Jordi Benet | Universitat Autònoma de Barcelona (original) (raw)
Papers by Jordi Benet
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.
23 3.1.3. Mètode de biòpsia del 1r corpuscle polar 24 3.1.4. Fixació de cèl•lules aïllades, 1r co... more 23 3.1.3. Mètode de biòpsia del 1r corpuscle polar 24 3.1.4. Fixació de cèl•lules aïllades, 1r corpuscle polar i metafase II, anàlisi usant contrast de fases i tractament previ a la hibridació in situ fluorescent 28 3.1.5. Hibridació in situ fluorescent en extensions del 1r corpuscle polar i metafase II 29 3.2. Material i mètodes de l'objectiu 2 32 3.2.1. Pacients amb cariotip normal. Característiques i antecedents clínics 33 3.2.2. Pacients portadores de translocacions. Característiques i antecedents clínics 33 3.2.3. Mètode utilitzat per al diagnòstic genètic preimplantacional analitzant el 1r corpuscle polar 35 3.3. Material i mètodes de l'objectiu 3 39 3.3.1. Material objecte d'estudi 39 3.3.2. Mètode d' hibridació in situ fluorescent emprat 40 3.4. Material i mètodes de l'objectiu 4 41 3.4.1. Preparació d'un cultiu no sincronitzat de limfòcits estimulats i obtenció de cèl•lules de Sertoli 41 Anàlisi citogenètica preimplantacional Índex III 3.4.2. Mètode d'anàlisi: hibridació in situ fluorescent 42 4. Resultats 44 4.1. Anàlisi d'aneuploïdies en una sèrie d'oòcits descartats de cicles de FIV 45 Article publicat: Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies. European Journal of Human Genetics 2003; 11: 325-336 46 4.2. Anàlisi d'aneuploïdies en la línia germinal femenina 47 4.2.1. Pacients amb cariotip normal 47 4.2.2. Pacients portadores de translocacions 50 Article publicat: Multiple aneuploidies in the oocytes of balanced translocation carriers: a preimplantation genetic diagnosis study using first polar body. Reproduction 2003; 126: 701-711 53 4.3. Estudi d'aneuploïdies en cèl•lules embrionàries de pacients portadores de translocacions recíproques 54 Article pendent de publicació: The importance of aneuploidy screening in reciprocal translocation carriers 56 4.4. Estadi replicatiu de la cromatina en cèl•lules proliferants. Avaluació de la interpretació dels resultats de la tècnica d'hibridació in situ fluorescent 57 Article publicat: The use of a cell-cycle phase-marker may decrease the percentage of errors when using FISH in PGD. Cytogenetic and Genome Research 2004; 105: 29-35 59 5. Discussió 60 5.1. Anàlisi d'aneuploïdies en oòcits descartats de cicles de FIV 61 5.2. Anàlisi d'aneuploïdies en la línia germinal femenina 64 5.2.1. Pacients amb cariotip normal 64 5.2.2. Pacients portadores de translocacions 66 5.3. Estudi d'aneuploïdies en cèl•lules embrionàries de pacients portadores de translocacions recíproques 69 5.4. Estadi replicatiu de la cromatina en cèl•lules proliferants i errors en la interpretació dels resultats de la tècnica d'hibridació in situ fluorescent 73 6. Conclusions 76 7. Bibliografia
Human Fertility, Jan 9, 2020
Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) s... more Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) seems to be the best procedure for its repair and to reduce testicular oxidative stress (ROS). As ROS causes guanine modifications, we postulated that DNA damage could be more intense in telomeres due to their G-rich nature. We studied the effect of MV on sperm telomere length (TL), single-and double-strand DNA fragmentation (ssSDF and dsSDF) and seminal parameters. Sperm telomeres from 12 fertile donors and 20 varicocele patients before and nine months after MV were labelled using FITC-PNA qFISH (a new method to obtain absolute TL from relative fluorescence intensity using FITC-fluorescent spheres). Both ssSDF and dsSDF were analysed using the alkaline and neutral Comet assays, respectively. The results showed that varicocele and MV had no effect on TL. Seminal parameters, ssSDF and dsSDF of varicocele patients were altered. Although these parameters improved after MV, values did not reach those seen in fertile donors. A good estimation of absolute TL was developed based on FITC-fluorescent spheres. The results showed that TL is not affected by varicocele or surgery. However, MV is able to partially reduce altered seminal parameters, ssSDF and dsSDF values in varicocele patients.
Fertility and Sterility, Apr 1, 2019
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.
Asian Journal of Andrology, 2015
Biomedicines, Aug 19, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Prenatal Diagnosis, Oct 15, 2015
Objective Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still... more Objective Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still a challenging aspect to address. As aneuploidy can be one of the factors influencing the low implantation rates obtained, the aim of this work was to combine monogenic analysis with comprehensive aneuploidy screening (double factor) in order to transfer the selected (healthy and euploid) embryos in the same in-vitro fertilization (IVF) cycle. Method In the present double-factor PGD (DF-PGD) approach, a single blastomere was biopsied from each embryo, and the whole genome amplification DNA product obtained was successfully used for both monogenic analysis and metaphase comparative genomic hybridization cytogenetic screening. The developed DF-PGD was applied to 62 embryos from seven families at risk for monogenic-inherited diseases in a total of seven IVF-DF-PGD cycles. Results While 68.2% of the diagnosed embryos were healthy for the monogenic diseases, only 43.3% of them were chromosomally normal considering aneuploidies and/or segmental chromosome imbalances. Six out of seven families had transferrable embryos according to DF-PGD results. Two healthy babies were born from the 11 selected embryo transfers. Conclusion In families at risk for monogenic diseases, the DF-PGD is a useful tool to select healthy and potentially viable embryos for transfer, according to their chromosome complement.
Biology, Dec 30, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
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...
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En una de les primeres converses que vaig tenir respecte al meu projecte de tesi algú em va dir q... more En una de les primeres converses que vaig tenir respecte al meu projecte de tesi algú em va dir que la CGH en cèl•lules úniques era impossible i que no acabaria la tesi ni en sis ni en set anys.... Bé, no diré que ha estat fàcil, de fet, no ho ha estat gens. No diré tampoc que no he tingut moments de: "ho deixo tot", sobretot en aquell primer any i mig en que no sortia res. Es va seguir endavant, però, i el 12 d'abril de 2002 varem analitzar per primera vegada la CGH d'un corpuscle, on es veia el resultat d'una segregació desequilibrada d'una translocació 1;5. Jo ja no crec en impossibles. M'agradaria agrair a tota la gent que, d'una manera o altra, ha contribuït a que aquesta tesi s'hagi pogut realitzar: A la Quima i al Jordi, per donar-me l'oportunitat de treballar en reproducció i fer la tesi d'un tema que m'apassiona. Per dirigir aquest treball i recolzar-me en moments difícils, fins i tot quan vaig marxar als Estats Units. Per creure tant en aquest projecte i per aquest últim mes tan dur de correccions, gràcies. A mis padres, por su sacrificio para poder darme una buena educación (aunque a veces ya sabéis que soy una maleducada). Por ayudarnos tanto este último año. Porque aunque escuchar una charla mía debe ser un tostón, siempre os presentáis voluntarios y no os dormís!!! Por confiar en mi y apoyar las decisiones que he tomado. Por animarme en mi nuevo proyecto aunque se que os dejo con el corazón triste... y yo me voy igual... Os quiero muchísimo. A mi hermano David, por intentar solucionar mi caos informático, criticar constructivamente mis diapos y mis figuras. Por ser cómo eres (un monstruo, por supuesto), te doy las grácias de una manera especial: albaricoque. Al meu Jorge, que et tinc davant, revisant per enèsima vegada el manuscrit, amb els ulls infladets de totes les hores que no hem dormit aquesta setmana. I tot i així encara em somrius. Per fer-me riure, per ensenyar-me tantes coses, per aquell 8 de Juliol, per totes les coses que hem fet i farem, per ajudar-me tant i aguantar els meus grunyits (si, aquesta paraula existeix...), per Estats Units, per estimar-me... perquè sense tu no ho hagués aconseguit... A mi prima, mis tios y mi abuelito, por ayudarnos con la mudanza y por las risas del karaoke. A la Cristina Hernando i a l'Esther Prat per ensenyar-me a fer CGHs i a cariotipar. A l'Imma Ponsa per tenir sempre un moment per ajudar als altres, per la teva generositat i per aquelles converses tècniques que van tan bé. A la Montse Codina per aguantar-me aquests darrers mesos d'escriptura. Per agafar-te amb filosofia els meus nombrosos dubtes ortogràfics i els meus esbufecs quan el word feia alguna de les seves i havia de començar de nou. A la Mònica per la teva manera d'agafar-te les coses, per les converses de ciència i del que no és ciència, per passar amb mi els nervis de la suficiència i per ensenyar-me les barraques de Girona. A l'Anna, per la teva solidaritat i per ajudar-me sempre que em veies un pel alterada. Al Javi, por hacerme reir tanto, por tus tortillas de patatas estupendas y por tu ayuda con el photoshop. Gracias por llamar aquella noche del caso clínico a las 2 de la mañana para dar ánimos. A la Laia, per ser tan bona "alumna", per fer preguntes intel•ligents i per fer-nos companyia tota la nit del cas clínic. Gràcies als teus DVD's no ens varem adormir!!! A l'Angels Niubó, per la nevera, per les comandes i per les receptes de les solucions. Però sobretot per recolzar tant la nostra aventura americana i per les nostres converses per arreglar el món. A l'Ignasi i al Jordi, per ajudar-me sempre que els hi ho demanat. A Pedro, por hacerme más llevadera las comidas y por nuestras charlas con la boca llena.
Human Reproduction Update, 2001
Preimplantation genetic diagnosis (PGD) using the ®rst polar body (1PB) is a modality of PGD that... more Preimplantation genetic diagnosis (PGD) using the ®rst polar body (1PB) is a modality of PGD that can be used when the woman is the carrier of a genetic disease or of a balanced chromosomal reorganization. PGD using 1PB biopsy in carriers of balanced chromosome reorganizations has not become generalized. Here, we describe our experience based on the analysis of unfertilized or fresh, non-inseminated control oocytes, by ®xing separately the 1PB and the corresponding oocyte, and on the study of six clinical cases of PGD using 1PB biopsy (four Robertsonian translocations and two reciprocal translocations). In fresh oocytes, the chromosome morphology of the 1PB was well preserved, and the results were always concordant for each oocyte±1PB pair. This indicates that the 1PB can be reliably used for the diagnosis of chromosome reorganizations. In these studies the technical problems encountered when performing PGD using 1PB biopsies for chromosome studies are also addressed. Three different strategies of 1PB biopsy (laser beam, partial zona dissection and acid Tyrode's) and two different protocols (intracytoplasmic sperm injection before or after 1PB biopsy) and their effect on the percentage of oocytes diagnosed and the fertilization rate, are discussed. In reciprocal translocation cases, published in the literature or studied by us, in which at least nine oocytes had been diagnosed, a correlation has been found between the frequency of nondisjunction observed and the theoretical recombination rate. To date, PGD by 1PB analysis alone or combined with blastomere biopsies in female carriers of chromosomal rearrangements has been used in 18 cases, with a further six cases reported here. A total of 325 cumulus±oocyte complexes have been obtained, of which 294 were biopsied and 224 were diagnosed. A total of 52 embryos was transferred, 19 of which implanted and 17 produced full-term pregnancies.
Fertility and Sterility, 2011
Objective: To apply a comprehensive chromosomal screening through short comparative genomic hybri... more Objective: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. Design: Clinical research study. Setting: A PGD laboratory and two IVF clinics. Patient(s): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a doubletranslocation carrier. Intervention(s): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. Main Outcome Measure(s): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. Result(s): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. Conclusion(s): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.
Clinical Genetics, 2012
Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-... more Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-gene disorders over the last 20 years. The aim of this work was to perform a double-factor preimplantation genetic diagnosis (DF-PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF-PGD approach in which two blastomeres from each cleavage-stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non-affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short-comparative genomic hybridization (CGH). The improved DF-PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single-gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.
Journal of Clinical Medicine
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 ...
International Journal of Molecular Sciences
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
Dipòsit Digital de Documents de la UAB, May 4, 2022
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.
23 3.1.3. Mètode de biòpsia del 1r corpuscle polar 24 3.1.4. Fixació de cèl•lules aïllades, 1r co... more 23 3.1.3. Mètode de biòpsia del 1r corpuscle polar 24 3.1.4. Fixació de cèl•lules aïllades, 1r corpuscle polar i metafase II, anàlisi usant contrast de fases i tractament previ a la hibridació in situ fluorescent 28 3.1.5. Hibridació in situ fluorescent en extensions del 1r corpuscle polar i metafase II 29 3.2. Material i mètodes de l'objectiu 2 32 3.2.1. Pacients amb cariotip normal. Característiques i antecedents clínics 33 3.2.2. Pacients portadores de translocacions. Característiques i antecedents clínics 33 3.2.3. Mètode utilitzat per al diagnòstic genètic preimplantacional analitzant el 1r corpuscle polar 35 3.3. Material i mètodes de l'objectiu 3 39 3.3.1. Material objecte d'estudi 39 3.3.2. Mètode d' hibridació in situ fluorescent emprat 40 3.4. Material i mètodes de l'objectiu 4 41 3.4.1. Preparació d'un cultiu no sincronitzat de limfòcits estimulats i obtenció de cèl•lules de Sertoli 41 Anàlisi citogenètica preimplantacional Índex III 3.4.2. Mètode d'anàlisi: hibridació in situ fluorescent 42 4. Resultats 44 4.1. Anàlisi d'aneuploïdies en una sèrie d'oòcits descartats de cicles de FIV 45 Article publicat: Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies. European Journal of Human Genetics 2003; 11: 325-336 46 4.2. Anàlisi d'aneuploïdies en la línia germinal femenina 47 4.2.1. Pacients amb cariotip normal 47 4.2.2. Pacients portadores de translocacions 50 Article publicat: Multiple aneuploidies in the oocytes of balanced translocation carriers: a preimplantation genetic diagnosis study using first polar body. Reproduction 2003; 126: 701-711 53 4.3. Estudi d'aneuploïdies en cèl•lules embrionàries de pacients portadores de translocacions recíproques 54 Article pendent de publicació: The importance of aneuploidy screening in reciprocal translocation carriers 56 4.4. Estadi replicatiu de la cromatina en cèl•lules proliferants. Avaluació de la interpretació dels resultats de la tècnica d'hibridació in situ fluorescent 57 Article publicat: The use of a cell-cycle phase-marker may decrease the percentage of errors when using FISH in PGD. Cytogenetic and Genome Research 2004; 105: 29-35 59 5. Discussió 60 5.1. Anàlisi d'aneuploïdies en oòcits descartats de cicles de FIV 61 5.2. Anàlisi d'aneuploïdies en la línia germinal femenina 64 5.2.1. Pacients amb cariotip normal 64 5.2.2. Pacients portadores de translocacions 66 5.3. Estudi d'aneuploïdies en cèl•lules embrionàries de pacients portadores de translocacions recíproques 69 5.4. Estadi replicatiu de la cromatina en cèl•lules proliferants i errors en la interpretació dels resultats de la tècnica d'hibridació in situ fluorescent 73 6. Conclusions 76 7. Bibliografia
Human Fertility, Jan 9, 2020
Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) s... more Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) seems to be the best procedure for its repair and to reduce testicular oxidative stress (ROS). As ROS causes guanine modifications, we postulated that DNA damage could be more intense in telomeres due to their G-rich nature. We studied the effect of MV on sperm telomere length (TL), single-and double-strand DNA fragmentation (ssSDF and dsSDF) and seminal parameters. Sperm telomeres from 12 fertile donors and 20 varicocele patients before and nine months after MV were labelled using FITC-PNA qFISH (a new method to obtain absolute TL from relative fluorescence intensity using FITC-fluorescent spheres). Both ssSDF and dsSDF were analysed using the alkaline and neutral Comet assays, respectively. The results showed that varicocele and MV had no effect on TL. Seminal parameters, ssSDF and dsSDF of varicocele patients were altered. Although these parameters improved after MV, values did not reach those seen in fertile donors. A good estimation of absolute TL was developed based on FITC-fluorescent spheres. The results showed that TL is not affected by varicocele or surgery. However, MV is able to partially reduce altered seminal parameters, ssSDF and dsSDF values in varicocele patients.
Fertility and Sterility, Apr 1, 2019
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.
Asian Journal of Andrology, 2015
Biomedicines, Aug 19, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Prenatal Diagnosis, Oct 15, 2015
Objective Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still... more Objective Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still a challenging aspect to address. As aneuploidy can be one of the factors influencing the low implantation rates obtained, the aim of this work was to combine monogenic analysis with comprehensive aneuploidy screening (double factor) in order to transfer the selected (healthy and euploid) embryos in the same in-vitro fertilization (IVF) cycle. Method In the present double-factor PGD (DF-PGD) approach, a single blastomere was biopsied from each embryo, and the whole genome amplification DNA product obtained was successfully used for both monogenic analysis and metaphase comparative genomic hybridization cytogenetic screening. The developed DF-PGD was applied to 62 embryos from seven families at risk for monogenic-inherited diseases in a total of seven IVF-DF-PGD cycles. Results While 68.2% of the diagnosed embryos were healthy for the monogenic diseases, only 43.3% of them were chromosomally normal considering aneuploidies and/or segmental chromosome imbalances. Six out of seven families had transferrable embryos according to DF-PGD results. Two healthy babies were born from the 11 selected embryo transfers. Conclusion In families at risk for monogenic diseases, the DF-PGD is a useful tool to select healthy and potentially viable embryos for transfer, according to their chromosome complement.
Biology, Dec 30, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
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...
tdx.cat
En una de les primeres converses que vaig tenir respecte al meu projecte de tesi algú em va dir q... more En una de les primeres converses que vaig tenir respecte al meu projecte de tesi algú em va dir que la CGH en cèl•lules úniques era impossible i que no acabaria la tesi ni en sis ni en set anys.... Bé, no diré que ha estat fàcil, de fet, no ho ha estat gens. No diré tampoc que no he tingut moments de: "ho deixo tot", sobretot en aquell primer any i mig en que no sortia res. Es va seguir endavant, però, i el 12 d'abril de 2002 varem analitzar per primera vegada la CGH d'un corpuscle, on es veia el resultat d'una segregació desequilibrada d'una translocació 1;5. Jo ja no crec en impossibles. M'agradaria agrair a tota la gent que, d'una manera o altra, ha contribuït a que aquesta tesi s'hagi pogut realitzar: A la Quima i al Jordi, per donar-me l'oportunitat de treballar en reproducció i fer la tesi d'un tema que m'apassiona. Per dirigir aquest treball i recolzar-me en moments difícils, fins i tot quan vaig marxar als Estats Units. Per creure tant en aquest projecte i per aquest últim mes tan dur de correccions, gràcies. A mis padres, por su sacrificio para poder darme una buena educación (aunque a veces ya sabéis que soy una maleducada). Por ayudarnos tanto este último año. Porque aunque escuchar una charla mía debe ser un tostón, siempre os presentáis voluntarios y no os dormís!!! Por confiar en mi y apoyar las decisiones que he tomado. Por animarme en mi nuevo proyecto aunque se que os dejo con el corazón triste... y yo me voy igual... Os quiero muchísimo. A mi hermano David, por intentar solucionar mi caos informático, criticar constructivamente mis diapos y mis figuras. Por ser cómo eres (un monstruo, por supuesto), te doy las grácias de una manera especial: albaricoque. Al meu Jorge, que et tinc davant, revisant per enèsima vegada el manuscrit, amb els ulls infladets de totes les hores que no hem dormit aquesta setmana. I tot i així encara em somrius. Per fer-me riure, per ensenyar-me tantes coses, per aquell 8 de Juliol, per totes les coses que hem fet i farem, per ajudar-me tant i aguantar els meus grunyits (si, aquesta paraula existeix...), per Estats Units, per estimar-me... perquè sense tu no ho hagués aconseguit... A mi prima, mis tios y mi abuelito, por ayudarnos con la mudanza y por las risas del karaoke. A la Cristina Hernando i a l'Esther Prat per ensenyar-me a fer CGHs i a cariotipar. A l'Imma Ponsa per tenir sempre un moment per ajudar als altres, per la teva generositat i per aquelles converses tècniques que van tan bé. A la Montse Codina per aguantar-me aquests darrers mesos d'escriptura. Per agafar-te amb filosofia els meus nombrosos dubtes ortogràfics i els meus esbufecs quan el word feia alguna de les seves i havia de començar de nou. A la Mònica per la teva manera d'agafar-te les coses, per les converses de ciència i del que no és ciència, per passar amb mi els nervis de la suficiència i per ensenyar-me les barraques de Girona. A l'Anna, per la teva solidaritat i per ajudar-me sempre que em veies un pel alterada. Al Javi, por hacerme reir tanto, por tus tortillas de patatas estupendas y por tu ayuda con el photoshop. Gracias por llamar aquella noche del caso clínico a las 2 de la mañana para dar ánimos. A la Laia, per ser tan bona "alumna", per fer preguntes intel•ligents i per fer-nos companyia tota la nit del cas clínic. Gràcies als teus DVD's no ens varem adormir!!! A l'Angels Niubó, per la nevera, per les comandes i per les receptes de les solucions. Però sobretot per recolzar tant la nostra aventura americana i per les nostres converses per arreglar el món. A l'Ignasi i al Jordi, per ajudar-me sempre que els hi ho demanat. A Pedro, por hacerme más llevadera las comidas y por nuestras charlas con la boca llena.
Human Reproduction Update, 2001
Preimplantation genetic diagnosis (PGD) using the ®rst polar body (1PB) is a modality of PGD that... more Preimplantation genetic diagnosis (PGD) using the ®rst polar body (1PB) is a modality of PGD that can be used when the woman is the carrier of a genetic disease or of a balanced chromosomal reorganization. PGD using 1PB biopsy in carriers of balanced chromosome reorganizations has not become generalized. Here, we describe our experience based on the analysis of unfertilized or fresh, non-inseminated control oocytes, by ®xing separately the 1PB and the corresponding oocyte, and on the study of six clinical cases of PGD using 1PB biopsy (four Robertsonian translocations and two reciprocal translocations). In fresh oocytes, the chromosome morphology of the 1PB was well preserved, and the results were always concordant for each oocyte±1PB pair. This indicates that the 1PB can be reliably used for the diagnosis of chromosome reorganizations. In these studies the technical problems encountered when performing PGD using 1PB biopsies for chromosome studies are also addressed. Three different strategies of 1PB biopsy (laser beam, partial zona dissection and acid Tyrode's) and two different protocols (intracytoplasmic sperm injection before or after 1PB biopsy) and their effect on the percentage of oocytes diagnosed and the fertilization rate, are discussed. In reciprocal translocation cases, published in the literature or studied by us, in which at least nine oocytes had been diagnosed, a correlation has been found between the frequency of nondisjunction observed and the theoretical recombination rate. To date, PGD by 1PB analysis alone or combined with blastomere biopsies in female carriers of chromosomal rearrangements has been used in 18 cases, with a further six cases reported here. A total of 325 cumulus±oocyte complexes have been obtained, of which 294 were biopsied and 224 were diagnosed. A total of 52 embryos was transferred, 19 of which implanted and 17 produced full-term pregnancies.
Fertility and Sterility, 2011
Objective: To apply a comprehensive chromosomal screening through short comparative genomic hybri... more Objective: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. Design: Clinical research study. Setting: A PGD laboratory and two IVF clinics. Patient(s): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a doubletranslocation carrier. Intervention(s): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. Main Outcome Measure(s): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. Result(s): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. Conclusion(s): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.
Clinical Genetics, 2012
Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-... more Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single-gene disorders over the last 20 years. The aim of this work was to perform a double-factor preimplantation genetic diagnosis (DF-PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF-PGD approach in which two blastomeres from each cleavage-stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non-affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short-comparative genomic hybridization (CGH). The improved DF-PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single-gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.
Journal of Clinical Medicine
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 ...
International Journal of Molecular Sciences
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
Dipòsit Digital de Documents de la UAB, May 4, 2022