Aislamiento, cultivo, caracterización y maduración de células indiferenciadas desde biopsias testiculares humanas

En el testículo existe una población de células pluripotentes, las Espermatogonias Stem Cells (SSC), responsables de la autorrenovación y la diferenciación, manteniendo así la espermatogénesis. La espermatogénesis es el proceso en el cual la espermatogonia sufre una serie de divisiones hasta originar el espermatozoide, que son las células fundamentales para transmitir la información genética a las generaciones futuras. Recientemente, han sido publicados una serie de estudios mostrando la posibilidad de generar células pluripotentes a partir de biopsias testiculares humanas de individuos adultos (Conrad et al., 2008; Kossack et al. ,2008). Los resultados obtenidos en éstos trabajos en humanos parecen indicar que las células obtenidas de las biopsias testiculares (las espermatogonias) sufren una reprogramación en cultivo, pasando a expresar marcadores embrionarios y pudiéndose diferenciar a las tres capas germinales y formar teratomas. En el presente proyecto proponemos demostrar la generación de líneas celulares de espermatogonias humanas desde las biopsias testiculares diagnósticas realizadas rutinariamente a pacientes con azoospermia obstructiva. Se trata de demostrar la prueba de concepto para su aplicación clínica en la preservación de la fertilidad en los niños que debido a su tratamiento oncológico deben recibir quimio/radioterapia resultando en la detención de la espermatogénesis. Para ello, se obtendrán líneas de espermatogonias autólogas mediante biopsia previa a su tratamiento oncológico, para luego después de su curación, ser inyectadas en testículo con el fin de recuperar la espermatogénesis en aquellos casos que se hubiera destruido. Durante el periodo de 2009-2010, se han recogido cerca de 50 muestras de biopsias testiculares de pacientes sometidos a tratamientos de Fertilización in vitro (FIV) en la Clínica IVI - Valencia. Las biopsias son realizadas con el objetivo de encontrar espermatozoides para el tratamiento de la infertilidad (ICSI). El material restante de las biopsias es donado mediante un consentimiento informado firmado por los pacientes. La muestra es transportada en hielo hasta en laboratorio , donde se empieza la digestión del material con el uso de enzimas. En primer lugar, realizamos el cultivo de células de Sertoli y a continuación, se lleva a cabo una incubación de 96 horas con factores que mantienen un ambiente favorable para la supervivencia de las espermatogonias. Tras ese período, se purifica la muestra con la utilización del anticuerpo CD49f (Conrad et al., 2008) presente en las células adheridas a la membrana basal de los túbulos seminíferos (espermatogonias). Por último, las células positivas son sembradas y mantenidas en cultivo con diferentes medios a los que se le añaden factores (GDNF, b-FGF, LIF) que mantienen la indiferenciación celular. Las líneas obtenidas poseen características similares: - Expresión de los marcadores de pluripotencia: OCT4, NANOG, SOX2 (determinado por PCR) - Expresión de los marcadores de células germinales: c-Kit, STELLA, BOULE, BLIMP1(determinados por PCR) - Ausencia de expresión de los marcadores de células germinales: GAF, VASA, DAZL, PIWIL2, PRM1, TNP2 (determinados por PCR) - Expresión de los marcadores de espermatogonias: CD49f, Thy-1 (CD90), GPR125, SSEA-1 (determinados por PCR e inmunocitoquímica) - Cultivo de Clusters celulares (medio serum-free + factores) - Microscopia Electrónica: análisis de los clusters en cultivo - Análisis del Cariotipo celular: Normal (46, XY) - FISH: presencia de 90% de células 2n. - Purificación de las células de Sertoli con Nile Red en el Cell SORTER - Análisis de Citometria: Nile Red positivas - Inmunocitoquímica: Oil Red positivas - Expresión de los marcadores de células de Sertoli: WT1, GATA-4 (determinados por PCR) - Ausencia de expresión del receptor hormonal: rFSH (determinado por PCR) - Transfección de las Líneas celulares con la proteína GFP/RPF. Con la obtención de líneas celulares de espermatogonias y células de Sertoli a partir de biopsias testiculares humanas de individuos adultos y el cultivo in vitro en laboratorio, hemos podido establecer un protocolo para aislar éstas células y mantenerlas en un ambiente favorable para su crecimiento y mantenimiento. Posteriormente, empezaremos los experimentos de co-cultivo celular, cultivando las células de Sertoli sobre una matriz de colágeno para dar base a su crecimiento, añadiremos las espermatogonias, además de hormonas como FSH y testosterona, para probar la capacidad de división celular que éstas células poseen in vitro (espermatogénesis).OBJECTIVE: To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells. DESIGN: Prospective analysis. SETTING: Research center. PATIENT(S): Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients. INTERVENTION(S): TESE, with enzymatic dissociation of samples to obtain a cell suspension, which was cultured for 4 days with 4 ng/mL GDNF. The CD49f+ cells were sorted using fluorescence-activated cell sorting (FACS) as a marker to identify spermatogonial stem cells (SSCs), which were cocultured with Sertoli cells expressing red fluorescent protein (RFP) in knockout serum replacement (KSR) media with addition of 1,000 IU/mL of follicle-stimulating hormone (FSH), 1 μM testosterone, 40 ng/mL of GDNF, and 2 μM retinoic acid (RA) for 15 days in culture at 37°C and 5% CO(2) to induce meiotic progression. Cells were collected and analyzed by immunofluorescence for meiosis progression with specific markers SCP3 and CREST, and they were confirmed by fluorescence in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Isolation of CD49f+ cells and coculture with Sertoli cells, meiosis progression in vitro, assessment of SSCs and meiotic markers real-time polymerase chain reaction (RT-PCR), immunohistochemical analysis, and FISH. RESULT(S): The CD49f+ isolated from the of total cell count in the TESE samples of azoospermic patients varied from 5.45% in OA to 2.36% in NOA. Sertoli cells were obtained from the same TESE samples, and established protocols were used to characterize them as positive for SCF, rGDNF, WT1, GATA-4, and vimentin, with the presence of tight junctions and lipid droplets shown by oil red staining. After isolation, the CD49f+ cells were cocultured with RFP Sertoli cells in a 15-day time-course experiment. Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from one NOA patient. A FISH analysis for chromosomes 13, 18, 21, X, and Y confirmed the presence of haploid cells on day 5 of the coculture. CONCLUSION(S): In vitro coculture of SSCs from TESE samples of NOA patients along with Sertoli cells promoted meiosis induction and resulted in haploid cell generation. These results improve the existing protocols to generate spermatogenesis in vitro and open new avenues for clinical translation in azoospermic patients

Primeiro bebê nascido no Brasil após diagnóstico simultâneo por PGT-A não-invasivo e convencional

Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culture media is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. hey did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGT-A and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one false-negative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGT-A, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other non-invasive approaches like morphology and morphokinetics selection.Abordagens para o teste genético pré-implantacional não-invasivo para aneuploidias (non-invasive preimplantation genetic testing for aneuploidies, niPGT-A, em inglês) com o objetivo de avaliar o DNA embrionário livre são promissoras, especialmente porque estas podem reverter as menores taxas de implantação causadas por inadequada biópsia de trofectoderma (TE). Nesse contexto, nosso centro é parte do maior estudo atual que avalia as taxas de concordância entre PGT-A convencional e niPGT-A, e relatamos aqui o nascimento do primeiro bebê brasileiro após niPGT-A. Os pais do bebê foram admitidos no nosso centro em 2018. Eles não apresentavam histórico de infertilidade, e estavam interessados em utilizar os tratamentos de fertilização in vitro (FIV) e PGT-A para evitar anomalias congênitas na progênie. Um total de 11 blastocistos expandidos (3 do dia-5 e 8 do dia-6) foram submetidos a biópsia, e os meios de cultivo condicionados (cultivo do dia-4 ao dia-6) de 8 blastocistos do dia-6 foram coletados para niPGT-A. No total, resultados informativos para as amostras de TE e dos meios foram obtidos para sete embriões. Entre os embriões com resultado informativo, 5 apresentaram diagnóstico concordante entre PGT-A convencional e niPGT-A, e 2 apresentaram diagnóstico discordante (1 falso positivo e 1 falso negativo). O Blastocisto 4, diagnosticado como 46, XY por ambos niPGT-A e PGT-A convencional, foi desvitrificado e transferido, o que resultou no nascimento de um menino saudável, que pesava 3,8 kg, em fevereiro de 2020. Com base em nossos resultados e literatura contemporânea, acreditamos que a aplicação atual mais segura do niPGT-A seria como método de seleção embrionária para pacientes sem indicação ao PGT-A convencional. A confiabilidade aproximada de 80% do niPGT-A para determinação da ploidia ainda é superior àquela obtida com abordagens não invasivas, como seleção morfológica ou morfocinética

The embryo mosaicism profile of next-generation sequencing PGT-A in different clinical conditions and their associations

IntroductionUniform chromosome abnormalities are commonly seen in early pregnancy loss, with analyses of the product of conception suggesting the presence of mosaic autosomal trisomy in ∼10% of cases. Although chromosomal mosaicism occurs in a minority of embryos, their relative commonality and uncertainty regarding associated transfer outcomes have created discussion at both the clinical and research levels, highlighting the need to understand the clinical conditions associated with the incidence of embryo mosaicism.MethodsWe took advantage of a preimplantation genetic testing for aneuploidy (PGT-A) database created from 2019 to 2022 in more than 160 in vitro fertilization (IVF) clinics in Brazil, the second-largest world market for IVF. We carried out descriptive statistical and associative analyses to assess the proportions of mosaicism associated with clinical conditions and reported incidence by chromosome, clinic origin, and biopsy operator.ResultsChromosomal analysis revealed that most mosaic aneuploidies occurred in the last three chromosomes, with 78.06% of cases having only one chromosome affected. Low mosaicism in trisomy represented the most ordinary form, followed by low mosaicism in monosomy. We identified associations between low (negatively-associated) and high mosaicism (positively-associated) and maternal age, indication (male factor and uterus/ovarian factor negatively associated with low and high mosaic, respectively), day of blastocyst development (day five has an overall better outcome), morphology grade (lower quality increased the chances of low and high mosaicism), origin (vitrified oocyte and embryo increased the rates of low and high mosaicism, respectively), and embryo sex (male embryos negatively associated with low mosaic).DiscussionWith these results, we hope to foster an improved understanding of the chromosomal mosaicism linked with distinct clinical conditions and their associations in Brazil