RESUMEN
The present study aimed to investigate a concentration-response curve of human recombinant FSH (hrFSH) for in vitro culture of isolated preantral and early antral follicles of goats. Isolated follicles were cultured for 18 days using the following treatments: basic culture medium (control); or control medium supplemented with 10, 50, and 100 mIU/mL of hrFSH. At the end of the culture, cumulus-oocyte complexes were recovered and subjected to in vitro maturation. The following endpoints were evaluated: follicle morphology, growth rate and antrum formation, oocyte viability and meiotic stage, and estradiol production, as well as relative expression of FSH receptor (FSHR), and steroidogenic enzyme (3ß-HSD, CYP17, and CYP19A1) genes. In antral follicles, the FSH addition at 50 mIU/mL increased follicular diameter and growth rate, percentage of fully developed oocytes, and oocyte diameter (P < 0.05), and tended to increase the percentage of MII oocytes when compared to the control (P = 0.07). With preantral follicles, FSH addition at 100 mIU/mL increased relative abundance of mRNA for CYP19A1 when compared to the control (P < 0.05). At the same FSH concentrations of 100 and 50 mIU/mL, there was a greater relatively abundance of mRNA for 3ß-HSD and CYP17 in preantral than in antral follicles (P < 0.05). For preantral and antral follicle comparisons when the same treatments were imposed, there were greater concentrations of estradiol for antral follicles (P < 0.05). In conclusion, hrFSH enhanced in a concentration-dependent manner the in vitro development of caprine antral follicles; however, there was no positive effect in the culture of preantral follicles.
Asunto(s)
Hormona Folículo Estimulante Humana/farmacología , Cabras , Folículo Ovárico/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Hormona Folículo Estimulante , Humanos , Oocitos , Folículo Ovárico/fisiologíaRESUMEN
ANTECEDENTES: La hiperestimulación ovárica controlada (HEOC) es uno de los componentes fundamentales de los ciclos de fecundación in-vitro (FIV). HEOC se ha realizado con gonadotrofinas exógenas de uso diario, agregando un componente de estrés adicional al tratamiento. La aparición de una FSH recombinante de depósito (corifollitropin alfa) permite disminuir el estrés asociado. Los estudios publicados no han mostrados diferencias en el número de ovocitos recuperados ni en las tasas de embarazo clínico. Los estudios existentes han sido financiados en su mayoría por la industria farmacéutica. OBJETIVO: Presentar la experiencia de la Unidad de Medicina Reproductiva Monteblanco con el uso de corifollitropin alfa en ciclos de FIV autólogos en pacientes no seleccionadas. MÉTODO: Se analizaron el número de ovocitos recuperados y la proporción de ovocitos en metafase 2 en pacientes sometidas a HEOC con corifollitropin alfa, FSH recombinante diaria (rFSH), y la combinación de FSH recombinante y urinaria. RESULTADOS: Se analizaron 727 ciclos de FIV: 270 con corifollitropin alfa, 33 con rFSH y 333 con combinación de FSH. No hubo diferencias significativas en la recuperación de ovocitos ni en la proporción de ovocitos en metafase 2. Al corregir por edad de la mujer y tipo de esquema de HEOC, encontramos que la edad de la mujer se asoció negativamente con el número de ovocitos recuperados, no así el tipo de esquema de HEOC. CONCLUSION: El uso de corifollitropin alfa en ciclos de FIV autólogos, no se diferencia significativamente de los otros esquemas de HEOC en la media de ovocitos recuperados ni en la media de ovocitos en metafase 2 obtenidos.
BACKGROUND: Controlled ovarian hyperstimulation (COH) is a main component of in-vitro fertilization (IVF) cycles. COH have been performed with daily exogenous gonadotropins administered, adding to treatment an additional component of stress. The appearance of a depot recombinant FSH (corifollitropin alfa) helps to reduce stress in patients undergoing IVF. No studies have shown differences in the number of retrieved oocytes or clinical pregnancy rates; however these studies have been funded by the pharmaceutical industry. AIMS: To show the experience of Reproductive Medicine Unit Monteblanco with the use of corifollitropin alpha in autologous IVF cycles, in unselected patients. METHODS: Our main outcome was the mean number of oocytes retrieved, and the proportion of oocytes in metaphase 2 in patients undergoing COH with corifollitropin alpha, daily recombinant FSH (rFSH), and a combination of rFSH and urinary gonadotropin. RESULTS: We analyzed 727 IVF cycles: 270 cycles with corifollitropin alpha, 33 exclusive rFSH and 333 cycles with rFSH combination and urinary gonadotropins. We did not found any statistically significant difference in the mean number of oocytes recovered nor the proportion of metaphase two oocytes obtained. After adjusting for age, we did not find that the COH protocol influenced the mean number of oocytes recovered. CONCLUSION: We concluded that the use of corifollitropin alpha in autologous IVF cycles does not alter the mean number of oocytes recovered, nor the proportion of oocytes in metaphase 2.
Asunto(s)
Humanos , Femenino , Inducción de la Ovulación/métodos , Fertilización In Vitro/métodos , Hormona Folículo Estimulante Humana/administración & dosificación , Hormona Folículo Estimulante Humana/farmacología , Recuperación del Oocito/métodos , Infertilidad , Modelos Lineales , Medicina ReproductivaRESUMEN
The aim of this study was to analyse the biological response to different recombinant human FSH (rhFSH) glycosylation variants on the endocrine activity and gene expression at whole-genome scale in human granulosa-like tumor cell line, KGN. The effects of differences in rhFSH sialylation and oligosaccharide complexity were determined on steroid hormone and inhibin production. A microarray approach was used to explore gene expression patterns induced by rhFSH glycosylation variants. Set enrichment analysis revealed that hormone sialylation and oligosaccharide complexity in rhFSH differentially affected the expression of genes involved in essential biological processes and molecular functions of KGN cells. The relevance of rhFSH oligosaccharide structure on steroidogenesis was confirmed assessing gene expression by real time-PCR. The results demonstrate that FSH oligosaccharide structure affects expression of genes encoding proteins, growth factors and hormones essential for granulosa cells function.
Asunto(s)
Sistema Endocrino/metabolismo , Hormona Folículo Estimulante Humana/química , Hormona Folículo Estimulante Humana/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Polisacáridos/química , Proteínas Recombinantes/química , Línea Celular Tumoral , Sistema Endocrino/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Glicosilación/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Humanos , Inhibinas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Polisacáridos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Esteroides/metabolismo , Relación Estructura-ActividadRESUMEN
Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.
Asunto(s)
Hormona Folículo Estimulante Humana/análisis , Hormona Folículo Estimulante Humana/farmacología , Tecnología Farmacéutica , Algoritmos , Alternativas a las Pruebas en Animales , Animales , Bioensayo , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Tamaño de los Órganos/efectos de los fármacos , Ovario/efectos de los fármacos , Ovario/crecimiento & desarrollo , Proyectos Piloto , Control de Calidad , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/análisis , Proteínas Recombinantes/farmacología , Reproducibilidad de los Resultados , Albúmina SéricaRESUMEN
PURPOSE: to test the hypothesis that the anti-müllerian hormone (AMH) serum level reflects the antral follicles' response to the administration of FSH. METHODS: prospective study, including 116 normo-ovulatory infertile patients submitted to controlled ovarian hyperstimulation with GnRH and FSH agonists. The AMH serum level was measured after reaching the pituitary suppression and before the FSH administration (basal day). The number of antral follicles was determined by ultrasonography at the basal day (precocious antral follicles; 2 to 8 mm) and at the day of hCG administration (dhCG; pre-ovulatory follicles; >16 mm). The follicle response to FSH was determined by the percentage of precocious antral follicles which reached pre-ovulatory stage in response to FSH (maturation rate). The correlation of AMH with the patients' age, the total number of precocious antral and pre-ovulatory follicles, collected oocytes, total dose of FHS in the controlled ovarian stimulation and the rate of follicular maturation was studied. For the statistical analysis, it simple regression analysis and the Spearman's test were used, at a 5% significance level. RESULTS: The serum level of AMH was positively correlated with the number of precocious antral follicles at the basal day (r=0.64; p<0.0001) and pre-ovulatory follicles in dhCG (r=0.23; p=0.01). Exceptionally, the serum level of AMH was negatively correlated with the maturation ratio (r=-0.24; p<0.008). CONCLUSIONS: AMH attenuates the follicular development caused by FSH administration.
Asunto(s)
Hormona Antimülleriana/sangre , Hormona Antimülleriana/fisiología , Hormona Folículo Estimulante Humana/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Adulto , Femenino , Humanos , Estudios ProspectivosRESUMEN
OBJETIVO: examinar a hipótese de que o nível sérico do hormônio antimülleriano (AMH) reflete a resposta dos folículos antrais à administração do FSH. MÉTODOS: estudo prospectivo, no qual foram incluídas 116 pacientes normo-ovulatórias inférteis submetidas à hiperestimulação ovariana controlada com agonista de GnRH e FSH. Depois de atingir a supressão pituitária e antes da administração de FSH (dia basal), o nível sérico de AMH foi mensurado. O número de folículos antrais foi determinado pela ultrassonografia no dia basal (folículos antrais precoces; 2 a 8 mm) e no dia da administração do hCG (dhCG; folículos pré-ovulatórios; >16 mm). A resposta folicular ao FSH foi determinada pela percentagem de folículos antrais precoces que atingiram os estágios pré-ovulatórios em resposta ao FSH (taxa de maturação). Foram estudadas as correlações do AMH com a idade das pacientes, número total de folículos antrais precoces e pré-ovulatórios, oócitos coletados, dose total de FSH na estimulação ovariana controlada e a taxa de maturação folicular. Para análise estatística, foram usados a regressão simples e o teste de Spearman's, com nível de significância de 5%. RESULTADOS: o nível sérico de AMH foi positivamente correlacionado com o número de folículos antrais precoces no dia basal (r=0,64; p<0,0001) e folículos pré-ovulatórios em dhCG (r=0,23; p=0,01). Excepcionalmente, o nível sérico de AMH foi negativamente correlacionado com a taxa de maturação (r=-0,24; p<0,008). CONCLUSÕES: o AMH atenua o desenvolvimento folicular em resposta à administração do FSH.
PURPOSE: to test the hypothesis that the anti-müllerian hormone (AMH) serum level reflects the antral follicles' response to the administration of FSH. METHODS: prospective study, including 116 normo-ovulatory infertile patients submitted to controlled ovarian hyperstimulation with GnRH and FSH agonists. The AMH serum level was measured after reaching the pituitary suppression and before the FSH administration (basal day). The number of antral follicles was determined by ultrasonography at the basal day (precocious antral follicles; 2 to 8 mm) and at the day of hCG administration (dhCG; pre-ovulatory follicles; >16 mm). The follicle response to FSH was determined by the percentage of precocious antral follicles which reached pre-ovulatory stage in response to FSH (maturation rate). The correlation of AMH with the patients' age, the total number of precocious antral and pre-ovulatory follicles, collected oocytes, total dose of FHS in the controlled ovarian stimulation and the rate of follicular maturation was studied. For the statistical analysis, it simple regression analysis and the Spearman's test were used, at a 5% significance level. RESULTS: The serum level of AMH was positively correlated with the number of precocious antral follicles at the basal day (r=0.64; p<0.0001) and pre-ovulatory follicles in dhCG (r=0.23; p=0.01). Exceptionally, the serum level of AMH was negatively correlated with the maturation ratio (r=-0.24; p<0.008). CONCLUSIONS: AMH attenuates the follicular development caused by FSH administration.
Asunto(s)
Adulto , Femenino , Humanos , Hormona Antimülleriana/sangre , Hormona Antimülleriana/fisiología , Hormona Folículo Estimulante Humana/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Estudios ProspectivosRESUMEN
The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.
Asunto(s)
Hormona Folículo Estimulante Humana/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular , Conexina 26 , Conexina 43/metabolismo , Conexinas/metabolismo , Estrógenos/metabolismo , Femenino , Hormona Folículo Estimulante Humana/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Técnicas de Cultivo de Órganos , Folículo Ovárico/citología , Adenohipófisis/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologíaRESUMEN
OBJECTIVE: To compare double homologous versus only one intrauterine insemination (IUI) by stimulated cycle. PATIENTS AND METHOD: In a prospective, comparative study 138 patients were included, who underwent to intrauterine insemination; not inclusion parameters were: patients with cancelled cycles by poor response or by ovarian hyperestimulation syndrome risk. Patients were divided in two groups: Group 1 (N = 69) one insemination at 36 hours after human gonadotrophin chorionic hormone (hCG) and group 2 (N = 69) two inseminations 24 and 48 hours after hCG. Sperm separation was done with swim up, wash and resuspended or isolate techiques, and ovarian stimulation was done with recombinant FSH, menotrophins or clomiphene citrate. Statistical analysis was made by T Student and chi2 tests. RESULTS: The overall pregnancy rate was 13.04% per cycle; and seminal characteristics after seminal preparation were similar. We observed better pregnancy rates in the patients with ovulatory dysfunction and endometriosis (14.9% and 18.2%). No differences were established in pregnancy rates when patients were compared in function to seminal preparation. CONCLUSIONS: Double IUI at 24 and 48 hours after hCG has the same results in pregnancy rates than only one IUI correctly indicated 36 hours after hCG, but the costs increases in double insemination.