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1.
J Vis Exp ; (167)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33522510

RESUMO

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth's culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.


Assuntos
Ovário/fisiologia , Técnicas de Cultura de Tecidos/métodos , Animais , Bovinos , Quimiocinas/metabolismo , Meios de Cultura , Feminino , Imageamento Tridimensional , Metaboloma , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Coloração e Rotulagem , Esteroides/metabolismo
2.
Methods Mol Biol ; 2273: 1-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604842

RESUMO

The mammalian ovary is a large source of oocytes organized into follicles at various stages of folliculogenesis. However, only a limited number of them can be used for in vitro embryo production (IVEP), while most have yet to complete growth and development to attain full meiotic and embryonic developmental competence. While the in vitro growth of primordial follicles in the ovarian cortex has the potential to produce mature oocytes, it is still at an experimental stage. The population of early antral follicles (EAFs), instead, may represent a reserve of oocytes close to completing the growth phase, which might be more easily exploited in vitro and could increase the number of female gametes dedicated to IVEP.Here we present in vitro culture strategies that have been developed utilizing physiological parameters to support the specific needs of oocytes at distinct stages of differentiation, in order to expand the source of female gametes for IVEP by maximizing the attainment of fertilizable oocytes. Furthermore, these culture systems provide powerful tools to dissect the molecular processes that direct the final differentiation of the mammalian oocyte.


Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Animais , Bovinos , Diferenciação Celular/fisiologia , Cromatina , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Mamíferos , Meiose , Oogênese , Folículo Ovariano
3.
Methods Mol Biol ; 2273: 75-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604845

RESUMO

The technological revolution in reproductive biology that started with artificial insemination procedures and embryo transfer led to the development of assisted reproduction techniques such as in vitro fertilization or even cloning of domestic animals by nuclear transfer from somatic cells. Currently, procedures of isolated immature ovarian follicles in vitro culture are becoming the prominent technology aimed to preserve or restore fertility especially of young oncological patients or those at risk of premature ovarian failure.Here, we describe a protocol that can be applied for in vitro growth of porcine, preantral ovarian follicles in three-dimensional (3D) culture conditions. After enzymatic isolation from the ovarian cortex, preantral follicles are suspended in a drop of medium and enclosed with fluorinated ethylene propylene (FEP) powder particles (microbioreactors). Such microbioreactors maintain the 3D structure of the follicles during the whole process of in vitro growth what is crucial to ensure proper folliculogenesis progression and their ability to survive.


Assuntos
Técnicas de Cultura de Células/métodos , Fertilização In Vitro/métodos , Folículo Ovariano/crescimento & desenvolvimento , Animais , Meios de Cultura/química , Transferência Embrionária/métodos , Feminino , Humanos , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia , Suínos
4.
Methods Mol Biol ; 2218: 1-9, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606218

RESUMO

Zebrafish ovarian follicles are mainly composed of the oocyte and a thin layer of follicle cells. Recent studies have demonstrated extensive cell-cell interactions between the oocyte and surrounding follicle layer and that the two compartments communicate mostly through paracrine factors. To understand the paracrine communication within the follicle, it is essential to know the spatial expression patterns of genes in the two compartments. However, since the follicle layer is extremely thin and the oocytes are enormous in size in fish, it is often difficult to detect gene expression by traditional methods such as in situ hybridization. Separation of the oocyte and surrounding follicle layer followed by RT-PCR detection provides a sensitive way to reveal the expression of individual genes in the two compartments of the follicle. This chapter introduces a method for mechanic separation of the oocyte and follicle layer at full-grown stage for expression analysis. Since fish have similar follicle structure, this method may also be used in other species as well.


Assuntos
Expressão Gênica/fisiologia , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Peixe-Zebra/fisiologia , Animais , Feminino , Ovário/fisiologia , Comunicação Parácrina/fisiologia
5.
Methods Mol Biol ; 2218: 169-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606231

RESUMO

In some animal species, fertilization occurs through a funnel-like canal called the "micropyle." In teleost fishes, the micropyle is formed by a very specialized follicle cell, called the micropylar cell (MC). Very little is known about the mechanisms underlying the specification and differentiation of the MC, a unique cell among hundreds that compose the follicle cell layer. The Hippo pathway effector Taz is essential for this process and is the first reported MC marker. Here, we describe a method to identify and mark the micropylar cell following the immunostaining procedure on cryosections or combining it with the RNA in situ hybridization on whole-mount follicles.


Assuntos
Folículo Ovariano/fisiologia , Peixe-Zebra/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Fertilização/fisiologia , Masculino , Oócitos/metabolismo , Oócitos/fisiologia , Folículo Ovariano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
6.
Ecotoxicol Environ Saf ; 209: 111733, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33385679

RESUMO

Both essential and non-essential elements have been associated with female reproductive function in epidemiologic investigations, including among IVF populations. To date, most investigators have used blood or urine to assess biomarkers of exposure, with few employing ovarian follicular fluid (FF). FF may offer a more direct "snapshot" of the oocyte microenvironment than blood or urine, however previous studies report follicle-to-follicle variability in FF constituents that may contribute to exposure misclassification. Our objectives were to investigate sources of trace element variability, to estimate FF biomarker reliability among women undergoing IVF (n = 34), and to determine the minimum number of follicles required to estimate subject-specific mean concentrations. We measured As, Hg, Cd, Pb, Cu, Mn, Se, and Zn in FF samples using inductively coupled plasma tandem mass spectrometry. Inter-subject (between-women) variability contributed most of the variability in FF element concentrations, with ovarian, follicular, and analytical as smaller sources of variability. The proportion of variability attributable to sources between-follicles differed by age, body mass index (BMI), race, and cigarette smoking for Cu, Se, and Zn, by BMI and cigarette smoking for As, by primary infertility diagnosis for Hg, Cu, Se, and Zn, and by ovarian stimulation protocol for Mn and Se. Four to five individual follicles were sufficient to estimate subject-specific mean Cu, Se, and Zn concentrations, while >14 were necessary for As, Hg, Cd, Pb, and Mn. Overall, our results suggest that FF is a suitable source of biomarkers of As and Hg exposure in ovarian follicles. Although limited in size, our study offers the most comprehensive exploration of biological variation in FF trace elements to date and may provide guidance for future studies of ovarian trace element exposures.


Assuntos
Líquido Folicular/metabolismo , Reprodutibilidade dos Testes , Oligoelementos/metabolismo , Adulto , Feminino , Fertilização In Vitro , Líquido Folicular/química , Humanos , Mercúrio/análise , Oócitos , Folículo Ovariano , Oligoelementos/análise
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(1): 93-99, 2021 Jan 30.
Artigo em Chinês | MEDLINE | ID: mdl-33509759

RESUMO

OBJECTIVE: To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism. METHODS: Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured in vitro. The cultured cells were treated with 0, 1, 10, 50, 100, 150, 200 and 500 µmol/L BPA, and the changes in cell proliferation, cell cycle, apoptosis and mitochondrial membrane potential were analyzed with CCK-8 method and flow cytometry. The protein expressions of Bcl-2, Bax, p53 and cyclin D1 in the treated cells were determined with Western blotting. Pregnant ICR mice were treated for a week with BPA at the concentration that produced significant effects on the preantral follicular granulosa cells, and the weight changes of the pregnant mice were recorded. The ovarian tissues of the offspring female mice were weighed at PND 10, 17, 21 and 42 followed by histological observation with HE staining and examination of Bcl-2 mRNA expression level with RT-qPCR. RESULTS: Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 µmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (P < 0.05). Exposure to a higher BPA concentration at 200 µmol/L obviously enhanced cell apoptosis by reducing the mitochondrial membrane potential and repressed the cell proliferation (P < 0.05). BPA exposure at 50 µmol/L and 200 µmol/L produced opposite effects on the protein expressions of Bcl-2 (P < 0.01), Bax (P < 0.05) and p53 (P < 0.05) in mouse ovarian preantral follicular granulosa cells. BPA exposure at the doses of 10 and 35 mg/kg caused rapid weight increment of the pregnant mice and changes in ovarian index of the offspring female mice. In the offspring female mice, the changes in Bcl-2 mRNA expression in the ovarian tissue showed a similar pattern to that of ovarian index. Exposure of the pregnant mice to a high BPA concentration at 35 mg/kg resulted in accelerated follicular development into antral follicular stage in PND 21 offspring female mice. CONCLUSIONS: BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.


Assuntos
Células da Granulosa , Folículo Ovariano , Animais , Apoptose , Compostos Benzidrílicos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Fenóis , Gravidez
8.
Trop Anim Health Prod ; 53(1): 102, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33417076

RESUMO

The present study was aimed at evaluating the differences of ovarian follicular dynamics and circulating progesterone (P4) concentrations between crossbred Holstein heifers that ovulated and did not ovulate after a P4-based synchronization protocol. Twenty-one crossbred (Holstein × Thai native) heifers with random stages of the oestrous cycle were subjected to the ovulation synchronization protocol, using an intravaginal P4-releasing device (Eazi-Breed CIDR®) for 7 days. Out of 21 CIDR-treated heifers, 14 ovulating heifers were classified as the ovulatory group and 7 non-ovulating heifers were considered the anovulatory group. The heifers having new wave emergence in ovulatory and anovulatory groups were 11/14 (78.6%) and 4/7 (57.1%), respectively. In ovulating heifers, the mean (± SEM) diameter of preovulatory follicle (PF, mm) was significantly larger, compared to non-ovulating heifers (7.21 ± 0.32 versus 4.04 ± 0.44; P = 0.001), while the mean (± SEM) follicular growth rates (mm/d) in non-ovulating heifers tended to be lower, compared to ovulating heifers (0.73 ± 0.17 versus 1.06 ± 0.08; P = 0.07). The mean (± SEM) circulating P4 concentration (ng/ml) throughout the CIDR protocol (0-10 days) in non-ovulating heifers was significantly higher, in comparison with ovulating heifers (2.82 ± 0.27 versus 1.83 ± 0.16; P = 0.02). However, no significant difference in the mean corpus luteum volume between groups was observed. In conclusion, the present results suggested that elevated circulating P4 concentrations and smaller PF diameters could cause ovulation failure in crossbred Holstein heifers, following a 7-day CIDR-based synchronization protocol.


Assuntos
Bovinos/fisiologia , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Progesterona/sangue , Animais , Ciclo Estral , Sincronização do Estro , Feminino
9.
Am J Surg Pathol ; 45(1): 59-67, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32815828

RESUMO

Thirty-eight ovarian Sertoli-Leydig cell tumors that contained follicles are described; in 33 of them follicles imparted a microscopic appearance resembling that of the juvenile granulosa cell tumor. The average age of the patients (28 y), frequency of androgenic manifestations (40%), and dominant histopathologic features were all typical of Sertoli-Leydig cell tumor, mostly (80%) of intermediate differentiation. The remaining tumors were poorly differentiated; none were well differentiated. The follicles that mimicked juvenile granulosa cell tumor accounted for ∼5% to 40% of the tumor volume. They typically arose out of the characteristic lobules seen in Sertoli-Leydig cell tumors of intermediate differentiation. There appeared to be a gradual loosening of the stroma imparting a pale appearance to the lobules and on that background follicles emerged. The follicles were mostly relatively regular and round to oval with basophilic or eosinophilic secretion and when fully formed perfectly mimicked juvenile granulosa cell morphology. In 18 of these cases, and 5 others, follicles were present that had a nonspecific morphology and had a random, nonlobule-associated distribution. The presence of a juvenile granulosa-like appearance often raised consideration of the diagnosis of a sex cord-stromal tumor of mixed forms (so-called gynandroblastoma) but a multifocal origin within lobules of otherwise typical Sertoli-Leydig cell tumors, and overall tumor characteristics indicates aberrant differentiation within the latter tumor of a nature only sporadically noted in the prior literature. Such neoplasms should, in our opinion, not be placed in the grouping of a sex cord-stromal tumor of mixed forms but rather in the Sertoli-Leydig category.


Assuntos
Folículo Ovariano/patologia , Tumor de Células de Sertoli-Leydig/patologia , Adolescente , Adulto , Idoso , Feminino , Tumor de Células da Granulosa/patologia , Humanos , Pessoa de Meia-Idade , Tumores do Estroma Gonadal e dos Cordões Sexuais/patologia , Adulto Jovem
10.
J Dairy Sci ; 104(2): 2369-2383, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33309353

RESUMO

The objective of the current study was to evaluate the relationship of body condition score (BCS) at 35 d in milk (DIM), milk production, diseases, and duration of the dry period with prevalence of anovulation at 49 DIM and then, specifically, with the prevalence of each anovular phenotype. We hypothesized that anovular follicular phenotypes, classified based on maximal size of the anovular follicle, have different etiologies. A total of 942 lactating Holstein cows (357 primiparous and 585 multiparous) from 1 herd had ovaries evaluated by ultrasonography at 35 ± 3 and 49 ± 3 DIM to detect the absence of a corpus luteum (CL), and to measure the diameter of the largest follicle. Cows were classified as cyclic at 49 DIM if a CL was observed in at least 1 of the 2 examinations, or anovular if no CL was observed at either examination. Cows considered anovular were divided into 3 groups based on the largest diameter of the largest follicle as follows: ranging from 8 to 13 mm, 14 to 17 mm, or ≥18 mm. Cows were evaluated for the following diseases: retained placenta, metritis, hyperketonemia, mastitis, lameness, respiratory problem, and digestive problem. At 35 DIM, BCS was determined, and milk yield for individual cows was recorded. A total of 28.5% (268/942) of cows were classified as anovular. Anovular cows had longer dry periods (90 vs. 71 d) and smaller BCS than cyclic cows (2.83 vs. 2.99). Cows with a single disease or multiple diseases had 2 and 3-fold increase in odds of being anovular, respectively. Anovular cows had follicles that ranged from 4 to 50 mm. The prevalence of anovular phenotype, among anovular cows, that had the diameter of the largest follicle ranging from 8 to 13 mm, 14 to 17 mm, and ≥18 mm was 29.9 (79/264), 37.5 (99/264), and 32.6% (86/264), respectively. Anovular cows with follicles of 8 to 13 mm had longer dry periods than those with follicles ≥18 mm (104 vs. 74 d), whereas anovular cows with medium size follicles had intermediate days dry (99 d). Cows with small and medium anovular follicles had smaller BCS and greater prevalence of multiple diseases than cyclic cows. For almost all risk factors, the cows with large anovular follicles (≥18 mm) were similar to cyclic cows and different from cows with smaller anovular follicles (8-13 mm). Thus, longer dry periods, less BCS at 35 DIM, and diseases were risk factors for anovulation. Moreover, the risk factors for the 3 distinct anovular follicle phenotypes differed.


Assuntos
Anovulação/veterinária , Doenças dos Bovinos/epidemiologia , Animais , Anovulação/epidemiologia , Anovulação/etiologia , Bovinos , Doenças dos Bovinos/etiologia , Corpo Lúteo/anormalidades , Feminino , Lactação , Leite , Folículo Ovariano , Fenótipo , Gravidez , Prevalência , Fatores de Risco
11.
Transl Res ; 227: 15-29, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32640290

RESUMO

Endometriosis is a common gynecological disease in which ovarian dysfunction can be an important cause of infertility. Elevated progesterone (P4) levels during the follicular phase is possibly associated with impaired oocyte quality and pregnancy outcome in endometriosis. Beclin-1 (BECN1), an essential mediator of autophagy, has been shown to be related to the development and progression of endometriosis. This study aimed to investigate the autophagic activity in ovarian granulosa cells (GCs) of patients with endometriosis and to clarify the role of BECN1 in preovulatory P4 elevation. Our results demonstrated that serum P4/estradiol (E2) ratio and P4-to-follicle index (the average P4 secretion per follicle) on the day of human chorionic gonadotropin administration were elevated in women with ovarian endometriosis. Increased expression of BECN1 and enhanced autophagy were observed in GCs of patients with ovarian endometriomas. In cultured GCs, BECN1 knockdown reduced P4 secretion and the expression of key steroidogenic enzymes; whereas overexpression of BECN1 resulted in induced P4 production with activated biosynthesis pathway. Moreover, inhibition of autophagy by BECN1 knockdown significantly attenuated low-density lipoprotein (LDL)-induced P4 synthesis. These findings provide new insights into the role of BECN1 in late follicular P4 elevation in patients with endometriosis by promoting the degradation pathway of LDL for P4 biosynthesis via lysosome activation in GCs, and have potential therapeutic implications for the improvement of oocyte quality in women affected by endometriosis.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/fisiologia , Endometriose/metabolismo , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Progesterona/metabolismo , Colesterol/metabolismo , Feminino , Humanos , Gravidez , Transdução de Sinais , Injeções de Esperma Intracitoplásmicas , Regulação para Cima
12.
Nature ; 589(7841): 264-269, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33328630

RESUMO

During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.


Assuntos
Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Epigênese Genética , Feminino , Fertilização , Meiose , Metilação , Camundongos , Oócitos/citologia , Folículo Ovariano/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
13.
PLoS Biol ; 18(12): e3001025, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33351795

RESUMO

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas , Células da Granulosa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Folículo Ovariano/fisiologia , Ovário/citologia , Gravidez , Análise de Célula Única/métodos , Transcriptoma/genética
14.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333986

RESUMO

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Folículo Ovariano/metabolismo , Síndrome do Ovário Policístico/metabolismo , Transdução de Sinais , Sequência de Bases , Feminino , Líquido Folicular/metabolismo , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Humanos , Oócitos/metabolismo , Síndrome do Ovário Policístico/etiologia
15.
Anim Sci J ; 91(1): e13465, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33222358

RESUMO

The present study was conducted to investigate the effect of cold storage time on apoptosis of cumulus cells (CCs) from porcine ovaries, and to compare the sensitivity of four apoptosis-detection methods. Porcine ovaries were stored in physiological saline solution at 4°C for 0, 7, 24 and 48 hr, and then cumulus cells or granulosa cells (GCs) in antral follicles were retrieved to detect cell apoptosis. Cumulus cells isolated from stored ovaries for 24 hr presented obvious apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labeling (TUNEL) assay. A typical DNA ladder pattern of apoptosis was observed in GCs 24 hr post storage treatment. The mean Olive Tail Moment of CCs was significantly increased after 24 hr using comet assay; however, the mean tail migration and mean tail DNA increased gradually after 7 hr of storage. In addition, annexin V/PI staining assay showed an obvious increase in apoptotic CCs (Annexin V positive, PI negative) 7 hr after treatment, and the apoptotic rate reached to a peak at 24 hr followed by a decline after 48 hr of storage to the level at 7 hr. In conclusion, cold storage of porcine ovary in physiological saline solution induced a time-dependent increase in apoptosis of cumulus cells, and annexin V/PI staining combined with comet assay provided a sensitive and reliable method to detect early damages in cumulus cells induced by cold storage of ovary.


Assuntos
Apoptose , Temperatura Baixa/efeitos adversos , Células do Cúmulo/patologia , Preservação de Órgãos/efeitos adversos , Preservação de Órgãos/métodos , Folículo Ovariano/citologia , Ovário , Animais , Separação Celular , Células Cultivadas , Células do Cúmulo/fisiologia , DNA , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Ovário/citologia , Suínos , Fatores de Tempo
16.
An Acad Bras Cienc ; 92(suppl 2): e20180935, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33146258

RESUMO

To compare the effects of two-(2D, microplate) and three-dimensional (3D, alginate) culture systems on the in vitro growth of small antral follicles in cattle, individual follicles were separately cultured in the two culture systems for 8 days. Half of the culture medium was replaced by fresh medium every 2 days; the former medium was used to assess the amount of follicular hormone secretion using ELISA. Individual follicle morphology, diameter, and survival rate were recorded every alternate day. The results showed that in 4 days, there was no significant difference between the two systems, except that the growth rate of follicles in 2D system was relatively faster. After 4 days, estradiol concentration in 3D system was higher than that in 2D system. However, progesterone concentration was lower than that in the 2D system. The survival rate and oocyte quality of follicles in 2D system were significantly lower than those in 3D system on day 8. The follicle diameter slightly increased (30-60 µm) in the entire process. Taken together, for in vitro culture of follicles within 4 days, the 2D culture system is more suitable. However, when the culture duration is >4 days, the 3D culture system is more suitable.


Assuntos
Estradiol , Folículo Ovariano , Animais , Bovinos , Meios de Cultura , Feminino , Hormônio Foliculoestimulante , Progesterona
17.
PLoS One ; 15(10): e0239779, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33044971

RESUMO

BACKGROUND: The conditions of diminished ovarian reserve and primary ovarian insufficiency, characterized by poor fertility outcomes, currently comprise a major challenge in reproductive medicine, particularly in vitro fertilization. Currently in the IVF industry, blastocyst developmental success rate per treatment is routinely overlooked when a live birth results from treatment. Limited data are available on this significant and actionable variable of blastocyst development optimization, which contributes to improvement of treatment success Women with elevated basal FSH concentration are reported to still achieve reasonable pregnancy rates, although only a few studies report correlations with blastocysts development. Diagnostic values of AMH/basal FSH concentrations can be useful for determining the optimal stimulation protocol as well as identification of individuals who will not benefit from IVF due to poor prognosis. The objective of this study is to identify actionable clinical and culture characteristics of IVF treatment that influence blastocyst developmental rate, with the goal of acquiring optimal success. METHODS AND FINDINGS: A retrospective observational study was performed, based on 106 women undergoing IVF, regardless of prognosis, over a six-month period from January 1, 2015 to June 31, 2015. Rate of high-quality blastocyst production, which can be used for embryo transfer or vitrification, per normally fertilized oocyte, was evaluated. Treatment was determined successful when outcome was ≥ 40% high-quality blastocysts. The data were initially evaluated with the Evtree algorithm, a statistical computational analysis which is inspired by natural Darwinian evolution incorporating concepts such as mutation and natural selection (see Supplementary Material). The analysis processes all variables simultaneously against the outcome, aiming to maximize discrimination of each variable to then create a "branch" of the tree which can be used as a decision in treatment. The final model results in only those variables which are significant to outcomes. Generalized linear model (GLM) employing logistic regression and survival analysis with R software was used and the final fitting of the model was determined through the use of random forest and evolutionary tree algorithms. Individuals presenting with an [AMH] of >3.15 ng/ml and a good prognosis had a lower success per treatment (n = 11, 0% success) when total gonadotropin doses were greater than 3325 IU. Individuals that presented with an [AMH] of <1.78 ng/ml and a poor prognosis exhibited a greater success per treatment (n = 11, 80% success). AMH emerged as a superior indicator of blastocyst development compared to basal FSH. The accuracy of the prediction model, our statistical analysis using decision tree, evtree methodology is 86.5% in correctly predicting outcome based on the significant variables. The likelihood that the outcome with be incorrect of the model, or the error rate is 13.5%. CONCLUSIONS: [AMH] is a superior indicator of ovarian stimulation response and an actionable variable for stimulation dose management for optimizing blastocyst development in culture. Women whose [AMH] is ≥3.2 mg/ml, having a good prognosis, and developing >12 mature follicles result in <40% blastocysts when gonadotropin doses exceed 3325 IU per treatment. IVF treatments for poor responders that present with infertility due to diminished ovarian reserve, if managed appropriately, can produce more usable blastocyst per IVF treatment, thus increasing rate of blastocyst developmental success and ultimately increasing live birth rates. Future studies are needed to investigate the intra-follicular and the intra-cellular mechanisms that lead to the inverse relationship of blastocysts development and total gonadotropin doses in good responders in contrast to poor responders.


Assuntos
Hormônio Antimülleriano/sangue , Blastocisto/metabolismo , Blastocisto/fisiologia , Desenvolvimento Embrionário/fisiologia , Hormônio Foliculoestimulante/sangue , Adulto , Transferência Embrionária/métodos , Feminino , Fertilização In Vitro/métodos , Humanos , Infertilidade/sangue , Infertilidade/terapia , Nascimento Vivo , Masculino , Folículo Ovariano/metabolismo , Reserva Ovariana/fisiologia , Ovário/metabolismo , Ovário/fisiologia , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos
18.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2109-2112, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018422

RESUMO

Quantification of ovarian and follicular volume and follicle count are performed in clinical practice for diagnosis and management in assisted reproduction. Ovarian volume and Antral Follicle Count (AFC) are typically tracked over the ovulation cycle. Volumetric analysis of ovary and follicle is manual and largely operator dependent. In this manuscript, we have proposed a deep-learning method for automatic simultaneous segmentation of ovary and follicles in 3D Transvaginal Ultrasound (TVUS), namely S-Net. The proposed loss function restricts false detection of follicles outside the ovary. Additionally, we have used multi-layered loss to provide deep supervision for training the network. S-Net is optimized for inference time and memory while utilizing 3D context in the 2D deep-learning network. 66 3D TVUS volumes (13,200 2D image slices) were acquired from 66 subjects in this Institutional Review Board (IRB) approved study. The segmentation framework provides approximately 92% and 87% average DICE overlap with the ground truth annotations for ovary and follicles, respectively. We have obtained state-of-the-art results with a detection rate of 88%, 91% and 98% for follicles of size 2-4mm, 4-12mm and >12mm.


Assuntos
Aprendizado Profundo , Ovário , Feminino , Humanos , Imageamento Tridimensional , Folículo Ovariano/diagnóstico por imagem , Ovário/diagnóstico por imagem , Ultrassonografia
19.
Medicine (Baltimore) ; 99(36): e21979, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899038

RESUMO

Aim of the present study was to explore the evaluative effectiveness of age, ovarian volume (OV), antral follicle count (AFC), serum follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), AFC/Age ratio, AMH/Age ratio, FSH/luteinizing hormone (LH) ratio, and ovarian response prediction index (ORPI) to determine which could more advantageously assess ovarian reserve and response.This research enrolled 319 consecutive infertile women who had undergone in vitro fertilization-ET/intracytoplasmic sperm injection (IVF-ET/ICSI) treatments. Abovementioned variables were measured and calculated. Receiver operating characteristic (ROC) curve analysis was used to analyze the predictive accuracy of variables and to calculate cut-off values and corresponding sensitivity and specificity.Our study revealed that the significant variables for evaluating a decline in ovarian reserve include age, OV, FSH, AFC/Age ratio, AMH/Age ratio, and ORPI. Moreover, the area under the curve (AUC) of AFC/Age ratio was higher than other 5 variables (AUC = 1.000), and the cut-off value of AFC/Age ratio was 0.111 (sensitivity 100.00%, specificity 100.00%). The significant variables forecasting excessive ovarian response were age, AFC, AMH, FSH, AFC/Age ratio, AMH/Age ratio, FSH/LH ratio, and ORPI, and the significant variables forecasting poor ovarian response were AMH, LH, OV, AFC/Age ratio, AMH/Age ratio, and FSH/LH ratio. When ORPI was used to predict excessive response, the cut-off value of ORPI was 0.880 (sensitivity 84.72%, specificity 67.32%) and ORPI presented better effectiveness. When used to predict poor response, the evaluative effectiveness of 6 variables was almost similar, although the AUC of AFC/Age ratio presented the largest value.Regarding the infertile women, AFC/Age ratio performed better than did the other variables in evaluating ovarian reserve, and it offered excellent effectiveness in predicting poor ovarian response, however, ORPI presented better effectiveness in predicting excessive ovarian response.


Assuntos
Infertilidade Feminina/diagnóstico , Folículo Ovariano , Reserva Ovariana , Adulto , Fatores Etários , Feminino , Hormônios/sangue , Humanos , Infertilidade Feminina/sangue , Estudos Retrospectivos , Índice de Gravidade de Doença
20.
Ecotoxicol Environ Saf ; 205: 111338, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32956867

RESUMO

Lead (Pb) is well-recognized for its great hazards to human and wildlife health. It has negative influences on multiple organs and systems of birds. Especially, lead exposure caused adverse impacts on bird reproduction. In this study, one week old female Japanese quails were randomly allocated into four groups and each group was respectively fed with 0, 50 ppm, 500 ppm and 1000 ppm Pb in drinking water for 36 days to determine the effects of chronic lead exposure on ovarian development and function. The results showed that Pb did accumulate in the ovary and ovarian development was delayed by high dose lead exposure (500 ppm and 1000 ppm). Moreover, high Pb dosage induced ovarian histopathological damages characterized by granulosa cells disorganization, follicle atresia and interstitial cell degeneration. Meanwhile, the concentration of estradiol (E2) was significantly decreased and mRNA levels of genes involved with ovarian steroidogenesis were significantly down-regulated by high concentration Pb. In addition, Pb exposure caused increasing cell apoptosis and significant changes of the expression of genes involved with cell death in the ovary. High dose Pb exposure also inhibited thyroid hormone release and disrupted ovarian thyroid deiodination apart from causing thyroid histopathological injury such as follicular deformation and atrophy. The study indicated that Pb might cause ovarian malfunction by inducing ovary and thyroid microstructural damages, thyroid hormone and estrogen release inhibition and ovarian steroidogenesis disruption.


Assuntos
Coturnix/metabolismo , Poluentes Ambientais/toxicidade , Estradiol/metabolismo , Expressão Gênica/efeitos dos fármacos , Chumbo/toxicidade , Ovário/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Adolescente , Animais , Apoptose/efeitos dos fármacos , Coturnix/genética , Coturnix/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Poluentes Ambientais/metabolismo , Estradiol/genética , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/patologia , Humanos , Chumbo/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/patologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ovário/patologia , Distribuição Aleatória , Reprodução/efeitos dos fármacos , Reprodução/genética , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Hormônios Tireóideos/genética
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