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Fatemi, F.

Publications and source records attributed to Fatemi, F..

3 recordsLinked to original sources

In vivo human uterine temperature, pH, and uterine fluid composition analysis

Study questionWhat are the temperature, pH and uterine fluid composition in the human uterus three days following a positive LH test or ovum pick-up? Summary answerThe mean uterine temperature was 36.94{+/-}0.26{degrees}C, the mean uterine pH was 6.76{+/-}0.22, and the concentrations of 37 components in aspirated uterine fluid were successfully determined. What is known alreadyEmbryo culture conditions in the laboratory impact key outcomes of IVF/ICSI treatments, such as the quality of the embryos and the live birth rate after treatment, and child outcomes, such as birth weight. Currently used conditions, including temperature, pH, and culture medium composition, are largely derived from clinical experience and experimental studies using animal models. Limited studies have been performed to determine the natural human preimplantation embryo environment in vivo during the physiologically relevant time of the menstrual cycle. This type of fundamental knowledge is required for evidence-based optimization of the in vitro embryo culture environment and improving IVF/ICSI outcomes. Study design, size, durationIn this cross-sectional study, conducted between April 2015 and March 2016, temperature and pH were measured in the human uterine cavity on the third day following a positive LH test or ovum pick-up, and uterine fluid was simultaneously aspirated for composition analysis. Uterine temperature was measured in fifty eight women, uterine pH was determined in fifty three women, and twenty two samples of aspirated uterine fluid were analysed for the concentrations of thirty-seven components. Participants/materials, setting, methodsThis study involved 61 healthy reproductive-aged women: 53 without ovarian stimulation and 8 who underwent ovarian stimulation. We measured uterine temperature using a probe inserted into the uterine cavity directly, and uterine pH after inserting a probe through the outer sheath of an IVF catheter. Uterine fluid was then aspirated using this outer IVF catheter and a 10 ml syringe, and subsequently analysed with a Cobas 8000 chemistry analyser and ultra-performance liquid chromatography-tandem mass spectrometry. Main results and the role of chanceThe mean uterine temperature on the third day following a positive LH test or ovum pick-up was 36.94 {+/-} 0.26{degrees}C and correlated with the womens core body temperature. The mean pH in the uterine cavity was pH 6.76 {+/-} 0.22, clearly lower than the standard pH used for human preimplantation embryo culture in vitro (pH 7.3 {+/-} 0.1). Concentrations of important energy sources were 0.8 {+/-} 0.02 mM pyruvate, 5.1 {+/-} 1.78 mM glucose and 6.60 {+/-} 1.12 mM lactate. Glutamic acid (1162 {+/-} 183 M), glycine (955 {+/-} 156 M) and alanine (513 {+/-} 82 M) were the most abundant amino acids in uterine fluid. Limitations, reasons for cautionIn absence of a preimplantation embryo, synergistic influences on the uterine environment may be overlooked. Single centre and specific population limitations may hinder broader generalization of the results. Uterine fluid likely contains additional components. Wider implications of the findingsThe in vivo uterine characteristics identified in this study are foundational to develop an in vivo evidence-based culture medium for human embryos. Further research is necessary to evaluate whether such a medium can improve human preimplantation embryo development and quality, thereby increasing cumulative live birth rates and improving child outcomes.

developmental biology↗

Single molecule analysis reveals positive allosteric modulators are required to stabilize the active state of a metabotropic glutamate receptor

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity, and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

biophysics↗

Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

Conjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Forster resonance energy transfer (smFRET) - can lead to substantial changes of the dyes photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

biochemistry↗