La aplicacion de una nueva tecnica, basada en la fecundacion in vitro heterologa de espermatozoid... more La aplicacion de una nueva tecnica, basada en la fecundacion in vitro heterologa de espermatozoides humanos y oocitos de hamster hace posible el analisis citogenetico directo del espermatozoide humano. La frecuencia media de anomalias cromosomicas obtenida en el estudio citogenetico de espermatozoides de tres individuos normales ha sido de 17.2% (6.9% de anomalias estructurales, 2.0% de hiperhaploidias, 9.5% de hipohaploidias). El elevado porcentaje de hipohaploidia podria explicarse por artefactos o por perdidas anafasicas. Si el nivel de aneuploidia se toma como el doble de la frecuencia de hiperhaploidia, la incidencia de anomalias cromosomicas en esta serie control era de 10.9%. Nuestros resultados indican que la seleccion por motilidad de los espermatozoides no comporta ningun tipo de seleccion cromosomica. Se ha realizado por primera vez un estudio de la expresion de la fragilidad cromosomica en espermatozoides de individuos normales. La frecuencia de expresion de lesiones cromosomicas fue de 5.9%. El 80% del total de lesiones cromosomicas fueron localizadas en lugares fragiles. Se ha llevado a termino un estudio preliminar a microscopia electronica de la ultraestructura cromosomica, especialmente de los gaps centromericos y de las regiones descondensadas. Finalmente, se ha realizado el estudio citogenetico de espermatozoides de un hombre 47,xyy. No se ha encontrado ningun gameto disomico, hecho que refuerza la hipotesis de la eliminacion de un cromosoma extra y durante la espermatogenesis. La incidencia de anomalias cromosomicas en este individuo estaba dentro del intervalo de la poblacion normal.
OBJECTIVE To analyze the effect of single- and double-stranded sperm DNA fragmentation (ssSDF and... more OBJECTIVE To analyze the effect of single- and double-stranded sperm DNA fragmentation (ssSDF and dsSDF) on human embryo kinetics monitored under a time-lapse system. DESIGN Observational, double blind, prospective cohort study. SETTING University spin-off and private center. PATIENT(S) One hundred ninety-six embryos from 43 infertile couples were included prospectively. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) SsSDF and dsSDF were analyzed in the same semen sample used for intracytoplasmic sperm injection. Embryo kinetics was then monitored using time-lapse technology, and the timing of each embryo division was obtained. RESULT(S) When comparing embryos obtained from semen samples with low dsSDF and high dsSDF, splitting data using a statistically significant delay in high dsSDF was observed in second polar body extrusion, T4, T8, morula, and starting blastocyst and embryo implantation rates were impaired. Embryo kinetics and implantation rates are not significantly affected when high values of ssSDF are present. Different patterns of delay in embryo kinetics were observed for these different types of DNA damage: dsSDF caused a delay along all stages of embryo development; however, its major effect was observed at the second polar body extrusion and morula stages, coinciding with embryo DNA damage checkpoint activation as described before; ssSDF had its major effect at the pronucleus stage, but embryo kinetics was then restored at all following stages. The results show that dsSDF could be the main type of DNA damage that affects embryo development in intracytoplasmic sperm injection cycles, probably due to motility-based sperm selection in this assisted reproduction procedure. CONCLUSION(S) Double-stranded sperm DNA damage caused a delay in embryo development and impaired implantation, while single-stranded DNA damage did not significantly affect embryo kinetics and implantation.
El programa HyperCell es una eina informatica que dona informacio actualitzada de la majoria de l... more El programa HyperCell es una eina informatica que dona informacio actualitzada de la majoria de les arees de la biologia cel·lular
Desde el punto de vista metodologico esta tesis aporta, primero una mejora en la tecnica de fusio... more Desde el punto de vista metodologico esta tesis aporta, primero una mejora en la tecnica de fusion heterologa de ovocitos de hamster con esperma humano mediante el uso de ionoforo lo que mejora el proceso de capacitacion espermatica. En segundo lugar el uso de vinblastina, como agente antimitotico, es un hecho relevante que mejora sustancialmente la calidad de la extension cromosomica y en consecuencia incrementa el numero de complementos cromosomicos de mejor morfologia para ser estudiados. Una tercera mejora importante ha consistido en un metodo original para el pintado de cromosomas enteros. Permite distinguir los complementos cromosomicos de ovocito de hamster de los complementos de pronucleo masculino humano. Ademas, usando sondas de ADN marcadas con fluorescencia, especificas para los cromosomas preseleccionados, se han estudiado cromosomas humanos especificos. Esta aproximacion metodologica mejorada hace posible estudiar con exito la segregacion meiotica en portadores de reor...
Increasing intrauterine insemination (IUI) success rates is essential to improve the quality of c... more Increasing intrauterine insemination (IUI) success rates is essential to improve the quality of care for infertile couples. Additionally, straight referral of couples with less probability of achieving a pregnancy through IUI to more complex methods such as in vitro fertilization is important to reduce costs and the time to pregnancy. The aim of the present study is to prospectively evaluate the threshold values for different parameters related to success in intrauterine insemination in order to provide better reproductive counseling to infertile couples, moreover, to generate an algorithm based on male and female parameters to predict whether the couple is suitable for achieving pregnancy using IUI. For that, one hundred ninety-seven infertile couples undergoing 409 consecutive cycles of intrauterine insemination during a two-year period were included. The first year served as a definition of the parameters and thresholds related to pregnancy achievement, while the second year was ...
Since the first description of a commensal seminal microbiome using sequencing, less than a decad... more Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of 16S rRNA gene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the 16S rRNA gene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina’s MiSeq sequencing platform. Comparison between th...
<p>DNA molecular weight markers consisting of Low Range PFG Marker (M1) and Lambda ladder P... more <p>DNA molecular weight markers consisting of Low Range PFG Marker (M1) and Lambda ladder PFG marker (M2) are detailed. Negative controls in B, lane 1 show a thin compression zone. Positive controls in B, lanes 2, 3 and 4 show DNA digestion into sizes of around the 48 Kb. Sperm DNA fragmentation of the specific samples of this figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044679#pone-0044679-t002" target="_blank">Table 2</a>.</p
La aplicacion de una nueva tecnica, basada en la fecundacion in vitro heterologa de espermatozoid... more La aplicacion de una nueva tecnica, basada en la fecundacion in vitro heterologa de espermatozoides humanos y oocitos de hamster hace posible el analisis citogenetico directo del espermatozoide humano. La frecuencia media de anomalias cromosomicas obtenida en el estudio citogenetico de espermatozoides de tres individuos normales ha sido de 17.2% (6.9% de anomalias estructurales, 2.0% de hiperhaploidias, 9.5% de hipohaploidias). El elevado porcentaje de hipohaploidia podria explicarse por artefactos o por perdidas anafasicas. Si el nivel de aneuploidia se toma como el doble de la frecuencia de hiperhaploidia, la incidencia de anomalias cromosomicas en esta serie control era de 10.9%. Nuestros resultados indican que la seleccion por motilidad de los espermatozoides no comporta ningun tipo de seleccion cromosomica. Se ha realizado por primera vez un estudio de la expresion de la fragilidad cromosomica en espermatozoides de individuos normales. La frecuencia de expresion de lesiones cromosomicas fue de 5.9%. El 80% del total de lesiones cromosomicas fueron localizadas en lugares fragiles. Se ha llevado a termino un estudio preliminar a microscopia electronica de la ultraestructura cromosomica, especialmente de los gaps centromericos y de las regiones descondensadas. Finalmente, se ha realizado el estudio citogenetico de espermatozoides de un hombre 47,xyy. No se ha encontrado ningun gameto disomico, hecho que refuerza la hipotesis de la eliminacion de un cromosoma extra y durante la espermatogenesis. La incidencia de anomalias cromosomicas en este individuo estaba dentro del intervalo de la poblacion normal.
OBJECTIVE To analyze the effect of single- and double-stranded sperm DNA fragmentation (ssSDF and... more OBJECTIVE To analyze the effect of single- and double-stranded sperm DNA fragmentation (ssSDF and dsSDF) on human embryo kinetics monitored under a time-lapse system. DESIGN Observational, double blind, prospective cohort study. SETTING University spin-off and private center. PATIENT(S) One hundred ninety-six embryos from 43 infertile couples were included prospectively. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) SsSDF and dsSDF were analyzed in the same semen sample used for intracytoplasmic sperm injection. Embryo kinetics was then monitored using time-lapse technology, and the timing of each embryo division was obtained. RESULT(S) When comparing embryos obtained from semen samples with low dsSDF and high dsSDF, splitting data using a statistically significant delay in high dsSDF was observed in second polar body extrusion, T4, T8, morula, and starting blastocyst and embryo implantation rates were impaired. Embryo kinetics and implantation rates are not significantly affected when high values of ssSDF are present. Different patterns of delay in embryo kinetics were observed for these different types of DNA damage: dsSDF caused a delay along all stages of embryo development; however, its major effect was observed at the second polar body extrusion and morula stages, coinciding with embryo DNA damage checkpoint activation as described before; ssSDF had its major effect at the pronucleus stage, but embryo kinetics was then restored at all following stages. The results show that dsSDF could be the main type of DNA damage that affects embryo development in intracytoplasmic sperm injection cycles, probably due to motility-based sperm selection in this assisted reproduction procedure. CONCLUSION(S) Double-stranded sperm DNA damage caused a delay in embryo development and impaired implantation, while single-stranded DNA damage did not significantly affect embryo kinetics and implantation.
El programa HyperCell es una eina informatica que dona informacio actualitzada de la majoria de l... more El programa HyperCell es una eina informatica que dona informacio actualitzada de la majoria de les arees de la biologia cel·lular
Desde el punto de vista metodologico esta tesis aporta, primero una mejora en la tecnica de fusio... more Desde el punto de vista metodologico esta tesis aporta, primero una mejora en la tecnica de fusion heterologa de ovocitos de hamster con esperma humano mediante el uso de ionoforo lo que mejora el proceso de capacitacion espermatica. En segundo lugar el uso de vinblastina, como agente antimitotico, es un hecho relevante que mejora sustancialmente la calidad de la extension cromosomica y en consecuencia incrementa el numero de complementos cromosomicos de mejor morfologia para ser estudiados. Una tercera mejora importante ha consistido en un metodo original para el pintado de cromosomas enteros. Permite distinguir los complementos cromosomicos de ovocito de hamster de los complementos de pronucleo masculino humano. Ademas, usando sondas de ADN marcadas con fluorescencia, especificas para los cromosomas preseleccionados, se han estudiado cromosomas humanos especificos. Esta aproximacion metodologica mejorada hace posible estudiar con exito la segregacion meiotica en portadores de reor...
Increasing intrauterine insemination (IUI) success rates is essential to improve the quality of c... more Increasing intrauterine insemination (IUI) success rates is essential to improve the quality of care for infertile couples. Additionally, straight referral of couples with less probability of achieving a pregnancy through IUI to more complex methods such as in vitro fertilization is important to reduce costs and the time to pregnancy. The aim of the present study is to prospectively evaluate the threshold values for different parameters related to success in intrauterine insemination in order to provide better reproductive counseling to infertile couples, moreover, to generate an algorithm based on male and female parameters to predict whether the couple is suitable for achieving pregnancy using IUI. For that, one hundred ninety-seven infertile couples undergoing 409 consecutive cycles of intrauterine insemination during a two-year period were included. The first year served as a definition of the parameters and thresholds related to pregnancy achievement, while the second year was ...
Since the first description of a commensal seminal microbiome using sequencing, less than a decad... more Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of 16S rRNA gene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the 16S rRNA gene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina’s MiSeq sequencing platform. Comparison between th...
<p>DNA molecular weight markers consisting of Low Range PFG Marker (M1) and Lambda ladder P... more <p>DNA molecular weight markers consisting of Low Range PFG Marker (M1) and Lambda ladder PFG marker (M2) are detailed. Negative controls in B, lane 1 show a thin compression zone. Positive controls in B, lanes 2, 3 and 4 show DNA digestion into sizes of around the 48 Kb. Sperm DNA fragmentation of the specific samples of this figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044679#pone-0044679-t002" target="_blank">Table 2</a>.</p
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