Qirong Tablets inhibits apoptosis of ovarian granulosa cells via PI3K/Akt/ HIF-1 signaling pathway / 中国中药杂志

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.

Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice / 生理学报

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.

Evaluation of gonadotropin-replacement therapy in male patients with hypogonadotropic hypogonadism / 亚洲男科学杂志(英文版)

Hypogonadotropic hypogonadism (HH) is a rare disease in which medical treatment has a high success rate to achieve fertility. This study aimed to analyze the efficacy of hormone replacement therapy and determine predictive factors for successful spermatogenesis and spontaneous pregnancy in patients with idiopathic HH. A total of 112 patients with low testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and normal prolactin levels were diagnosed with HH and administered LH and FSH analogs as hormone replacement therapy. During treatment, 96 (85.7%) patients had sperm present in ejaculate samples. Among these patients, 72 were married and wanted a child. Of these 72 patients, 48 (66.7%) of couples had pregnancies from natural conception. After initiation of treatment, the mean time for the appearance of sperm in semen was 9.48 months. There were no significant differences between baseline FSH, T, and LH levels; however, older age, larger testicular size, and low rate of undescended testes were favorable factors for successful spermatogenesis. Larger testicular size and older age were also the main predictive factors for natural conception. We found that patients with undescended testes had a younger age, smaller testes, and lower T levels compared with patients exhibiting descended testes. The rate of sperm found in the ejaculate was not significantly decreased in patients with undescended compared with descended testis (73.7% vs 87.6%, P = 0.261). The medical approach for males with HH and azoospermia provides a successful treatment modality in regard to successful spermatogenesis and achievement of pregnancy.

Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech

Gonadotropin therapy plays an integral role in ovarian stimulation for infertility treatments. Efforts have been made over the last century to improve gonadotropin preparations. Undoubtedly, current gonadotropins have better quality and safety profiles as well as clinical efficacy than earlier ones. A major achievement has been introducing recombinant technology in the manufacturing processes for follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin. Recombinant gonadotropins are purer than urine-derived gonadotropins, and incorporating vial filling by mass virtually eliminated batch-to-batch variations and enabled accurate dosing. Recombinant and fill-by-mass technologies have been the driving forces for launching of prefilled pen devices for more patient-friendly ovarian stimulation. The most recent developments include the fixed combination of follitropin alfa + lutropin alfa, long-acting FSH gonadotropin, and a new family of prefilled pen injector devices for administration of recombinant gonadotropins. The next step would be the production of orally bioactive molecules with selective follicle-stimulating hormone and luteinizing hormone activity.

Oocyte Maturity in Relation to Woman's Age in In Vitro Fertilization Cycles Stimulated by Single Regimen

PURPOSE: During stimulated in vitro fertilization (IVF) cycle, up to 30% of the recovered oocytes are immature ones which have poor fertilization capacity; however, the precise influencing factors are largely unknown. Here, we analyzed the association of oocyte immaturity with woman's age in IVF cycles stimulated by single regimen. MATERIALS AND METHODS: A total of one-hundred ninety five IVF cycles stimulated by recombinant FSH and GnRH antagonist protocol between 2003 and 2009 were analyzed retrospectively. The mean age of women was 34.2+/-4.0 (26-45 years). After triggering by exogenous hCG, an ultrasound-guided retrieval of oocytes was performed 35-36 hours later. All clinical data were stratified by woman's age; group I: or =41 (n=19). RESULTS: The total retrieved oocytes, as well as immature oocytes, were significantly lower in group IV, however, the mean % of immature oocytes was significantly higher in group IV than other age groups. Oocyte immaturity tended to decrease as increasing age in women aged 40 years or less. CONCLUSION: In stimulated IVF cycle, much higher oocyte immaturity was noted in women aged 41 years or more.

Comparison of Clinical Efficacy between a Single Administration of Long-Acting Gonadotrophin-Releasing Hormone Agonist (GnRHa) and Daily Administrations of Short-Acting GnRHa in In Vitro Fertilization-Embryo Transfer Cycles

This study was aimed to evaluate the efficacy of a single administration of long-acting gonadotrophin-releasing hormone agonist (GnRHa) as compared with daily administrations of short-acting GnRHa in controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET) cycles. The mean dosage of recombinant follicle-stimulating hormone (rFSH) required for COH (2,354.5+/-244.2 vs. 2,012.5+/-626.1 IU) and the rFSH dosage per retrieved oocyte (336.7+/-230.4 vs. 292.1+/-540.4 IU) were significantly higher in the long-acting GnRHa group (N= 22) than those in the short-acting GnRHa group (N=28) (p<0.05). However, the mean number of visit to the hospital that was required before ovum pick-up (3.3+/-0.5 vs. 22.2+/-2.0) and the frequency of injecting GnRHa and rFSH (12.8+/-1.2 vs. 33.5+/- 3.5) were significantly decreased in the long-acting GnRHa group (p<0.0001). The clinical pregnancy rate, implantation rate, and early pregnancy loss rate were not significantly different between the 2 groups. So, we suggest that a single administration of long-acting GnRHa is a useful alternative for improving patient's convenience with clinical outcomes comparable to daily administrations of short-acting GnRHa in COH for IVF-ET cycles.

Uso de la hormona folículo estimulante humana recombinante en la infertilidad anovulator / Recombinant human follicle-stimulating hormone in anovulatory infertility

Objetivo: estudiar los efectos usando dosis múltiples de la FSH humana recombinante en nueve mujeres con hipogonadismo hipogonadotrófico, analizando su capacidad de inducir el crecimiento folicular y síntesis de esteroides ováricos. Diseño: Trabajo clínico abierto. Material y método: De nueve casos de hipogonadismo gonadotrófico, una tenía diagnóstico de síndrome de Kallman , una deficiencia aislada de gonadotropinas y siete panhipopituitarismo secundario a hipofisectomía. Se administró inyecciones IM de hFSHr (Folitropina-alfa Instituto Ares-Serono, Ginebra) en dosis crecientes (semana 1:75 UI/día, semana 2:150 UI/día, semana 3:225 UI/día) durante un promedio de 17 días (rango: 15-19). Se efectuó toma de muestras de sangre regularmente y estudios ultrasonográficos frecuentes para monitorizar el desarrollo folicular. Para las mediciones hormonales se recurrió al radioinmunoanálisis. Resultados: Las concentraciones séricas iniciales de FSH y de hormona luteinizantes (LH) fueron 0,48 (0,09-1,22) UI/L y 0,26 (menor 0,15-0,41) UI/L, respectivamente. Los valores máximos de FSH durante la prueba llegaron a 10,3 (7,9-13,6) UI/L, en tanto que las concentraciones séricas de LH significativamente permanecieron estables 0,33 (0,19-0.52) UI/L. Los niveles séricos de androstenediona y testosterona no mostraron cambios significativos durante la administración de hFSHr y el estradiol sérico reveló sólo un aumento leve: 131.7 (51-239) pmol/L. Se desarrollaron folículos múltiples que llegaron a dimensiones pre-ovulatorias (mayor 14 mm), con un índice de desarrollo folicular de 1,92 más menos 0.7 mm/día. Conclusiones: Del presente trabajo se deduce que la hFSHr resultó efectiva en mujeres con deficiencia de gonadotropinas para estimular el crecimiento folicular normal hasta la etapa preovulatoria e inducción de ovulación, sin impedirlo el aumento leve observado del estradiol. Asimismo, los niveles séricos de LH endógeno extremadamente bajos cursaron con secreción androgénica deficitaria, indicando la necesidad de contar con dicha gonadotropina para inducir una apropiada síntesis de esteroides.

Eficácia do FSH puro e FSH recombinante em fertilizaçäo in vitro: análise preliminar / Recombinant and urinary follicle-stimulating hormone in in-vitro fertilization: preliminary data

OBJETIVO: Comparar a eficácia do FSH puro (Metrodin©) com FSH recombinante (Puregon©). CASUíSTICA E METODOLOGIA: Sete pacientes atendidas no Ambulatório de Esterilidade Conjugal do HCFMRP-USP entre 29 e 39 anos foram submetidas a dois ciclos de induçäo da ovulaçäo (com FSH puro e com FSH recombinante, näo necessariamente nesta ordem, na dose de 200 a 250 UI/dia), onde a própria paciente foi controle dela mesma. Foram avaliados além do custo de cada tratamento, o número de dias de induçäo da ovulaçäo, unidades internacionais (UI) de FSH utilizadas, folículos puncionados (maior ou igual 12 mm), oócitos captados e oócitos fertilizados. RESULTADOS: Näo houve diferença no número de dias da ovulaçäo, no número de folículos puncionados e oócitos captados. Entretanto, quando se utilizou FSH recombinante, uma menor quantidade de mediçäo foi necessária em relaçäo ao FSH puro (1464 UI ñ 284 UI/ciclo e 2014 UI ñ 407 UI/ciclo, respectivamente -p < 0,05) e houve um maior número de oócitos fertilizados (5,14ñ2,2 e 2,28ñ2,16 respectivamente -p < 0,05). CONCLUSöES: Apesar do maior custo do FSH recombinante (FSH recombinante=US$0.85/UI e FSH puro=US$0.55/UI), näo houve diferença entre o custo total dos tratamentos, já que foi necessária uma quantidade menor de medicaçäo para se obter a ovulaçäo quando se usou o FSH recombinante. O achado de um maior número de oócitos fertilizados com o FSH recombinante sugere que ele foi mais eficaz no tratamento da infertilidade conjugal, embora a casuística seja ainda limitada.

Gravidez com FSH-recombinante: relato de caso / Recombinant FSH and pregnancy: case report

OBJETIVO: relatar um caso de gravidez obtida após tratamento de fertilizaçäo "in vitro" usando FSH-recombinante. DESENHO: estudo restrospectivo de caso clínico. MATERIAL E MÉTODO: foi realizada técnica de fertilizaçäo in vitro (FIV), associada a técnica de injeçäo intracitoplasmática de espermatozóide (ICSI) em paciente de 42 anos de idade. Foram obtidos dois óvulos após induçäo da ovulaçäo com protocolo longo usando acetato de leuprolide e FSH-recombinante. Após a transferência dos embriöes o suporte da fase lútea foi feito com estradiol transdérmico e progesterona oleosa. RESULTADO: foram aspirados 2 oócitos M2 com 100 por cento de fertilizaçäo após o uso da ICSI. No 12§ dia pós transferência de 02 embriöes com 0 por cento de fragmentaçäo, foi constatada gravidez através de dosagem sérica de b-HCG e confirmada através de ultra-sonografia transvaginal realizada no 26§ dia pós transferência quando visualizou-se saco gestacional único e tópico com embriäo com comprimento crânio nádega de 2,8 mm, atividade cardíaca presente e vesícula vitelínica normal, compatível com gestaçäo única de 5 semanas de 3 dias. CONCLUSÃO: os autores relatam um caso de gravidez obtida através da técnica de FIV-ICSI em paciente de 42 anos onde foi utilizado FSH recombinante na induçäo da ovulaçäo.

Inducción de la ovulación con FSH pura en pacientes anovulatorias resistentes a citrato de clomifeno / ovulation induction with pure FSH in anovulatory patientas resistant to clomiphene cytrate

El obejtivo de este estudio fue evaluar la utilidad de la FSH para el tratamiento de pacientes anovulatorias con resistencia al citrato de clomiferno. Se trataron 72 pacientes con anovulación crónica resistente al citrato de clomifeno. La inducción de la ovulación fue iniciada el día tres del ciclo menstrual con 75 UI de FSH pura, se realizó control ultrasonográfico transvaginal del desarrollo folicular, las dosis posteriores de FSH se ajustaron según los resultados del ultrasonido transvaginal. Fueron administradas 10,000 UI de gonadotropina coriónica humana cuando el folículo dominante tuvo un diámetro ò 16 mm. La tasa de embarazo por cilo fue de 18.0 por ciento, mientras que la tasa acumulada de embarazos fue de 72.2 por ciento. No hubo diferencia significativa en la tasa de embarazos entre los grupos de esterilidad primaria y secundaria. La tasa de abortos espontáneos fue similar a la encontrada en la población general. Como conclusión, se puede afirmar que el tratamiento con FHS pura en mujeres resistentes al citrato de clomifeno es efectivo en la mayoría de ellas. Los resistentes a las gonadotropinas, pueden ser candidatos a la inducción quirúrgica de la ovulación

Síndrome de ovarios poliquísticos. Inducción de ovulación con FSH pura / Polycysric ovarian syndrome. Pure FSH ovulation induction

Objetivo. Adquirir experiencia institucional en el uso de FSH "pura" para inducir ovulación en mujeres con síndrome de ovarios poliquísticos. Material y métodos. Se estudiaron durante el periodo comprendido del 1o. de junio de 1990 al 31 de mayo de 1993, 110 mujeres con diagnóstico de esterilidad anovulatoria, secundaria a síndrome de ovario poliquísticos, sin ningún otro factor causal de esterilidad, en quienes previamente ya se había intentado y fracasado la inducción de ovulación con citrato de clomifen. Se realizaron 284 ciclos de tratamiento con FSH pura, con un protocolo en el que se inicia la administración de 150 UI por vía IM del medicamento a partir del primer día del ciclo menstrual espontáneo o inducido, implementando vigilancia estrecha mediante determinaciones seriadas de estradiol sérico y seguimiento del crecimiento folicular por ultrasonografía a partir del séptimo u octavo día del ciclo, y al encontrar datos inminentes de ruptura folicular, se administraron de 5 a 10,000 UI de Hormona Gonadotropina Coriónica para favorecer la ovulación. Resultados: Se logró ovulación en 205 ciclos (72.1 por ciento), con un total de 62 embarazos que corresponde al 56.3 por ciento del total de pacientes. De estas gestaciones, 13 fueron embarazos múltiples (un quíntuple, un cuádruple, cuatro triples y siete dobles). Se perdieron 15 embarazos (24.1 por ciento) en abortos del primer y segundo trimestre y un embarazo ectópico. El síndrome de hiperestimulación ovárica se presentó en 12 casos, constituyendo el 5.8 por ciento. Conclusión. La FSH pura es el medicamento de segunda elección para inducir la ovulación en el síndrome de ovarios poliquísticos, en aquellas mujeres en quienes previamente se haya fracasado con el citrato de clomifen

Uso de la urofoliculotropina en los trastornos menstruales de la adolescencia / Use of urofoliculotropin in the menstrual disorders of adolescence

Los autores discuten la conducta a adoptar en las alteraciones menstruales frecuentes en la adolescencia, especialmente la oligomenorrea. Creen que deben ser tratadas activamente en base a la comprobación de LH aumentada con FSH normal o disminuida sobre todo si se acompañan de obesidad, acné o hipertricosis. Presentan su experiencia personal proponiendo a la urofoliculotropina como posible tratamiento de elección, aun cuando reconocen la necesidad de ulteriores observaciones