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Prenat Diagn ; 39(5): 361-368, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30740743


OBJECTIVES: To determine the association between medications intake in early pregnancy and variation in the fetal fraction (FF) in pregnant women undergoing cell-free DNA (cfDNA) testing. METHODS: We performed a retrospective cohort study of women (n = 1051) undergoing cfDNA testing at an academic center. The exposed group included women taking medications (n = 400; 38.1%), while the nonexposed group consisted of women taking no medications (n = 651; 61.9%). Our primary outcome was FF. We performed univariate and multivariate analyses as appropriate. RESULTS: The FFs were 8.8% (6.6-12.1), 8.7% (6.3-11.6), and 7.7% (5.1-9.3) among women taking 0, 1, and two or more medications, respectively (P < 0.01). Using multivariable linear mixed effects model, the mean FF was significantly lower among those taking two or more medications compared with the nonexposed group. FF was directly correlated with gestational age at the time of cfDNA testing and inversely correlated with maternal obesity. Exposure to metformin was associated with 1.8% (0.2-3.4) lower mean FF when compared with the nonexposed group (P = 0.02). Obesity and intake of two or more medications were associated with higher hazard ratio of having a low FF less than 4%. CONCLUSIONS: Exposure to metformin or two or more medications was associated with decreased FF, and obesity is associated with delay in achieving adequate FF percentage. These findings should be considered while counseling patients on test limitations.

J Clin Endocrinol Metab ; 104(7): 2796-2800, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30759233


CONTEXT: Ovarian hyperandrogenism from polycystic ovary syndrome (PCOS) and hyperinsulinemia from insulin resistance are modulators of ovarian follicle development. We report on a woman with PCOS and hyperandrogenism and severe insulin resistance from metabolic syndrome who received long-term GnRH analogue therapy preceding bilateral salpingo-oophorectomy for massive ovarian enlargement. Ovarian histological examination showed proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas, demonstrating a unique link between hyperinsulinemia and granulosa cell mitogenesis. CASE DESCRIPTION: A 30-year-old woman with PCOS with hyperandrogenism, severe insulin resistance from metabolic syndrome, and nonalcoholic steatohepatitis experienced abdominal pain from bilaterally enlarged ovaries. She had previously experienced a pulmonary embolism while taking oral contraceptives and hepatotoxicity from metformin and spironolactone therapies. Long-term GnRH analogue therapy to induce pituitary desensitization to GnRH successfully decreased gonadotropin-dependent steroidogenesis without improving insulin resistance. Despite GnRH analogue therapy, progressive ovarian enlargement in the presence of hyperinsulinemia from worsening metabolic function eventually required bilateral salpingo-oophorectomy for removal of massively enlarged ovaries. Histological examination showed both ovaries contained proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas. CONCLUSIONS: In women with PCOS and hyperinsulinemia from severe insulin resistance due to metabolic syndrome, granulosa cell proliferation within antral follicles can occur despite long-term GnRH analogue therapy, implicating hyperinsulinemia as a granulosa cell mitogen in the absence of gonadotropin-dependent ovarian function.

J Clin Endocrinol Metab ; 99(4): 1299-306, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24423334


CONTEXT: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown. OBJECTIVE: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA for 21-hydroxylase and the mineralocorticoid receptor. DESIGN: This was a prospective cohort study. SETTING: The study was conducted at an academic center. PATIENTS: LGC lipid content and follicle fluid (FF) hormone analysis was performed on 27 nonobese IVF women. LGCs from six additional nonobese IVF women were used for gene expression studies. INTERVENTION: At oocyte retrieval, FF was aspirated from the first follicle (≥16 mm in size) of each ovary and pooled LGCs were collected. MAIN OUTCOME MEASURES: FF steroid analysis was performed by liquid chromatography-tandem mass spectrometry. LGCs were stained with lipid fluorescent dye BODIPY FL C16 to estimate lipid content by confocal microscopy as a cholesterol source for steroidogenesis in vivo. Quantitative real-time PCR was performed using LGCs to detect 21-hydroxylase and mineralocorticoid receptor mRNA expression. Pearson correlation coefficients determined associations between FF steroid levels and LGC lipid content. RESULTS: FF levels of the 21-hydroxylase-derived steroids, 11-deoxycorticosterone [DOC, 39.97, median (13.94-63.02) ng/mL] and 11-deoxycortisol [11DOC, 2.07 (0.69-5.01) ng/mL], along with the 21-hydroxylase precursor 17-hydroxyprogesterone [1268.21 (493.26-3558.39) ng/mL], positively correlated with LGC lipid content (84 ± 43 fluorescent units/sample) (P ≤ .05, all steroids). 21-Hydroxylase and mineralocorticoid receptor mRNA expression was detected in LGCs. CONCLUSIONS: Human LGCs likely synthesize 21-hydroxylase-derived mineralocorticoids from cholesterol-containing lipid in vivo to promote postovulatory luteinization via mineralocorticoid receptor-mediated events.

Fertilização In Vitro , Células da Granulosa/metabolismo , Mineralocorticoides/metabolismo , Folículo Ovariano/metabolismo , Indução da Ovulação , Esteroide 21-Hidroxilase/metabolismo , Adulto , Colesterol/metabolismo , Estudos de Coortes , Feminino , Líquido Folicular/química , Líquido Folicular/metabolismo , Células da Granulosa/química , Humanos , Peso Corporal Ideal , Metabolismo dos Lipídeos , Lipídeos/análise , Luteinização/fisiologia , Mineralocorticoides/análise , Folículo Ovariano/química , Esteroide 21-Hidroxilase/genética
PLoS One ; 8(6): e64752, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23755141


Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse) Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal). When compared to adipose stem cells (ASCs), microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell homing. Being highly resistant to severe cellular stress, Muse-AT cells have the potential to make a critical impact on the field of regenerative medicine and cell-based therapy.

Células-Tronco Pluripotentes/fisiologia , Estresse Fisiológico , Gordura Subcutânea Abdominal/citologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Forma Celular , Células Cultivadas , Ectoderma/citologia , Endoderma/citologia , Feminino , Hepatócitos/metabolismo , Humanos , Mesoderma/citologia , Miócitos de Músculo Liso/metabolismo , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Medicina Regenerativa , Transcriptoma , Regulação para Cima
Steroids ; 78(9): 920-6, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23707571


Androgens play a pivotal role in the regulation of body fat distribution. Adipogenesis is a process whereby multipotent adipose stem cells (ASCs) initially become preadipocytes (ASC commitment to preadipocytes) before differentiating into adipocytes. Androgens inhibit human (h) subcutaneous (SC) abdominal preadipocyte differentiation in both sexes, but their effects on hASC commitment to preadipocyte formation is unknown. We therefore examined whether androgen exposure to human (h) ASCs, isolated from SC abdominal adipose of nonobese women, impairs their commitment to preadipocyte formation and/or subsequent differentiation into adipocytes. For this, isolated hASCs from SC abdominal lipoaspirate were cultured in adipogenesis-inducing medium for 0.5-14days in the presence of testosterone (T, 0-100nM) or dihydrotestosterone (DHT, 0-50nM). Adipogenesis was determined by immunofluorescence microscopy and by quantification of adipogenically relevant transcriptional factors, PPARγ, C/EBPα and C/EBPß. We found that a 3-day exposure of hASCs to T (50nM) or DHT (5nM) in adipogenesis-inducing medium impaired lipid acquisition and decreased PPARγ, C/EBPα and C/EBPß gene expression. The inhibitory effects of T and DHT at this early-stage of adipocyte differentiation, were partially and completely reversed by flutamide (F, 100nM), respectively. The effect of androgens on hASC commitment to a preadipocyte phenotype was examined via activation of Bone Morphogenic Protein 4 (BMP4) signaling. T (50nM) and DHT (5nM) significantly inhibited the stimulatory effect of BMP4-induced ASC commitment to the preadipocyte phenotype, as regards PPARγ and C/EBPα gene expression. Our findings indicate that androgens, in part through androgen receptor action, impair BMP4-induced commitment of SC hASCs to preadipocytes and also reduce early-stage adipocyte differentiation, perhaps limiting adipocyte numbers and fat storage in SC abdominal adipose.

Adipócitos/metabolismo , Adipogenia , Células-Tronco Adultas/fisiologia , Androgênios/fisiologia , Di-Hidrotestosterona/farmacologia , Testosterona/fisiologia , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Feminino , Flutamida/farmacologia , Expressão Gênica , Humanos , Metabolismo dos Lipídeos , PPAR gama/genética , PPAR gama/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Testosterona/farmacologia
J Assist Reprod Genet ; 30(5): 733-40, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23512091


PURPOSE: To quantify intracellular lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF), based upon different cell preparation methods. METHODS: CCs and MGCs from 16 lean women undergoing ovarian stimulation for IVF were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method 1) and subsequent lipid accumulation in vitro (Method 2). Cells for initial lipid content were immediately fixed at the time of the oocyte retrieval with 4% paraformaldehyde in suspension, while those for subsequent lipid accumulation in vitro were cultured for 4 h with 5% fetal calf serum and then fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C16 and nuclear marker DAPI. Intracellular lipid was quantified by confocal microscopy, using ImageJ software analysis. RESULTS: There was no significant effect of cell type (P = 0.2) or cell type-cell preparation method interaction (P = 0.8) on cell area (Method 1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; Method 2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 µm(2)). The mean area of all cells combined was significantly less for cells prepared by Method 1 (116.2 ± 4.9 µm(2)) vs. Method 2 (243.5 ± 22.5 µm(2), P < 0.00005). Intracellular lipid level, however, was significantly altered by cell preparation method (P < 0.05; cell preparation method-cell type interaction, P < 0.00001). Initial lipid content was significantly lower in CC (74.5 ± 9.3) than MGC (136.3 ± 16.7 fluorescence/cell area, P < 0.00005), while subsequent lipid accumulation in vitro was significantly higher in CC (154.0 ± 9.1) than MGC (104.6 ± 9.9 fluorescence/cell area, P < 0.00001). The relatively diminished initial CC lipid content compared to subsequent CC lipid accumulation in vitro (P < 0.00001), and the opposite pattern for MGC (P < 0.05), significantly lowered the CC/MGC lipid ratio in Method 1 (0.55 ± 0.04) vs. Method 2 (1.58 ± 0.10, P < 0.00001). CONCLUSIONS: Differential uptake or utilization of lipid by CC and MGC occurs during oocyte maturation and steroidogenesis, respectively, with the amount of lipid present in ovarian cells a function of both the follicular microenvironment at the time of the oocyte retrieval and the capacity of these cells to accumulate lipid in vitro over time.

Células do Cúmulo/química , Fertilização In Vitro , Células da Granulosa/química , Lipídeos/análise , Ovário/citologia , Indução da Ovulação , Adulto , Células Cultivadas , Células do Cúmulo/metabolismo , Células do Cúmulo/ultraestrutura , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/ultraestrutura , Humanos , Metabolismo dos Lipídeos/fisiologia , Microscopia Confocal/métodos , Ovário/química , Ovário/metabolismo , Ovário/ultraestrutura , Gravidez