RESUMEN

Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05-0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of in vitro culture. Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.

Asunto(s)

Infertilidad Masculina , Inyecciones de Esperma Intracitoplasmáticas , Citratos , Medios de Cultivo , Femenino , Fertilización In Vitro/métodos , Glucosa , Histidina , Humanos , Infertilidad Masculina/patología , Infertilidad Masculina/terapia , Lisina , Espectroscopía de Resonancia Magnética , Masculino , Embarazo , Estudios Prospectivos , Piruvatos , Semen , Inyecciones de Esperma Intracitoplasmáticas/métodos , Valina

RESUMEN

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Asunto(s)

Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Medios de Cultivo/química , Fertilización In Vitro , Oxígeno

RESUMEN

Infertility affects approximately 15-20% of individuals of reproductive age worldwide. Over the last 40 years, assisted reproductive technology (ART) has helped millions of childless couples. However, ART is limited by a low success rate and risk of multiple gestations. Devising methods for selecting the best gamete or embryo that increases the ART success rate and prevention of multiple gestation has become one of the key goals in ART today. Special emphasis has been placed on the development of non-invasive approaches, which do not require perturbing the embryonic cells, as the current morphology-based embryo selection approach has shortcomings in predicting the implantation potential of embryos. An observed association between embryo metabolism and viability has prompted researchers to develop metabolomics-based biomarkers. Nuclear magnetic resonance (NMR) spectroscopy provides a non-invasive approach for the metabolic profiling of tissues, gametes and embryos, with the key advantage of having a minimal sample preparation procedure. Using NMR spectroscopy, biologically important molecules can be identified and quantified in intact cells, extracts or secretomes. This, in turn, helps to map out the active metabolic pathways in a system. The present review covers the contribution of NMR spectroscopy in assisted reproduction at various stages of the process.

Asunto(s)

Espectroscopía de Resonancia Magnética , Técnicas Reproductivas Asistidas , Biomarcadores , Técnicas de Cultivo de Embriones , Femenino , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Metabolómica/métodos , Especificidad de Órganos , Técnicas Reproductivas Asistidas/tendencias , Biología de Sistemas/métodos

RESUMEN

Confining heme protein in silico often leads to beneficial functionalities such as an enhanced electrochemical response from the heme center. This can be harnessed to design effective biosensors for medical diagnostics. Proteins under confinement, surface confinement on the electrode to be precise, have more ordered and monodisperse structure compared to the protein in bulk solution. As the electrochemical response of a protein comes from those protein molecules that are confined within the electrical double layer across the electrode-electrolyte interface, it is expected that restriction of conformational fluctuations of the polymeric protein will help in enhancement of the electrochemical response. This is probably the prima facie reason for electrochemical response enhancement under confinement. We examine the dynamic features of hemoglobin under confinement vis-à-vis that in bulk solution. We use a variety of spectroscopic techniques across a wide time-space window to establish the following facts: (a) hardening of the protein polypeptide backbone, (b) slowing down of protein diffusion, (c) increase in relaxation times in NMR, and (d) slowing down of dielectric relaxation times under confinement. This indicates an overall quenching of protein dynamics when the protein is confined inside silica matrix. Thus, we hypothesize that along with retention of secondary structure, this quenching of dynamics contributes to the enhancement of electrochemical response observed.

Asunto(s)

Hemoglobinas , Polímeros , Difusión , Estructura Secundaria de Proteína , Dióxido de Silicio

RESUMEN

Paternal genetic alterations may affect embryo viability and reproductive outcomes. Currently it is unknown whether embryo metabolism is affected by sperm-mediated abnormalities. Hence, using a mouse model, this study investigated the response to paternally transmitted DNA lesions on genetic integrity and metabolism in preimplantation embryos. Spent embryo culture media were analysed for metabolites by nuclear magnetic resonance spectroscopy and embryonic genetic integrity was determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay on embryonic Day 4.5 (E4.5). Metabolic signatures were compared between normally derived embryos (control) and embryos derived from spermatozoa carrying induced DNA lesions (SDL). SDL embryos showed a significant reduction in blastocyst formation on E3.5 and E4.5 (P<0.0001) and had an approximately 2-fold increase in TUNEL-positive cells (P<0.01). A cohort of SDL embryos showing delayed development on E4.5 had increased uptake of pyruvate (P<0.05) and released significantly less alanine (P<0.05) to the medium compared with the corresponding control embryos. On the other hand, normally developed SDL embryos had a reduced (P<0.001) pyruvate-to-alanine ratio compared with normally developed embryos from the control group. Hence, the difference in the metabolic behaviour of SDL embryos may be attributed to paternally transmitted DNA lesions in SDL embryos.

Asunto(s)

Apoptosis/efectos de los fármacos , Blastocisto/metabolismo , Daño del ADN/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Blastocisto/efectos de los fármacos , Cisplatino/farmacología , Medios de Cultivo , Fragmentación del ADN/efectos de los fármacos , Técnicas de Cultivo de Embriones , Masculino , Ratones

RESUMEN

We introduce a self-assembling polypeptide-based nanotube system having the ability to specifically target cancer cells. The nanotubes target the cancer cell surface through integrin engagement with the help of multiple RGD units present along their surface. While the nanotubes are non-toxic towards cells in general, they can be loaded with suitable drugs to be released in a sustained manner in cancer cells. In addition, the nanotubes can be utilized for cellular imaging using any covalently tagged fluorescent dye. They are stable over a wide range of temperature due to intermolecular disulphide bonds formed during the self-assembly process. At the same time, presence of disulphide bonds provides a redox molecular switch for their degradation. Taken together this system provides a unique avenue for multimodal formulation in cancer therapy.

Asunto(s)

Nanotubos/química , Neoplasias , Humanos , Terapia Molecular Dirigida/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Imagen Óptica/métodos , Oxidación-Reducción , Péptidos/química , Multimerización de Proteína

RESUMEN

The metabolomic profile of an embryo culture medium can aid in the advanced prediction of embryonic developmental potential and genetic integrity. But it is not known if this technology can be used to determine the in vitro potential of inner cell mass (ICM) in adherence and proliferation. Here, we investigated the developmental potential of mouse 2-cell embryos carrying cisplatin-induced DNA lesions (IDL), beyond blastocyst stage using ICM outgrowth assay. The genetic integrity of ICM cells was determined by comet assay. The metabolic signatures of spent medium were recorded 84 hours post injection of hCG (hpi-hCG), and after 96 hours of extended in vitro culture (Ex 96) by NMR spectroscopy. We observed that blastocysts that lack the ability to adhere in vitro had an increased requirement of pyruvate (p < 0.01), lactate (p < 0.01), and were accompanied by a significant reduction of pyruvate-alanine ratio in the culture medium. We propose that the aforementioned metabolites from 84 hpi-hCG spent medium be further explored using appropriate experimental models, to prove their potential as biomarkers in the prediction of implantation ability of in vitro derived human embryos in clinical settings.

Asunto(s)

Blastocisto/citología , Adhesión Celular , Medios de Cultivo/metabolismo , Embrión de Mamíferos/citología , Animales , Embrión de Mamíferos/metabolismo , Técnicas In Vitro , Ratones

RESUMEN

DNA-binding protein domains (DBDs) sample diverse conformations in equilibrium facilitating the search and recognition of specific sites on DNA over millions of energetically degenerate competing sites. We hypothesize that DBDs have co-evolved to sense and exploit the strong electric potential from the array of negatively charged phosphate groups on DNA. We test our hypothesis by employing the intrinsically disordered DBD of cytidine repressor (CytR) as a model system. CytR displays a graded increase in structure, stability and folding rate on increasing the osmolarity of the solution that mimics the non-specific screening by DNA phosphates. Electrostatic calculations and an Ising-like statistical mechanical model predict that CytR exhibits features of an electric potential sensor modulating its dimensions and landscape in a unique distance-dependent manner, while DNA plays the role of a non-specific macromolecular chaperone. Accordingly, CytR binds its natural half-site faster than the diffusion-controlled limit and even random DNA conforming to an electrostatic-steering binding mechanism. Our work unravels for the first time the synergistic features of a natural electrostatic potential sensor, a novel binding mechanism driven by electrostatic frustration and disorder, and the role of DNA in promoting distance-dependent protein structural transitions critical for switching between specific and non-specific DNA-binding modes.

Asunto(s)

ADN/química , Proteínas de Escherichia coli/química , Proteínas Intrínsecamente Desordenadas/química , Fosfatos/química , Proteínas Represoras/química , Secuencias de Aminoácidos , Sitios de Unión , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Electricidad Estática , Termodinámica