s measured with LDPI; 44 malignant melanomas, five melanoma metastases, 59 dysplastic nevi and eight basal cell carcinomas were studied before excision for precise histological diagnosis. RESULTS: There is a significantly higher perfusion of the malignant melanomas (3.15 +/- 1.87 AU) compared to dysplastic nevi (1.14 +/- 0.97 AU) (p < 0.01). By calculating a ratio of the mean perfusion in the tumour and the mean perfusion in adjacent healthy skin, the potential source of error because of regional differences in perfusion is eliminated. The ratio of malignant melanomas (10.78 +/- 9.18) is significantly higher than these of melanoma metastases (4.20 +/- 1.66), basal cell carcinomas (3.24 +/- 1.32) and dysplastic nevi (2.85 +/- 1.32). CONCLUSIONS: The high resolution LDPI has the potential to be a non-invasive screening method for preoperative differential diagnosis of pigmented skin tumours. Besides the epiluminescence microscopy and sonographic determination of the tumour thickness, we have now the possibility to get preoperative information about tumour vascularization.
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Carcinoma Basocelular/diagnóstico , Procesamiento de Imagen Asistido por Computador/instrumentación , Flujometría por Láser-Doppler/instrumentación , Melanoma/diagnóstico , Neovascularización Patológica/diagnóstico , Nevo Pigmentado/diagnóstico , Neoplasias Cutáneas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Basocelular/irrigación sanguínea , Carcinoma Basocelular/patología , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Melanoma/irrigación sanguínea , Melanoma/patología , Microcirculación/fisiología , Persona de Mediana Edad , Neovascularización Patológica/patología , Nevo Pigmentado/irrigación sanguínea , Nevo Pigmentado/patología , Sensibilidad y Especificidad , Piel/irrigación sanguínea , Piel/patología , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/patologíaRESUMEN
The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.
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Blastocisto/metabolismo , Ciclo del Ácido Cítrico , Fertilización In Vitro , Glucosa/metabolismo , Porcinos/metabolismo , Análisis de Varianza , Animales , Supervivencia Celular , Células Cultivadas , Femenino , Edad Gestacional , Glutamina/metabolismo , Embarazo , Ácido Pirúvico/metabolismo , Porcinos/embriologíaRESUMEN
In fall 1996, the University of Colorado at Boulder instituted a ban on beer sales at football games. To evaluate the effects of the ban, the authors collected two types of data: first, they examined the effects of the ban on game-day security incidents; second, they looked at survey data from season ticket holders and students. They administered the surveys after the first two postban seasons to assess ticket holders' attitudes about the new policy. The incident data they found indicated dramatic decreases in arrests, assaults, ejections from the stadium, and student referrals to the judicial affairs office following the ban. Survey data also indicated moderately negative attitudes about the ban among students and some season ticket holders. However, all fans were likely to renew their tickets regardless of their attitudes toward the policy. The study illustrates what can be achieved when alcohol is eliminated from a setting that frequently fosters disorderly and aggressive behaviors.
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Alcoholismo/prevención & control , Universidades/legislación & jurisprudencia , Violencia/estadística & datos numéricos , Adolescente , Adulto , Actitud , Colorado , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Deportes , Encuestas y CuestionariosRESUMEN
Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.
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Blastocisto/fisiología , Fertilización In Vitro/veterinaria , Porcinos/embriología , Animales , Blastocisto/metabolismo , Medios de Cultivo/farmacología , Femenino , Fertilización In Vitro/métodos , Glucosa/metabolismo , Masculino , Oocitos/metabolismo , Oocitos/fisiología , Embarazo , Ácido Pirúvico/metabolismo , Distribución Aleatoria , Estadísticas no Paramétricas , Porcinos/metabolismoRESUMEN
Effective activation protocols that can be used during nuclear transfer investigations in goats need to be developed. We compared the development of IVF goat embryos with those of nonfertilized parthogenetically developing oocytes activated by treatment with either ionomycin or ethanol, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP). Cumulus oocyte complexes (COCs) recovered from abattoir goat ovaries were either matured in a conventional laboratory incubator or placed in pre-equilibrated maturation medium and shipped overnight in a battery-operated dry incubator to another laboratory. Mature COCs were allocated randomly to one of three treatment groups. Group 1 oocytes (n=169 shipped, n=253 not shipped) were fertilized in vitro at 24 h postmaturation (hpm). The remaining COCs were activated at 28 hpm in either ionomycin (Group 2: n=362 shipped, n=202 not shipped), or ethanol (Group 3: n=263 shipped, n=249 not shipped). Activated oocytes were immediately incubated in 6-DMAP for 4 h. Blastocyst development was evaluated on Day 8 post-insemination/activation. Percent cleavage was comparable in shipped and nonshipped oocytes and in all treatment groups. In both shipped and nonshipped oocytes, parthenotes developing from ionomycin- and ethanol-activated oocytes had significantly greater blastocyst development (P<0.01) compared to IVF embryos (28.5 +/- 3.0, 27.4 +/- 2.8, 10.3 +/- 3.0, respectively for the nonshipped oocytes and 9.9 +/- 2.1, 10.3 +/- 2.4, 3.7 +/- 4.7 respectively for the shipped oocytes). Shipped oocytes had lower blastocyst development compared to nonshipped oocytes in the three treatment groups. The mean blastocyst cell number was not statistically different between shipped and nonshipped oocytes or among treatment groups, suggesting that all were equally viable.
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Fertilización In Vitro/veterinaria , Cabras/embriología , Partenogénesis , Animales , Blastocisto/efectos de los fármacos , Etanol/farmacología , Femenino , Ionomicina/farmacología , Masculino , Oocitos/efectos de los fármacos , Partenogénesis/efectos de los fármacos , Manejo de Especímenes/veterinariaRESUMEN
AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.
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Antifúngicos/química , Antifúngicos/metabolismo , Proteínas Bacterianas , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Quitina/metabolismo , Streptomyces/química , Secuencia de Aminoácidos , Sitios de Unión , Celulasa/química , Quitina/análogos & derivados , Quitina/química , Difusión , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Docilidad , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotación , Alineación de Secuencia , Soluciones , Electricidad Estática , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/químicaRESUMEN
Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties nikkomycin Cz, and nikkomycins Cx and Cz, respectively. The nikV mutants formed nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized nikkomycins I, J, X, and Z, but amounts of nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.
Asunto(s)
Aminoglicósidos , Antibacterianos/biosíntesis , Péptidos , Streptomyces/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Prueba de Complementación Genética , Modelos Químicos , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Fenotipo , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
AIM OF THE STUDY: Investigation of various laboratory parameters in stored whole blood, with respect to duration of storage and kind of product. METHODS: Whole blood was donated by 12 healthy volunteers using CPDA1 stabilisator. Six units were filtered with the Leukotrap A1-System (PALL Comp., Dreieich, Germany) for leukocyte depletion. The twelve units were stored for 49 days. Several hematological, biochemical and coagulatory parameters were analysed during storage. RESULTS: There was an adequate reduction of lycocytes by filtration (< 3 x 10(6) white cells per unit). ATP decreased during storage to 45% of initial value at the 49th day, without any influence of the kind of preparation. The course of other parameters such as lactate and free haemoglobin (increase), PH-value (decrease), antithrombin III (decrease), prothrombin, protein C, thrombin-antithrombin-complex, alpha-2-Anti-plasmin (decrease or indifferent) did not show any influence of the kind of preparation. Coagulation factors V and VIII decreased in both preparations, which was significantly less pronounced in whole blood with leukocyte depletion. In contrast parameters of activated coagulation such as D-Dimere and fibrinmonomeric did not change during storage after leukocyte reduction but increased at the end of storage time in CPDA1-blood. CONCLUSIONS: Several parameters indicating quality of stored blood were constant in whole blood independent of the kind of preparation during a storage of 49 days. This is in contrast to the main part of specific scientific communications. A beneficial influence of leukocyte depletion was observed for some coagulation parameters whereas increasing characteristics of activated coagulation in CPDA1-stored whole blood at the end of storage time had to be observed. The preparation of whole CPDA1-blood is recommended for autologous predonation, if storage time does not exceed 30 days. Storage time of > 40 days seems to be possible for autologous whole blood after filtration for leucocyte depletion.