Variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías / Amniotic fluid culture varietys for poliploydies in vitro diagnosis

RESUMEN Fundamento: El cultivo celular permite el análisis directo de las células vivas mediante un microscopio. El estudio de las células contenidas en el líquido amniótico, mediante técnicas de cultivo, detecta anomalías en número y morfología de los cromosomas, que pueden relacionarse con enfermedades genéticas. Objetivo: Caracterizar las variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías. Metodología: Se realizó un estudio descriptivo transversal, en el Centro Provincial de Genética Médica de Camagüey, en el periodo de noviembre de 2016 a abril de 2018.La población de estudio estuvo constituida por 1571 muestras útiles de líquido amniótico obtenidas por amniocentesis, en gestantes en el segundo trimestre, evaluadas en consulta multidisciplinaria con criterios clínicos de estudios cromosómicos según lo establecido en el diagnóstico prenatal citogenético, previo consentimiento informado. Se utilizaron 20 mL de líquido amniótico para la siembra de células fetales, y se aplicaron tres variantes de cultivo abierto (directo, centrifugado y expandido). Se determinó el complemento cromosómico en cada variedad. Resultados: Predominó el complemento cromosómico normal. Las tetraploidías prevalecieron en el cultivo expandido. El índice mitótico fue similar en las tres variedades de cultivo y el cultivo directo tuvo el más bajo índice de poliploidías. Conclusiones: El cariotipo normal fue predominante. Las tetrapolidías fueron las alteraciones más frecuentes y prevalecieron en el cultivo expandido. En el cultivo directo se presentó el más bajo índice de errores inducidos in vitro.

ABSTRACT Background: Cell culture allows direct analysis of live cells under a microscope. The cell study contained in amniotic fluid, by culture techniques, detects abnormalities in chromosome number and morphology, which can be related to genetic diseases. Objective: To describe amniotic fluid culture strains for the in vitro diagnosis of polyploidy. Methodology: A cross-sectional descriptive study was conducted at the Camagüey Provincial Center of Medical Genetics, from November 2016 to April 2018.The study population consisted of 1571 useful amniotic fluid samples obtained by amniocentesis, in pregnant women in the second trimester, evaluated by multidisciplinary discussion with clinical criteria for chromosomal studies as established in the cytogenetic prenatal diagnosis, prior informed consent. 20 mL of amniotic fluid were used for fetal cell seeding, and three open culture strains (direct, centrifuged and expanded) were applied. Chromosomal complement was determined in each variety. Results: Normal chromosome complement was predominant. Tetraploidy prevailed in the expanded culture. The mitotic index was similar in the three culture strains and the direct culture had the lowest polyploidy index. Conclusions: Normal karyotype was predominant. Tetraploidy were the most frequent modifications and prevailed in the expanded culture. Direct culture had the lowest rate of the in vitro induced errors.

Tetraploidia em Rhamdia quelen (Quoy e Gaimard, 1824) por choque térmico duplo (quente e frio) / Tetraploidy in Rhamdia quelen (Quoy & Gaimard 1824) by a dual thermal shock (heat and cold)

Avaliou-se a eficiência do choque térmico duplo para induzir tetraploidia em jundiá Rhamdia quelen em diferentes tempos pós-fertilização. Vinte mililitros de ovos (10, 15, 20, 25, 30 e 35 mpf minutos pós-fertilização) foram tratados com choque térmico quente (39±0,2ºC) durante 3 min seguido de choque térmico frio (1,0±0,1ºC) durante 30 min. Um grupo controle foi utilizado. As taxas de fertilização e de eclosão foram medidas 12 hpf (horas pós-fertilização) e 30 hpf, respectivamente. Decorridas 60 hpf, 25 larvas de cada unidade experimental foram fixadas para verificação da ploidia, por citometria de fluxo. A taxas de fertilização foram de 87,83% para o controle e de 23,4%; 28,5%; 30,4%; 20,0%; 30,3% e 36,7%, para os grupos tratados com choque térmico aos 10, 15, 20, 25, 30, 35 mpf, respectivamente. Encontraram-se tetraploides apenas nos grupos de ovos tratados aos 15 mpf e 20 mpf. Este é o primeiro trabalho de indução de tetraploidia em jundiá por choques térmicos quente e frio, o qual possibilitou o aperfeiçoamento dessa técnica.(AU)

The efficiency of double thermal shock to induce tetraploidy in Rhamdia quelen (jundiá) at different post-fertilization periods was assessed. Each test was performed with 20 mL of eggs (10, 15, 20, 25, 30 and 35 mpf minutes post-fertilization) exposed to hot thermal shock (39±0.2ºC) for 3 min, followed by cold thermal shock (1.0±0.1ºC) for 30 min. A control group was also employed. Fertilization and hatching rates were evaluated 12 and 30 hours post-fertilization (hpf), respectively. After 60 hpf, twenty-five larvae from each experimental unit were fixed to verify the ploidy by flow cytometry. Control fertilization rate was 87.83% and reached 23.4%, 28.5%, 30.4%, 20.0%, 30.3%, 36.7% respectively for 10, 15, 20, 25, 30, 35 mpf. Tetraploids were detected only at 15 mpf and 20 mpf. This is the first research of tetraploidy in jundiá obtained by hot and cold shocks, making possible the improvement of this technique.(AU)

Tetraploidia em Rhamdia quelen (Quoy e Gaimard, 1824) por choque térmico duplo (quente e frio) / Tetraploidy in Rhamdia quelen (Quoy & Gaimard 1824) by a dual thermal shock (heat and cold)

Avaliou-se a eficiência do choque térmico duplo para induzir tetraploidia em jundiá Rhamdia quelen em diferentes tempos pós-fertilização. Vinte mililitros de ovos (10, 15, 20, 25, 30 e 35 mpf – minutos pós-fertilização) foram tratados com choque térmico quente (39±0,2ºC) durante 3 min seguido de choque térmico frio (1,0±0,1ºC) durante 30 min. Um grupo controle foi utilizado. As taxas de fertilização e de eclosão foram medidas 12 hpf (horas pós-fertilização) e 30 hpf, respectivamente. Decorridas 60 hpf, 25 larvas de cada unidade experimental foram fixadas para verificação da ploidia, por citometria de fluxo. A taxas de fertilização foram de 87,83% para o controle e de 23,4%; 28,5%; 30,4%; 20,0%; 30,3% e 36,7%, para os grupos tratados com choque térmico aos 10, 15, 20, 25, 30, 35 mpf, respectivamente. Encontraram-se tetraploides apenas nos grupos de ovos tratados aos 15 mpf e 20 mpf. Este é o primeiro trabalho de indução de tetraploidia em jundiá por choques térmicos quente e frio, o qual possibilitou o aperfeiçoamento dessa técnica.

The efficiency of double thermal shock to induce tetraploidy in Rhamdia quelen (jundiá) at different post-fertilization periods was assessed. Each test was performed with 20 mL of eggs (10, 15, 20, 25, 30 and 35 mpf – minutes post-fertilization) exposed to hot thermal shock (39±0.2ºC) for 3 min, followed by cold thermal shock (1.0±0.1ºC) for 30 min. A control group was also employed. Fertilization and hatching rates were evaluated 12 and 30 hours post-fertilization (hpf), respectively. After 60 hpf, twenty-five larvae from each experimental unit were fixed to verify the ploidy by flow cytometry. Control fertilization rate was 87.83% and reached 23.4%, 28.5%, 30.4%, 20.0%, 30.3%, 36.7% respectively for 10, 15, 20, 25, 30, 35 mpf. Tetraploids were detected only at 15 mpf and 20 mpf. This is the first research of tetraploidy in jundiá obtained by hot and cold shocks, making possible the improvement of this technique.

Tetraploidía en Ceratophrys (Anura, Leptodactylidae), análisis del registro fósil

It has been confirmed the existence of the tetraploid counterpart of the genus Ceratophrys Wied, 1824 (extint at the present time) in Argentina and Brazil since the Pliocene, and the apparition of the octaploidy in the Upper Pleistocene - Holocene. Fossil material of the diploid form, distinctive of the Chacoan District, has been reported from the horizons of the Montehermosense Formation in south-east of the province of Buenos Aires, Argentina. The coexistence of diploid-tetraploid and tetraploid-octaploid forms in the same province was documented.

Tetraploidía en Ceratophrys (Anura, Leptodactylidae), análisis del registro fósil

It has been confirmed the existence of the tetraploid counterpart of the genus Ceratophrys Wied, 1824 (extint at the present time) in Argentina and Brazil since the Pliocene, and the apparition of the octaploidy in the Upper Pleistocene - Holocene. Fossil material of the diploid form, distinctive of the Chacoan District, has been reported from the horizons of the Montehermosense Formation in south-east of the province of Buenos Aires, Argentina. The coexistence of diploid-tetraploid and tetraploid-octaploid forms in the same province was documented.