Proporción sexual y hermafroditismo del molusco, Anadara tuberculosa (Bivalvia: Arcidae) en Panamá / Sexual proportion and hermaphroditism of the mollusc, Anadara tuberculosa (Bivalvia: Arcidae) in Panama

Introducción: El bivalvo Anadara tuberculosa es un recurso pesquero y económico importante en el Pacífico Este Tropical. La proporción sexual varia a lo largo de su área de distribución, con casos de aparición de hermafroditismo, ocasional para algunas poblaciones, normal en otras. Estas características podrían ser una respuesta a presiones antropogénicas y ambientales. Objetivo: Evaluar la proporción sexual y el hermafroditismo en la especie en el Pacífico de Panamá. Métodos: Se analizó información reproductiva de A. tuberculosa generada entre 1994 y 2020 para el Golfo de Montijo, así como datos de 2017 para el manglar de David. Se aplicaron procedimientos macroscópicos, contrastados con histológicos y se generó un factor de corrección para ajustar la proporción sexual macroscópica, debido al efecto, sobre la proporción sexual, que ocasiona los organismos indeterminados. Resultados: Para el Golfo de Montijo, la histología indicó una proporción sexual hembra a macho de 1:1 hasta 3.2:1, entre 1994 y 2017, mientras que la macroscopía varió de 1.9:1 a 5.1:1 entre 2004 y 2020. Para el manglar de David, la histología indicó una proporción sexual de 3.7:1, y de 7.9:1 por macroscopía en el 2017. En ambas localidades, especímenes más grandes mostraron mayor variación en la proporción sexual, pese a la técnica implementada, así como hermafroditismo protándrico no casual en el 2017. Conclusiones: La aparición del hermafroditismo protándrico y los cambios en la proporción sexual indican que la población de A. tuberculosa, en ambas localidades, está respondiendo a presiones antropogénicas y ambientales.

Introduction: The bivalve Anadara tuberculosa is an important fishery in the Eastern Tropical Pacific. The sex ratio varies along its geographic range and there are cases of hermaphroditism, occasional in some populations, normal in others. These characteristics may be a response to anthropogenic and environmental pressure. Objective: To evaluate sex ratio and hermaphroditism of the species in the Panamanian Pacific. Methods: Data on reproductive features of A. tuberculosa generated between 1994 and 2020 for Gulf of Montijo, as well as 2017 data from mangroves of David was analyzed. Macroscopic diagnosis of individual sex is sensitive to the problem of indeterminate individuals, which in turn can skew the sex ratio analysis; therefore, the macroscopic method was compared with histological sections to generate a correction factor to adjust the macroscopically determined sex ratio. Results: For Montijo, histology indicated that the female to male sex ratio ranged from 1:1 to 3.2:1 between 1994 and 2017, while macroscopy indicated 1.9:1 to 5.1:1 between 2004 and 2020. For David, histology indicated a ratio of 3.7:1, and macroscopy 7.9:1. At both localities, larger specimens had more sex ratio variation, independently of the technique, and there was non-casual protandric hermaphroditism in 2017. Conclusion: The occurrence of protandric hermaphroditism and sex ratio changes suggest anthropogenic and environmental pressure in all populations.

Interstitial disease associated with connective tissue disease and vasculitis.

Systemic autoimmune diseases comprise a complex, heterogeneous group of entities. Noteworthy among the pulmonary complications of these entities is interstitial involvement, which manifests with the same radiopathologic patterns as in idiopathic interstitial pneumonia. High-resolution computed tomography is the gold-standard imaging technique; it enables us to identify and classify the disease and to determine its extent, providing useful information about the prognosis. In this group of processes, the most common pattern of presentation is nonspecific interstitial pneumonia. It is essential for radiologists to work together in collaboration with other specialists to reach the correct diagnosis and enable appropriate, integrated treatment.

Estructura de tallas, distribución y abundancia de Anadara tuberculosa (Bivalvia: Arcidae) en dos sistemas de manglar del Pacífico de Panamá / Size structure, distribution, and abundance of Anadara tuberculosa (Bivalvia: Arcidae) in two mangrove systems from the Pacific Coast of Panama

Resumen Introducción: Anadara tuberculosa es un recurso pesquero y económico importante, propio de los ecosistemas de manglar en el Pacífico Orienta Tropical. Objetivo: El objetivo del estudio fue analizar la estructura de tallas, distribución y densidad de A. tuberculosa, en el Golfo de Montijo y Manglares de David, para evaluar el estado del recurso en los dos sistemas de manglar del Pacífico de Panamá. Métodos: Los datos corresponden a información histórica de tallas y densidad levantada entre el 2004 y 2020 para el Golfo de Montijo (GM), así como datos de tallas, densidades y distribución levantados en el 2016 de manera simultánea en 51 cuadrantes en GM y 56 cuadrantes en Manglares de David (MD), muestreados en temporada seca, transición, lluviosa y transición, donde además se registró la temperatura, la salinidad y el pH del agua del canal y del agua retenida en el manglar. En el 2016 se instalaron y muestrearon cuadrantes fijos para seguimiento de densidades, muestreo que se repitió en el 2019. En el 2019 se establecen transectos desde el borde del manglar con el canal principal, hasta límite con tierra firme y en cada transecto se instalaron tres cuadrantes, en los estratos inicial, medio y final. Resultados: Los ejemplares del Golfo de Montijo resultaron más grandes, pero la densidad resultó superior en Manglares de David. La comparación de tallas por periodo de muestreo en el 2016, dentro de cada localidad, resultó significativa solo para el Golfo de Montijo, donde las mayores tallas se presentaron en marzo y junio y las menores en octubre-diciembre. No se encontró asociación entre la temperatura, salinidad y pH, y la distribución del molusco. Para el Golfo de Montijo se encontró un descenso sostenido de las densidades desde 1.82 conchas/m2 en el 2004 hasta 0.6 conchas/m2 en el 2019, contrario a lo observado en la estructura de tallas, donde se observó predominio de las tallas superiores a los 50 mm de longitud total y un aumento de la talla promedio de captura de 52.62 mm en el 2004 a 59.28 mm en el 2020. Atendiendo a la distancia del canal principal, se determinó la presencia del molusco hasta poco más de dos kilómetros dentro del manglar y hasta más de 500 m del canal secundario más cercano, así como una disminución de la densidad y un aumento de la talla conforme nos acercamos a tierra firme. Conclusiones: Para A. tuberculosa, la combinación de una estructura de tallas sobre los 50 mm y de bajas densidades, indican que la población de concha negra en el Golfo de Montijo presenta un grado de deterioro, asociado a estresores pesqueros y ambientales.

Abstract Introduction: Anadara tuberculosa is an economically important fishery resource of mangrove ecosystems in the tropical Eastern Pacific. Objective: The objective of this study was to analyze the size structure, distribution, and density of A. tuberculosa, in the Gulf of Montijo (GM) and Mangroves of David (MD) in the Panamanian Pacific, to assess the state of the resource in Panama's two Pacific mangrove systems. Methods: We analyzed Gulf of Montijo historical clam size records and densities from 2004 and 2020. Hundred and seven 30 m2 permanent plots (51 from GOM and 56 from MOD) were set and sampled in 2016 at four time intervals (dry season, transition into rainy, rainy season and transition into dry season) to obtain clams sizes, densities and distributions. The plots were sampled again in 2019. Physical variables such as temperature (°C), salinity (PSU) and pH from the water retained in the mangrove channels were also recorded. Nine transects were installed and sampled in 2019. Three at the seaward edge of the mangrove fringe, three at the inshore edge, and three half-way between the first two. Three quadrats were sampled at the beginning, middle and end of each transect. Results: Gulf of Montijo A. tuberculosa were found to larger but at lower densities than those from Mangroves of David. At each site, size comparison by sampling period, were statistically significant only for GOM, where larger sizes were found in March and July and smaller sizes in October-December. There was no correlation between the distributions of A. tuberculosa and temperature, salinity and pH. A sustained decrease in A. tuberculosa densities was found for GOM (1.82 ind/m2 in 2004 to 0.6 ind/m2 in 2019). This contrast with the predominantly larger clam sizes above 50 mm in total length and an increase in mean size of collected individuals (52.62 mm in 2004 to 59.28 mm in 2020). Specimens of A. tuberculosa were found up to slightly more than 2 km inshore from the main channel and within slightly more than 500 m from a secondary channel. Clam densities decreased and sizes increased in a seaward-inshore direction. Conclusions: For A. tuberculosa, the combination of size classes larger than 50 mm and low densities of individuals is evidence of population deterioration associated to fishery and environmental stressors.

Functional analysis of mTERF5 and mTERF9 contribution to salt tolerance, plastid gene expression and retrograde signalling in Arabidopsis thaliana.

We previously showed that Arabidopsis mda1 and mterf9 mutants, defective in the chloroplast-targeted mitochondrial transcription termination factors mTERF5 and mTERF9, respectively, display altered responses to abiotic stresses and abscisic acid (ABA), as well as perturbed development, likely through abnormal chloroplast biogenesis. To advance the functional analysis of mTERF5 and mTERF9, we obtained and characterized overexpression (OE) lines. Additionally, we studied genetic interactions between sca3-2, affected in the plastid-RNA polymerase RpoTp, and the mda1-1 and mterf9 mutations. We also investigated the role of mTERF5 and mTERF9 in plastid translation and plastid-to-nucleus signalling. We found that mTERF9 OE reduces salt and ABA tolerance, while mTERF5 or mTERF9 OE alter expression of nuclear and plastid genes. We determined that mda1-1 and mterf9 mutations genetically interact with sca3-2. Further, plastid 16S rRNA levels were reduced in mda1-1 and mterf9 mutants, and mterf9 was more sensitive to chemical inhibitors of chloroplast translation. Expression of the photosynthesis gene LHCB1, a retrograde signalling marker, was differentially affected in mda1-1 and/or mterf9 compared to wild-type Col-0, after treatments with inhibitors of carotenoid biosynthesis (norflurazon) or chloroplast translation (lincomycin). Moreover, mterf9, but not mda1-1, synergistically interacts with gun1-1, defective in GUN1, a central integrator of plastid retrograde signals. Our results show that mTERF9, and to a lesser extent mTERF5, are negative regulators of salt tolerance and that both genes are functionally related to RpoTp, and that mTERF9 is likely required for plastid ribosomal stability and/or assembly. Furthermore, our findings support a role for mTERF9 in retrograde signalling.

Los documentos de voluntades anticipadas. La opinión de los representantes / Advance directives. Representatives' opinions

Introducción. El uso y utilidad de los documentos de voluntades anticipadas (DVA) es motivo de frecuente polémica en relación con su validez y eficacia, aspectos inexplorados en nuestro país desde la perspectiva de los representantes. Objetivo. Conocer la opinión de los representantes designados en un DVA registrado, sobre su utilización. Metodología. Estudio transversal mediante encuesta telefónica a los representantes de 146 personas fallecidas que, desde febrero de 2012, habían registrado un DVA. Resultados. Noventa y ocho (67,1%) de los entrevistados afirmaron que se consultó el DVA, 86 (58,9%) que se les consultó la opinión como representante y 120 (82,1%) creían que se respetó la voluntad del paciente. Ciento dos (69,9%) de las personas entrevistadas consideraron que los pacientes que previamente habían planificado su atención mediante un DVA tuvieron una buena muerte, 33 (22,4%) manifestaron que podría haber sido mejor y 10 (6,9%) creían que sufrieron mucho. Conclusión. Los DVA en su gran mayoría fueron consultados y respetados, y posiblemente este hecho se relaciona con que la mayoría de los representantes afirmasen que la muerte de sus representados fue percibida como confortable. Para finalizar sería conveniente otro tipo de estudios complementarios dirigidos a personal sanitario para conocer las percepciones de estos respecto a la utilización de los DVA en el proceso de muerte (AU)

Introduction. The use and usefulness of Advance Directives has led to a lot of controversy about their validity and effectiveness. Those areas are unexplored in our country from the perspective of representatives. Objective. To determine the opinion of the representatives appointed in a registered Statement of Advance Directives (SAD) on the use of this document. Methods. Telephone survey of representatives of 146 already dead people and who, since February 2012, had registered a SAD document. Results. More the two-thirds (98) of respondents recalled that the SAD was consulted, with 86 (58.9%) saying that their opinion as representative was consulted, and 120 (82.1%) believe that the patient's will was respected. Of those interviewed, 102 (69.9%) believe that patients who had previously planned their care using a SAD had a good death, with 33 (22.4%) saying it could have been better, and 10 (6.9%) believe they suffered greatly. Conclusion. The SAD were mostly respected and consulted, and possibly this is related to the fact that most of the representatives declare that the death of those they represented was perceived as comfortable. It would be desirable to conduct further studies addressed at health personnel in order to know their perceptions regarding the use of Advance Directives in the process of dying (AU)

[Advance directives. Representatives' opinions]. / Los documentos de voluntades anticipadas. La opinión de los representantes.

INTRODUCTION: The use and usefulness of Advance Directives has led to a lot of controversy about their validity and effectiveness. Those areas are unexplored in our country from the perspective of representatives. OBJECTIVE: To determine the opinion of the representatives appointed in a registered Statement of Advance Directives (SAD) on the use of this document. METHODS: Telephone survey of representatives of 146 already dead people and who, since February 2012, had registered a SAD document. RESULTS: More the two-thirds (98) of respondents recalled that the SAD was consulted, with 86 (58.9%) saying that their opinion as representative was consulted, and 120 (82.1%) believe that the patient's will was respected. Of those interviewed, 102 (69.9%) believe that patients who had previously planned their care using a SAD had a good death, with 33 (22.4%) saying it could have been better, and 10 (6.9%) believe they suffered greatly. CONCLUSION: The SAD were mostly respected and consulted, and possibly this is related to the fact that most of the representatives declare that the death of those they represented was perceived as comfortable. It would be desirable to conduct further studies addressed at health personnel in order to know their perceptions regarding the use of Advance Directives in the process of dying.

Carcinoma adenoide quístico traqueal en un paciente con tos crónica / No disponible

El carcinoma adenoide quístico es una neoplasia traqueal primaria poco frecuente cuya histología es la segunda más común después del carcinoma de células escamosas. Los síntomas están usualmente en relación con obstrucción de la vía aérea e incluyen estridor, hemoptisis, disnea y sibilancias. Muchos de estos pacientes son diagnosticados incorrectamente durante meses o años como bronquitis crónica o asma hasta que la lesión se diagnostica. Presentamos un caso de un paciente de 35 años estudiado por tos de seis meses de evolución con carcinoma adenoide quístico traqueal. La exploración física fue normal. La radiografía de tórax mostró una ligera impronta sobre la luz traqueal. La tomografía computarizada y la fibrobroncoscopia con biopsia aportaron el diagnóstico definitivo

Adenoid cystic carcinoma is an infrequent primary tracheal tumor that is second most common tracheal malignancy at histology after squamous cell carcinoma. Symptoms are usually related to airway obstruction including stridor, hemoptysis, shortness of breath and wheezing respiration. Patients may be incorrectly diagnosed and treated for chronic bronchitis or asthma for months or years before the lesion is detected. We present a 35-years-old patient with a 6-month history of cough diagnosed with adenoid cystic carcinoma. General physical examination did not reveal any significant abnormality. Chest radiograph demonstrated a slight narrowing of the tracheal air column. Computed tomography and fibrobronchoscopy with biopsy allowed the final diagnosis

Tratamiento antifúngico con posaconazol y anfotericina B en paciente con mucormicosis rinosinusal / Antifungal treatment with posaconazole and liposomal amphotericin B in patient with mucormycosis rhinosinusal

Introducción: La mucormicosis es una infección fúngica con elevada morbilidad y mortalidad. Se requiere tanto tratamiento quirúrgico como antifúngico, siendo el antimicótico de elección anfotericina B. Posaconazol es un antimicótico derivado de triazol que presenta actividad in vitro e in vivo frente a zygomicetes y que se ha usado en pacientes refractarios o intolerantes a anfotericina B. Descripción del caso: Varón de 70 años con recidiva de mucormicosis rinosinusal ocular izquierda. Había recibido anfotericina B liposomal asociada a posaconazol 8 semanas; tras este primer ingreso fue dado de alta con dosis infraterapéuticas de posaconazol. Volvió a ingresar por recidiva de la enfermedad, se practicó cirugía y recibió anfotericina B liposomal asociada a posaconazol 5 semanas. Siete meses después del alta, el paciente estaba clínicamente estable y continuaba tratamiento con posaconazol. Discusión/Conclusiones: Anfotericina B es el tratamiento de elección en la mucormicosis. Posaconazol es una alternativa aceptable en pacientes refractarios o intolerantes, presentando un perfil de seguridad favorable

No disponible

Neumonía lipoidea: A propósito de un caso / No disponible

La neumonía lipoidea es una enfermedad respiratoria poco frecuente, secundaria a la aspiración de materiales oleosos o bien por la liberación de lípidos desde los alvéolos destruidos por diferentes causas. La exposición crónica a sustancias grasas puede pasar inadvertida y producir síntomas respiratorios inespecíficos. Presentamos un caso de neumonía lipoidea crónica exógena en el que no se halló la sustancia de exposición, pero ante la densidad grasa de los infiltrados del TC y la ausencia de otras etiologías posibles se llegó al diagnóstico

Lipoid pneumonia is a rare respiratory disease, secondary to oil substances inhalation or by leakage of lipids of the alveolus cells which are destroyed in different causes. Chronic exposure to fatty substances can remain unnoticed and produce nonspecific respiratory symptoms. We present a case of chronic exogenous lipoid pneumonia in which case the causal agent were not found, but the fat density of the infiltrates in the TC and the absence of other etiologies allowed for the diagnosis

Presence of an extra chromosome alters meiotic double-stranded break repair dynamics and MLH1 foci distribution in human oocytes.

Studies performed on human trisomic 21 oocytes have revealed that during meiosis, the three homologues 21 synapse and, in some cases, achieve what looks like a trivalent. This implies that meiotic recombination takes place among the three homologous chromosomes 21, and to some extent, crossovers form between them. To see how meiotic recombination is in the presence of an extra chromosome 21, we analyzed the distribution of three recombination markers (γH2AX, RPA, and MLH1) on trisomic 21 oocytes at pachynema and, in particular, on chromosomes 21. Results clearly show how the presence of an extra chromosome 21 alters meiotic recombination progression, leading to the presence of a higher number of early recombination markers at pachynema. Moreover, the distribution on these chromosomes 21 of some of these markers is different in aneuploid oocytes. Finally, there is a substantial increase in the number of MLH1 foci, a marker of most crossovers in mammals, which is related to the number of synapsed chromosomes in pachynema. Thus, bivalents 21 had fewer MLH1 foci than partial or total trivalents, suggesting a close relationship between synapsis and crossover designation. All of the data presented suggest that the presence of an extra chromosome alters meiotic recombination globally in aneuploid human oocytes.

Validación, adaptación y traducción al castellano del MacCAT-T: herramienta para evaluar la capacidad en la toma de decisiones sanitarias / Validation, adaptation and translation of the MacCAT-T into Spanish: a tool to assess the ability to make health decisions

Introducción. La valoración de la capacidad es elemento esencial del proceso de consentimiento informado y un deber del médico. Una herramienta ampliamente desarrollada es el MacCAT-T que explora cuatro habilidades para consentir un tratamiento. No se dispone versión en español y el objetivo principal de este trabajo es validar, adaptar y traducir el MacCAT-T al castellano. Material y métodos. Se tradujo al español e inversamente al inglés. Se validó en su apariencia y contenido (a través de 15 expertos), en constructo (confiabilidad interevaluador y consistencia interna) y en criterio (la validez de un instrumento comparándola con algún criterio externo, en este caso el rendimiento cognoscitivo evaluado por el mini examen cognoscitivo de Lobo). Se incluyeron noventa pacientes ambulatorios médico-quirúrgicos, mayores de 18 años sin déficits de expresión y/o con graves alteraciones de conciencia que no permitieran la realización de la entrevista. Resultados. Los resultados han permitido valorar los diferentes tipos de validez. Su media de aplicación ha sido entre 9 y 13 minutos. Discusión. Los datos son coherentes con los obtenidos en otras aplicaciones del MacCAT-T en lengua inglesa y facilitan la disposición de una herramienta en castellano para valorar la capacidad en la toma de decisiones sanitarias(AU)

Introduction. Capacity assessment is an essential element of the informed consent process and is the duty of the physician. The MacCAT-T instrument explores four skills needed to consent a treatment. There is no Spanish version, and the main objective of this work is to validate, adapt and translate the MacCAT-T into Spanish. Material and methods. The MacCAT-T was translated into Spanish and then back-translated into English. It was validated as regards its appearance and content (by 15 experts), construct (inter-rater reliability and internal consistency) and criteria (the validity of an instrument by comparing it to some external criterion, in this case the Mini Examen Cognoscitivo de Lobo). Ninety medical and surgical outpatients over 18 years were included with no deficits of expression and/or severe disorders of consciousness that did not allow them to be interviewed. Results. They have been optimal considering different types of validity. The average application time was between 9 and 13minutes. Discussion. Data are consistent with those obtained in other applications of MacCAT-T in the English language and facilitate the provision of a Spanish tool for assessing capacity(AU)

[Validation, adaptation and translation of the MacCAT-T into Spanish: a tool to assess the ability to make health decisions]. / Validación, adaptación y traducción al castellano del MacCAT-T: herramienta para evaluar la capacidad en la toma de decisiones sanitarias.

INTRODUCTION: Capacity assessment is an essential element of the informed consent process and is the duty of the physician. The MacCAT-T instrument explores four skills needed to consent a treatment. There is no Spanish version, and the main objective of this work is to validate, adapt and translate the MacCAT-T into Spanish. MATERIAL AND METHODS: The MacCAT-T was translated into Spanish and then back-translated into English. It was validated as regards its appearance and content (by 15 experts), construct (inter-rater reliability and internal consistency) and criteria (the validity of an instrument by comparing it to some external criterion, in this case the Mini Examen Cognoscitivo de Lobo). Ninety medical and surgical outpatients over 18 years were included with no deficits of expression and/or severe disorders of consciousness that did not allow them to be interviewed. RESULTS: They have been optimal considering different types of validity. The average application time was between 9 and 13minutes. DISCUSSION: Data are consistent with those obtained in other applications of MacCAT-T in the English language and facilitate the provision of a Spanish tool for assessing capacity.

Hernia de Bochdalek: presentación de un caso / Bochdalek's hernia: a case report

No disponible

[Repair of a leaking bleb filtration with conjunctival and amniotic membrane transplantation]. / Comunicación corta. Reparación de ampolla filtrante postrabeculectomía con injerto de conjuntiva y membrana amniótica.

CLINICAL CASE: A 71-year-old woman presented with a leaking bleb after a combined phacotrabeculectomy performed 13 years ago. To construct a new filtering bleb, the necrotic area was fully excised. The amniotic membrane was inserted over the scleral flap underneath healthy conjunctiva edges. A conjunctiva-Tenon autograft from the contralateral eye was sutured and was sealed with fibrin adhesive. DISCUSSION: We propose a new surgical technique to repair late leak failures after trabeculectomy with mitomycin C in blebs with a large avascular area. Transplantation of amniotic membrane and conjunctival autograft may be used to repair late leak failures with inadequate conjunctiva to advance.

Comunicación corta. Reparación de ampolla filtrante postrabeculectomía con injerto de conjuntiva y membrana amniótica / Repair of a leaking bleb filtration with conjunctival and amniotic membrane transplantation

Caso clínico: Mujer de 71 años que presenta punto de fuga en la ampolla de una facotrabeculectomíarealizada 13 años antes. Se reseca área necrótica y se sutura trasplante demembrana amniótica sobre el tapete escleral hiperfiltrante. Sobre esta se sutura injerto deconjuntiva y Tenon del ojo contralateral.Discusión: Se propone nueva técnica quirúrgica para el tratamiento de fístula de filtracióntardía tras trabeculectomía con mitomicina C y área avascular extensa. Se aplica adhesivo defibrina para el sellado de incisiones. El doble trasplante de membrana amniótica y conjuntivacontralateral puede utilizarse para reparar puntos de fuga tardíos postrabeculectomía conconjuntiva insuficiente(AU)

Clinical Case: A 71-year-old woman presented with a leaking bleb after a combined phacotrabeculectomyperformed 13 years ago. To construct a new filtering bleb, the necrotic areawas fully excised. The amniotic membrane was inserted over the scleral flap underneathhealthy conjunctiva edges. A conjunctiva-Tenon autograft from the contralateral eye wassutured and was sealed with fibrin adhesive.Discussion: We propose a new surgical technique to repair late leak failures after trabeculectomywith mitomycin C in blebs with a large avascular area. Transplantation of amniotic membrane and conjunctival autograft may be used to repair late leak failureswith inadequate conjunctiva to advance(AU)

Human meiotic progression and recombination are affected by Bisphenol A exposure during in vitro human oocyte development.

BACKGROUND: Bisphenol A (BPA) is a 'weak' endocrine disruptor. The effect of BPA on human reproduction is controversial but has been related to meiotic anomalies, recurrent miscarriages and abnormal karyotypes. METHODS: To evaluate the effects of BPA on survival, pairing-synapsis and meiotic recombination of human fetal oocytes, 21 510 oocytes from 12 cultured fetal ovaries were analyzed. Ovaries were cultured for 7, 14 or 21 days in control medium, dimethylsulfoxide-medium, BPA-medium and estradiol (E(2))-medium. Meiotic pairing-synapsis and recombination were studied by immunofluorescence against lateral element protein, central element protein of the synaptonemal complex and chromosome axis cohesin REC8. Mismatch repair protein, MLH1, was used as a crossover (CO) marker. Meiotic progression was analyzed following the number of surviving oocytes at different meiotic stages found in each culture time and condition, and the total number of MLH1 foci found in oocytes from cultured ovaries. RESULTS: Oocyte survival in vitro decreased with the addition of BPA to the medium (1 µM or greater). Oocyte degeneration was up to five times higher when BPA was added to culture medium. Moreover, oocytes exposed to BPA concentrations of 10 µM or higher presented approximately two times more MLH1 foci than unexposed cultured oocytes (P = 0.01). This was also observed in chromosome 21 from BPA-exposed oocytes, which had double the average number of MLH1 foci found in control oocytes (P = 0.001). E(2) was used as a positive control of estrogen receptors activity, and E(2) addition to the medium had similar effects on meiotic progression of oocytes from cultured ovaries. CONCLUSIONS: Our findings show that BPA concentrations of 1 µM or higher decrease the survival of human fetal oocytes in vitro, and concentrations of 10 µM or higher increase MLH1 foci number. MLH1 is considered a CO marker, and thus an increase in MLH1 foci could indicate an increase in COs in BPA-exposed oocytes. These data suggest that BPA can act as a toxic substance, which has particular implications for human females and the critical events of meiotic prophase, such as pairing-synapsis and recombination processes, as well as oocyte survival.

Cytogenetic analyses of human oocytes provide new data on non-disjunction mechanisms and the origin of trisomy 16.

BACKGROUND: Nowadays, oocyte donation is an extended practise in IVF programmes. However, to date, little information on aneuploidy frequency in oocytes from donors is available. Aneuploidy is one of the major causes of embryo and fetal wastage as well as of congenital mental and developmental disabilities. It is known that most aneuploidies are due to non-disjunction events occurring in the maternal germ line. Linkage studies have associated abnormal patterns of meiotic recombination to the origin of the non-disjunction event in many aneuploid conditions. METHODS AND RESULTS: In the present study, we analyse the frequency of chromosome imbalances in a series of metaphase I (MI; n = 44) and metaphase II (MII; n = 103) oocytes from 140 young donors (aged from 18 to 35 years, mean age 26.6) after hormone-induced superovulation. The aneuploidy frequency found in MII oocytes was 12.6%, and both whole-chromosome non-disjunction (1.94%) and premature separation of sister chromatids (PSSC) (12.6%) have been found. The chromosomes involved have been identified by multiplex fluorescent in situ hybridization (FISH). Achiasmate chromosomes have been identified in MI oocytes (9.1%), with most of them corresponding to chromosome 16 (6.8%). For this reason, the meiotic recombination pattern of chromosome 16 has been analysed in prophase I oocytes (n = 81) by immunofluorescence staining against MLH1 protein and subsequent FISH with specific probes. Our results show a percentage of oocytes with non-crossover bivalent 16 (2.5%) and a high percentage of bivalents 16 with a single exchange (19.8%). CONCLUSIONS: In the present study, we report the finding of a considerable frequency of aneuploidy in oocytes from young donors, with the frequency of PSSC being higher than the frequency of whole-chromosome non-disjunction. In addition, we report vulnerable patterns of meiotic recombination in chromosome 16 that may be at risk of leading to a non-disjunction event. This gives new data on the susceptibility of the control population to conceive a trisomic 16 embryo.

A new culture technique that allows in vitro meiotic prophase development of fetal human oocytes.

BACKGROUND: Little is known about the mechanisms that regulate meiosis in the human female fetus as a result of the technical difficulties in obtaining samples. Currently, there is no technique for human fetal oocyte culture that permits the maintenance of fetal ovarian tissue in vitro which allows the progression of meiosis in a reproducible and standardized way. METHODS: Meiotic progression was analyzed following pairing-synapsis and recombination progress. A total of 7119 oocytes were studied and analyzed. The proteins used to evaluate meiotic progression were: REC8, SYCP1, SYCP3 and MLH1, studied by immunofluorescence. Four different sample disaggregating methods were used, two enzymatic (trypsin and collagenase + hyaluronidase) and two mechanical (puncture and ovarian fragments). Two different culture media were used, control media and stem cell factor (SCF)-supplemented media. The oocytes were studied at initial time T0, and then at T7, T14 and T21 days after culture. RESULTS: The mechanical methods increased the total number of oocytes found at the different times of culture and decreased the number of degenerated oocytes. Independently of the disaggregation method used, oocytes cultured with SCF-supplemented media showed a higher proportion of viable oocytes and fewer degenerated cells at all culture timepoints. No evidence of abnormal homologous chromosome synapsis was observed. Meiotic recombination was only observed in oocytes mechanically disaggregated and cultured with supplemented media. CONCLUSIONS: The oocytes obtained by mechanical disaggregating methods and cultured with SCF-supplemented media are able to follow pairing-synapsis and recombination, comparable to oocytes in vivo. The culture conditions described herein confirm the methodology as a standardized and reproducible method.

Analysis of recombination along chromosome 21 during human female pachytene stage.

It is accepted that recombination errors during human female meiotic prophase have some influence on the origin of trisomy 21. A total of 335 oocytes from four euploid fetuses were analysed by immunofluorescence and fluorescence in-situ hybridization in order to assess the recombination nodules along chromosome 21. Results based on the analysis of recombination points on the bivalent 21 during human female meiosis showed that both number [none (3.70%), one (79.01%) and two (17.29%)1 and distribution (always positioned interstitially on the q-arm) are different in males, ensuring that the two homologues more efficiently remain together until anaphase 1.Therefore, the mainly maternal origin of trisomy 21 appears not be linked to the first stages of oocyte development during fetal life, and this leads to the suggestion that the influence of environmental factors on the segregation of chromosome 21 homologues in later meiotic stages could have a significant role in the predominant maternal origin of trisomy 21.

ATR, BRCA1 and gammaH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes.

Asynapsis of homologous chromosomes at the pachytene stage has been associated with gametogenic failure and infertility, but the cellular mechanisms involved are currently unknown in human meiocytes. In mice, the protein encoded by the breast-cancer susceptibility gene Brca1 has been described to direct kinase ATR (ataxia telangiectasia and Rad3 related) to any unpaired DNA at the pachytene stage, where ATR triggers H2AX phosphorylation, resulting in the silencing of those chromosomes. In this study, the distribution of ATR, BRCA1 and the phosphorylated histone gammaH2AX is assessed by immunofluorescence in human oocytes and it is found that they localize at unpaired chromosomes at the pachytene stage. Evidence is shown to propose that BRCA1, ATR and gammaH2AX in the human may be part of a system such as the one previously described in mouse, which signals unsynapsed chromosomes at pachytene and may lead to their silencing.