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1.
Hum Reprod ; 37(12): 2757-2767, 2022 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-36287638

RESUMEN

STUDY QUESTION: Does the type of incubator used to culture human preimplantation embryos affect development to the blastocyst stage and alter amino acid utilization of embryos in assisted reproduction? SUMMARY ANSWER: Culturing embryos in a time lapse system (TLS) was associated with a higher Day 5 blastocyst formation rate and altered amino acid utilization when measured from Day 3 to Day 5 compared to the standard benchtop incubator. WHAT IS KNOWN ALREADY: Culture environment is known to be important for the developing preimplantation embryo. TLSs provide a stable milieu allowing embryos to be monitored in situ, whereas embryos cultured in standard benchtop incubators experience environmental fluctuations when removed for morphological assessment. STUDY DESIGN, SIZE, DURATION: A prospective clinical trial randomizing 585 sibling embryos to either the TLS (289 embryos) or the standard benchtop incubator (296 embryos) over a 23-month period in a UK University Hospital Fertility Clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were aged 42 years or under, had an antral follicle count of ≥12 and ≥6 2 pronucleate zygotes. Zygotes were cultured individually in 25 µl of medium. Randomized embryos were graded and selected for transfer or cryopreservation on Day 5. For those embryos produced by women who underwent stimulation with recombinant FSH injections and were triggered with hCG, spent medium was collected on Day 5 for amino acid analysis by high pressure liquid chromatography. Clinical pregnancy was defined as the presence of a foetal heart beat on ultrasound scan at 7 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, blastocyst formation rate on Day 5 was significantly higher in embryos cultured in the TLS (55%) compared to the standard incubator (45%; P = 0.013). Similarly, there was an increase in the number of blastocysts suitable for cryopreservation in the TLS (31%) compared to the standard incubator (23%; P = 0.032). There was a significant difference in the utilization of 12 amino acids by blastocysts cultured from Day 3 to Day 5 in the TLS compared to the standard incubator. Embryos cultured in the TLS displayed an increased total amino acid utilization (P < 0.001) and reduced amino acid production (P < 0.001) compared to those in the standard incubator. Irrespective of incubator used, embryos fertilized by ICSI depleted significantly more amino acids from the medium compared to those fertilized by conventional IVF. There was no difference in the mean score of blastocysts transferred, or the clinical pregnancy rate after transfer of embryos from either of the incubators. LIMITATIONS, REASONS FOR CAUTION: The study was not powered to discern significant effects on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The metabolism and development of preimplantation embryos is impacted by the type of incubator used for culture. Further research is required to investigate the long-term implications of these findings. STUDY FUNDING/COMPETING INTEREST(S): NIHR Southampton Biomedical Research Centre Commercial and Enterprise Incubator Fund funded this study. The TLS was provided on loan for the study by Vitrolife. The authors declare no conflict of interests. TRIAL REGISTRATION NUMBER: ISRCTN73037149. TRIAL REGISTRATION DATE: 12 January 2012. DATE OF FIRST PATIENT'S ENROLMENT: 21 January 2012.


Asunto(s)
Blastocisto , Desarrollo Embrionario , Embarazo , Humanos , Femenino , Estudios Prospectivos , Desarrollo Embrionario/fisiología , Blastocisto/metabolismo , Incubadoras , Aminoácidos/farmacología , Aminoácidos/metabolismo , Técnicas de Cultivo de Embriones
2.
Reproduction ; 161(1): F41-F51, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33258799

RESUMEN

Development of the preimplantation embryo is reliant on nutrients present in the milieu of the reproductive tract. While carbohydrates, amino acids, lipids, and micronutrients are often considered when discussing preimplantation embryo nutrition, environmental oxygen is frequently overlooked. Although oxygen is not classically considered a nutrient, it is an important component of the in vitro culture environment and a critical regulator of cellular physiology. Oxygen is required to sustain an oxidative metabolism but when oxygen becomes limited, cells mount a physiological response driven by a family of transcription factors termed 'hypoxia inducible factors' which promote expression of a multitude of oxygen sensitive genes. It is this hypoxic response that is responsible not only for the switch to a glycolytic metabolism but also for a plethora of other cellular responses. There has been much debate in recent years over which environmental oxygen tension is preferential for the culture of preimplantation embryos. The review will evaluate this question and highlights how research using human embryonic stem cells can inform our understanding of why culturing under physiological oxygen tensions may be beneficial for the development of embryos generated through clinical in vitro fertilisation.


Asunto(s)
Blastocisto/fisiología , Genitales Femeninos/fisiología , Células Madre Embrionarias Humanas/fisiología , Hipoxia , Oxígeno/fisiología , Femenino , Homeostasis , Humanos , Salud Reproductiva
3.
Clin Sci (Lond) ; 135(11): 1445-1466, 2021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-34008846

RESUMEN

During Ramadan, many pregnant Muslim women fast between dawn and sunset. Although the impacts of prolonged maternal intermittent fasting (IF) on fetal growth and placental function are under-researched, reported effects include reduced placental weight and birth weight. In the present study, pregnant Wistar rats were used to model repeated cycles of IF on fetal development and placental function and to examine sex-specific effects. In the IF group, food was withdrawn daily from 17:00 to 09:00 over 21 days of gestation, while the control group received food ad libitum. Both groups had free water access. IF dams consumed less food, had significantly reduced weight compared with controls, with reduced plasma glucose and amino acids. Both fetal sexes were significantly lighter in the IF group with reduced fetal plasma amino acids. Placental weights and morphology were unchanged. The profile of placental metabolites was altered in the IF group with sex-specific responses evident. Transplacental flux of 14C-methylaminoisobutyric acid (14C-MeAIB), a system A amino acid transporter substrate, was significantly reduced in both fetal sexes in the IF group. Sodium-dependent 14C-MeAIB uptake into isolated placental plasma membrane vesicles was unchanged. The gene expression of system A transporter Slc38a1, Slc38a2 and Slc38a4 was up-regulated in IF male placentas only. No changes were observed in placental SNAT1 and SNAT2 protein expression. Maternal IF results in detrimental impacts on maternal physiology and fetal development with changes in the placental and fetal metabolite profiles. Reduced placental system A transporter activity may be responsible for fetal growth restriction in both sexes.


Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Ayuno , Retardo del Crecimiento Fetal/metabolismo , Placenta/metabolismo , Animales , Femenino , Desarrollo Fetal/fisiología , Feto/metabolismo , Embarazo , Ratas Wistar
4.
Hum Reprod ; 30(4): 917-24, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25697730

RESUMEN

STUDY QUESTION: Do the amino acid levels of human uterine fluid vary with age, BMI, phase of menstrual cycle, benign pathology or diet? SUMMARY ANSWER: The levels of 18 amino acids in human uterine fluid were shown to be affected only by maternal diet. WHAT IS KNOWN ALREADY: Murine, bovine and ovine uterine amino acid content has been reported, but no reliable data on the human exist. Murine studies have demonstrated that the intrauterine periconceptional nutritional environment is affected by maternal diet. STUDY DESIGN, SIZE, DURATION: Uterine secretions were aspirated from 56 women aged 18-45 years. The women were recruited preoperatively from gynaecological theatre operating schedules or hysterosalpingo-contrast-sonography (HyCoSy) lists. A proportion of these women had proven fertility; however, the majority were being investigated for subfertility. The BMI, gynaecological history and dietary pattern of these women were also assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reverse phase high performance liquid chromatography was used to analyse the concentrations of 18 amino acids within the uterine fluid and blood serum. The results were analysed against the women's stage of cycle, age, BMI and diet. MAIN RESULTS AND THE ROLE OF CHANCE: The profile of 18 amino acids in uterine fluid was described. In total, human uterine fluid was observed to contain an amino acid concentration of 3.54 mM (interquartile range: 2.27-6.24 mM). The relative concentrations of 18 amino acids were not significantly altered by age, BMI, cycle phase or the presence of specific benign gynaecological pathologies. However, a diet identified by a validated scoring system as being less healthy was associated with higher concentrations of asparagine (P = 0.018), histidine (P = 0.011), serine (P = 0.033), glutamine (P = 0.049), valine (P = 0.025), phenylalanine (P = 0.019), isoleucine (P = 0.025) and leucine (P = 0.043) in the uterine fluid compared with a healthier diet, defined as one with a higher intake of fresh vegetables, fruit, whole-grain products and fish and a low intake of red and processed meat and high fat dairy products. There were no significant correlations between serum amino acid concentrations and those in the uterine fluid. LIMITATIONS, REASONS FOR CAUTION: Our results enabled us to detect the effect of diet on the concentrations of amino acids in human uterine fluid; however, the study may not have had sufficient numbers to detect mild effects of BMI or age. WIDER IMPLICATIONS OF THE FINDINGS: These findings increase our understanding of the nutritional environment encountered by the preimplantation embryo, and indicate how periconceptional diet may alter this. Given the importance of early embryo environment for programming of development and future health, this information may aid in the development of nutritional interventions aimed at optimizing the preimplantation phase of human embryo development in vivo. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the NIHR, the Medical Research Council (G0701153) and the University of Southampton and was supported by the NIHR BRC in Nutrition and Southampton University NHS Foundation Trust. The authors declare no conflicts of interest.


Asunto(s)
Aminoácidos/química , Líquidos Corporales/química , Útero/química , Útero/patología , Adolescente , Adulto , Índice de Masa Corporal , Dieta , Femenino , Humanos , Estilo de Vida , Ciclo Menstrual , Persona de Mediana Edad , Obesidad/complicaciones , Periodo Preoperatorio , Adulto Joven
5.
Cell Physiol Biochem ; 33(1): 237-46, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24496287

RESUMEN

BACKGROUND/AIMS: Human embryonic stem cells (hESCs) are a potential source of cells for treatment of many degenerative diseases, but in culture have a propensity to spontaneously differentiate, possibly due to suboptimal conditions. Culture at low oxygen tensions improves hESC maintenance and regulates carbohydrate metabolism. Hence, a greater understanding of the nutrient requirements of hESCs will allow production of more appropriate culture media. This study aims to investigate the effect of environmental oxygen tension on the amino acid metabolism of hESCs. METHODS: The production or depletion of amino acids by hESCs cultured at 5% or 20% oxygen in the presence or absence of FGF2 was measured by reversephase HPLC. RESULTS: Atmospheric oxygen, or removal of FGF2 from hESCs cultured at 5% oxygen, perturbed the uptake or release of individual amino acids and the total amino acid turnover compared to hESCs cultured at 5% oxygen. In particular, serine uptake was reduced at 20% oxygen and by removal of FGF2. CONCLUSIONS: Highly pluripotent hESCs, cultured at 5% oxygen, demonstrate a greater amino acid turnover than hESCs cultured at 20% oxygen, or without FGF2. These data suggest that amino acid turnover could be used as a measure of the self-renewal capacity of hESCs.


Asunto(s)
Aminoácidos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Oxígeno/farmacología , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Metaboloma/efectos de los fármacos , Factores de Tiempo
6.
Stem Cells ; 31(11): 2343-53, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23922292

RESUMEN

The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.


Asunto(s)
Reprogramación Celular/fisiología , Células Madre Embrionarias/fisiología , Oocitos/fisiología , Células Madre Pluripotentes/fisiología , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Transferencia Nuclear , Oocitos/citología , Oocitos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Tetraploidía
7.
BMC Womens Health ; 14: 130, 2014 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-25407227

RESUMEN

BACKGROUND: In vitro fertilisation (IVF) treatment provides an opportunity to study early developmental responses to periconceptional dietary interventions. Retrospective studies have suggested links between preconception diet and fertility, and more recently, a "Mediterranean" diet has been reported to increase pregnancy rates by up to 40%. In addition, a prospective study examining increased intake of omega-3 polyunsaturated fats demonstrated a quickened rate of embryo development after IVF. However, up to now, few prospective randomised controlled trials have investigated the impact of periconceptional dietary interventions on fertility outcomes. METHODS AND DESIGN: The study is a randomised controlled trial of a dietary intervention consisting of olive oil for cooking, an olive oil based spread, and a daily supplement drink enriched with Vitamin D (10 microgram daily) and marine omega-3 fatty acids (2 g daily) for 6 weeks preconception versus a control diet of sunflower seed oil for cooking, a sunflower oil based spread, and a daily supplement drink without added Vitamin D or marine omega-3 fatty acids. Couples undergoing IVF will be randomised to either the intervention or control group (55 in each arm). The primary endpoint is embryo developmental competency in vitro, measured by validated morphokinetic markers. Secondary outcomes will include the effect of the dietary intervention on the nutritional content of the intrauterine environment. DISCUSSION: This approach will enable rigorous examination of the impact of the dietary intervention on early embryo development, together with the influence of the peri-implantation intra-uterine nutritional environment. TRIAL REGISTRATION: ISRCTN50956936.


Asunto(s)
Dieta , Suplementos Dietéticos , Desarrollo Embrionario/efectos de los fármacos , Fertilización In Vitro/métodos , Atención Preconceptiva , Adolescente , Adulto , Culinaria , Dinoprost/análisis , Dinoprostona/análisis , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/análisis , Ácido Eicosapentaenoico/administración & dosificación , Ácido Eicosapentaenoico/análisis , Endometrio/química , Endometrio/citología , Endometrio/inmunología , Femenino , Ácido Fólico/análisis , Líquido Folicular/química , Humanos , Masculino , Aceite de Oliva , Aceites de Plantas/administración & dosificación , Embarazo , Índice de Embarazo , Proyectos de Investigación , Análisis de Semen , Aceite de Girasol , Vitamina B 12/análisis , Vitamina B 6/análisis , Vitamina D/administración & dosificación , Vitamina D/análisis , Adulto Joven
8.
Sci Rep ; 11(1): 18921, 2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34584110

RESUMEN

Articular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young's) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.


Asunto(s)
Cartílago Articular/crecimiento & desarrollo , Células Madre Embrionarias Humanas/metabolismo , Ingeniería de Tejidos/métodos , Agrecanos/metabolismo , Cartílago/metabolismo , Cartílago Articular/metabolismo , Diferenciación Celular , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Regeneración Tisular Dirigida/métodos , Células Madre Embrionarias Humanas/trasplante , Humanos , Células Madre Mesenquimatosas/metabolismo
9.
Lipids ; 56(2): 201-209, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33047321

RESUMEN

The fatty acid composition of human follicular fluid is important for oocyte development and for pregnancy following in vitro fertilization (IVF). This study investigated whether a dietary intervention that included an increase in marine omega-3 fatty acids, olive oil and vitamin D alters the fatty acid composition of human follicular fluid. The association of lifestyle factors with follicular fluid fatty acid composition was also investigated. Fifty-five couples awaiting IVF were randomized to receive the 6-week treatment intervention of olive oil for cooking, an olive oil-based spread, and a daily supplement drink enriched with vitamin D and the marine omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and 56 couples were randomized to receive placebo equivalents. Dietary questionnaires were completed, and samples of blood were taken before and after the intervention. Follicular fluid was collected at oocyte retrieval and the fatty acid profile assessed using gas chromatography. In the control group, individual fatty acids in red blood cells and follicular fluid were significantly correlated. Furthermore, a healthier diet was associated with a lower percentage of follicular fluid arachidonic acid. The follicular fluid of women in the treatment group contained significantly higher amounts of EPA and DHA compared to the control group, while the omega-6 fatty acids linoleic, γ-linolenic, dihomo-γ-linolenic, and arachidonic were lower. This is the first report of a dietary intervention altering the fatty acid composition of follicular fluid in humans. Further research is required to determine whether this intervention improves oocyte quality.


Asunto(s)
Ácidos Grasos Omega-3/sangre , Ácidos Grasos/sangre , Líquido Folicular/química , Adolescente , Adulto , Estudios de Cohortes , Suplementos Dietéticos , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Fertilización In Vitro , Humanos , Adulto Joven
10.
Mol Hum Reprod ; 16(8): 557-69, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20571076

RESUMEN

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.


Asunto(s)
Aminoácidos/metabolismo , Aneuploidia , Desarrollo Embrionario/fisiología , Adulto , Cromatografía Líquida de Alta Presión , Criopreservación , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Humanos , Edad Materna , Embarazo , Factores Sexuales
11.
Reproduction ; 139(1): 85-97, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19755485

RESUMEN

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.


Asunto(s)
Desdiferenciación Celular , Hipoxia de la Célula , Proliferación Celular , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Oxígeno/fisiología , Proteínas Reguladoras de la Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Línea Celular , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/citología , Antígeno Lewis X/genética , Antígeno Lewis X/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Transporte de Proteínas , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Represoras , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
12.
Fertil Steril ; 113(2): 260-269, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31870562

RESUMEN

OBJECTIVE: To study the impact of increased dietary intake of omega-3 fatty acids, vitamin D, and olive oil for 6 weeks before in vitro fertilization (IVF) or IVF-intracytoplasmic sperm injection (ICSI) on morphokinetic markers of early embryo development. DESIGN: A double-blinded randomized controlled trial. SETTING: Academic IVF unit. PATIENT(S): A total of 111 couples undergoing IVF or IVF-ICSI were recruited. INTERVENTIONS(S): Fifty-five couples received the 6-week study intervention of a daily supplement drink enriched with omega-3 fatty acids and vitamin D plus additional olive oil and olive oil-based spread, and 56 couples received the control intervention. MAIN OUTCOME MEASURE(S): The primary end point for the study was the time taken for completion of the second cell cycle after fertilization (CC2). Secondary end points included time to complete the third and fourth cell cycles (CC3 and CC4), the synchrony of the second and third cell cycles (S2 and S3), and the day 3 and day 5 Known Implantation Data Scores (KIDScores). RESULT(S): There was no difference in CC2 between the two groups. However, CC4 was accelerated in the study group compared with the control group, and a significantly shortened S3 as well as an increase in KIDScore on day 3 were observed, indicating improved embryo quality in the study group. CONCLUSION(S): This study demonstrates that a short period of dietary supplementation alters the rate of embryo cleavage. Further research is required to investigate the mechanisms that regulate this effect, and whether the impact on embryo development translates into improved clinical outcomes. CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN50956936.


Asunto(s)
Suplementos Dietéticos , Desarrollo Embrionario/fisiología , Ácidos Grasos Omega-3/administración & dosificación , Fertilización In Vitro , Aceite de Oliva/administración & dosificación , Vitamina D/administración & dosificación , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Cooperación del Paciente , Embarazo , Índice de Embarazo , Vitamina D/sangre
13.
J Cell Mol Med ; 13(9B): 3541-55, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19438813

RESUMEN

Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research.


Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Medio de Cultivo Libre de Suero , Activinas/metabolismo , Supervivencia Celular , Medio de Cultivo Libre de Suero/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Edad Gestacional , Humanos , Inmunohistoquímica/métodos , Osteogénesis , Fenotipo , Embarazo , Regeneración , Factores de Tiempo
14.
Stem Cell Reports ; 12(4): 728-742, 2019 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-30880076

RESUMEN

Glycolysis and hypoxia are key regulators of human embryonic stem cell (hESC) self-renewal, but how changes in metabolism affect gene expression is poorly understood. C-terminal binding proteins (CTBPs) are glycolytic sensors that through NADH binding link the metabolic state of the cell to its gene expression, by acting as transcriptional corepressors, or coactivators. However, the role of CTBPs in hESCs has not previously been investigated. A direct interaction between hypoxia-inducible factor 2α (HIF-2α) and the CTBP proximal promoters in hESCs cultured only under hypoxia was demonstrated. Decreasing the rate of flux through glycolysis in hESCs maintained under hypoxia resulted in a reduction of CTBPs, OCT4, SOX2, and NANOG, but also in the expression of HIF-2α. Silencing CTBP expression resulted in the loss of pluripotency marker expression demonstrating that CTBPs are involved in hESC maintenance. These data suggest that under hypoxia, glycolysis regulates self-renewal through HIF-2α and the induction of the metabolic sensors CTBPs.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Autorrenovación de las Células/genética , Proteínas Co-Represoras/genética , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Glucólisis , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Modelos Biológicos , Oxígeno/metabolismo , Regiones Promotoras Genéticas
15.
Regen Med ; 13(2): 189-206, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29557248

RESUMEN

The rise in the incidence of musculoskeletal diseases is attributed to an increasing ageing population. The debilitating effects of musculoskeletal diseases, coupled with a lack of effective therapies, contribute to huge financial strains on healthcare systems. The focus of regenerative medicine has shifted to pluripotent stem cells (PSCs), namely, human embryonic stem cells and human-induced PSCs, due to the limited success of adult stem cell-based interventions. PSCs constitute a valuable cell source for musculoskeletal regeneration due to their capacity for unlimited self-renewal, ability to differentiate into all cell lineages of the three germ layers and perceived immunoprivileged characteristics. This review summarizes methods for chondrogenic, osteogenic, myogenic and adipogenic differentiation of PSCs and their potential for therapeutic applications.

16.
Oncotarget ; 9(33): 23274-23288, 2018 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-29796188

RESUMEN

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells.

17.
Sci Rep ; 5: 17500, 2015 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-26639784

RESUMEN

Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs.


Asunto(s)
Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 3/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Proteínas de la Membrana/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Hormonas Tiroideas/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Silenciador del Gen/efectos de los fármacos , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Ácido Láctico/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oxígeno/farmacología , Transporte de Proteínas/efectos de los fármacos , Proteínas de Unión a Hormona Tiroide
18.
Chem Biol ; 22(7): 838-48, 2015 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-26144885

RESUMEN

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleótido Desaminasas/antagonistas & inhibidores , Purinas/biosíntesis , Ribonucleótidos/farmacología , Aminoimidazol Carboxamida/farmacología , Animales , Activación Enzimática , Células HCT116 , Humanos , Transferasas de Hidroximetilo y Formilo/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Multimerización de Proteína/efectos de los fármacos
19.
Eur J Obstet Gynecol Reprod Biol ; 115 Suppl 1: S92-6, 2004 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15196724

RESUMEN

In vitro fertilisation (IVF) has permitted many thousands of couples to conceive world-wide yet is limited by low success rates and a confounding high multiple birth rate due to the transfer of more than one embryo. These problems could be overcome if single embryos of known developmental competence could be selected for replacement. It is clear that current embryo selection methods, which rely on morphological appearance alone, are poor predictors of viability. Metabolism is intrinsic to embryo health and much research has concentrated on developing a non-invasive metabolic marker of developmental competence.


Asunto(s)
Blastocisto/metabolismo , Desarrollo Embrionario y Fetal , Femenino , Humanos , Embarazo
20.
PLoS One ; 9(10): e108309, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25271810

RESUMEN

Low O2 tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is unknown. HIF-2α was found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O2). This binding induced an array of histone modifications associated with gene transcription while a heterochromatic state existed at atmospheric O2. Interestingly, an enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This was sustained by HIF-2α which enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG promoter. Thus, these data have uncovered a novel role of HIF-2α as a direct regulator of key transcription factors controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation which enhances the regenerative potential of these cells.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Secuencias Reguladoras de Ácidos Nucleicos , Hipoxia de la Célula , Línea Celular , Células Madre Embrionarias/citología , Histonas/metabolismo , Humanos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo
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