Metabolic Activity of Human Embryos after Thawing Differs in Atmosphere with Different Oxygen Concentrations.

The vitrification of human embryos is more and more frequently being utilized as a method of assisted reproduction. For this technique, gentle treatment of the embryos after thawing is crucial. In this study, the balance of amino acids released to/consumed from the cultivation media surrounding the warmed embryos was observed in the context of a cultivation environment, which was with the atmospheric oxygen concentration ≈20% or with a regulated oxygen level-hysiological (5%). It is the first time that total amino acid turnover in human embryos after their freezing at post compaction stages has been evaluated. During this study, progressive embryos (developed to blastocyst stage) and stagnant embryos (without developmental progression) were analyzed. It was observed that the embryos cultivated in conditions of physiological oxygen levels (5% oxygen) showed a significantly lower consumption of amino acids from the cultivation media. Progressively developing embryos also had significantly lower total amino acid turnovers (consumption and production of amino acids) when cultured in conditions with physiological oxygen levels. Based on these results it seems that a cultivation environment with a reduced oxygen concentration decreases the risk of degenerative changes in the embryos after thawing. Therefore, the cultivation of thawed embryos in an environment with physiological oxygen levels may preclude embryonal stagnation, and can support the further development of human embryos after their thawing.

Study of metabolic activity of human embryos focused on amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Assisted reproduction is a quickly developing field of reproductive medicine whose importance is growing every year due to the increasing number of patients suffering from infertility. As a result, there is a need for the continuous development and/or improvement of assisted reproductive technologies. This paper presents a new method for the in vitro measurement of the amino acid turnover of developing embryos based on capillary electrophoresis with light-emitting diode-induced fluorescence detection. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde/NaCN, and the resulting fluorescent derivatives were baseline resolved within 25 min in a background electrolyte comprised of 50 mM sodium tetraborate, 73 mM sodium dodecyl sulphate, 5 mM sodium deoxycholate and 2.5 mM (2-hydroxypropyl)-ß-cyclodextrin (pH ≈ 9.3). The migration time and the peak area repeatability (n = 10) were below 0.5 and 4.3%, respectively. The limits of detection ranged from 12.6 nM (histidine) to 39.3 nM (taurine). The developed method, which only requires 2 µL of raw sample, was successfully applied for determining the metabolic activity of human embryos exposed to different environmental stress conditions.

Electrophoretically mediated microanalysis for simultaneous on-capillary derivatization of standard amino acids followed by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection.

Amino acids are crucial compounds involved in most biochemical processes essential for life. Since their dynamic turnover reflects the actual physiology of the cell/organism, a turnover assessment may provide valuable information related to multiple physiological and pathophysiological conditions. The sensitive determination of amino acids is predominantly associated with their derivatization which might be laborious, time-consuming and difficult to standardize. However, capillary electrophoresis offers the automatic injection and mixing of reactants, incubation of the reaction mixture, separation and detection of the reaction products in one on-capillary procedure. Among the on-capillary mixing strategies, electrophoretically mediated microanalysis (EMMA) is superior in terms of mixing efficiency. In this paper, we present an optimization of EMMA for the simultaneous derivatization of standard amino acids by naphthalene-2,3-dicarboxaldehyde/NaCN and its application to targeted human embryo metabolomics. For such a purpose, novel separation conditions were developed involving the background electrolyte, comprised of 73mM sodium dodecyl sulfate, 6.7 % (v/v) 1-propanol, 0.5mM (2-hydroxypropyl)-ß-cyclodextrin and 135mM boric acid/sodium hydroxide buffer (pH 9.00). Finally, the optimized EMMA was compared to a fundamentally different mixing strategy, namely the transverse diffusion of laminar flow profiles, and proved to be also suitable for human plasma analysis.

MEKC-LIF method for analysis of amino acids after on-capillary derivatization by transverse diffusion of laminar flow profiles mixing of reactants for assessing developmental capacity of human embryos after in vitro fertilization.

Evaluating the physiological state of an organism is of clinical importance. In assisted reproduction, knowledge of the embryo's physiology is crucial for selecting the embryo with the highest developmental capacity to ensure high pregnancy rates. Amino acids (AAs) are involved in many biochemical processes during embryo development, which means that the determination of AA fluctuations in the embryo's surroundings can determine the embryo's physiological state. Since current embryo selection methods are mainly based on visual assessment, which lacks proper accuracy, a novel method for the analysis of AAs in the embryo's surroundings was developed. AAs were analyzed by means of MEKC-LIF after on-capillary derivatization by naphthalene-2,3-dicarboxaldehyde. The reactants were injected under the three zone arrangement and mixed using the transverse diffusion of laminar flow profiles methodology. The resulting derivatives of all the standard AAs were baseline resolved in the BGE comprised of 35 mM sodium tetraborate, 55 mM SDS, 2.7 M urea, 1 mM BIS-TRIS propane, and 23 mM NaOH within 50 min. The method was applied on an analysis of spent culture media used in assisted reproduction to culture embryos after in vitro fertilization.

On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: A promising tool for drug development.

In this work, the combination of an immobilized enzyme microreactor (IMER) based on the clinically important isoform cytochrome P450 2C9 (CYP2C9) with capillary electrophoresis (CE) is presented. The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method. The formation of an IMER in the inlet part of the separation capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement. The resulting on-line system provides an integration of enzyme reaction mixing and incubation, reaction products separation, detection and quantification into a single fully automated procedure with the possibility of repetitive use of the enzyme and minuscule amounts of reactant consumption. The on-line kinetic and inhibition studies of CYP2C9's reaction with diclofenac as a model substrate and sulfaphenazole as a model inhibitor were conducted in order to demonstrate its practical applicability. Values of the apparent Michalis-Menten constant, apparent maximum reaction velocity, Hill coefficient, apparent inhibition constant and half-maximal inhibition concentration were determined on the basis of the calculation of the effective substrate and inhibitor concentrations inside the capillary IMER using a model described by the Hagen-Poisseulle law and a novel enhanced model that reflects the influence of the reactants' diffusion during the injection process.

Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection.

Human-assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human-assisted reproduction. A fused-silica capillary of 64.0 cm total length and 50 µm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to -24 kV, and capillary temperature to 15°C. The presented method requires only 2 µL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 µM, respectively. The results demonstrated the method's suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo.

On-line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV-absorption detection.

This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.