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

Abstract

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)-β-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.

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

“…Ovarian response was monitored by transvaginal ultrasonography and serum oestradiol. Oocyte collection was performed as previously described [20]. Oocytes were collected by follicular aspiration 36 h after hCG administration.…”

“…A total of 17 amino acids, Tau and Ala-Gln were determined using capillary electrophoresis with fluorescence detection. Sample preparation, derivatization and analysis of amino acids present in the media were done according to the procedure described in detail in a previously published paper [20]. In this procedure, 2 µL of spent cultivation media were mixed with 8 µL of acetonitrile for protein precipitation.…”

Homocyclic o‐dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis

“…More recently, our research group employed NDA/NaCN for the determination of AAs in spent culture media used in assisted reproduction for embryo cultivation in order to assess embryo viability [147, 153, 209]. Both the TDLFP [147] and plug‐plug mode of EMMA [153] on‐capillary derivatization approaches were designed (see Fig.…”

“…Finally, a precapillary derivatization approach was employed and revealed the strong effect of oxygen concentration during cultivation on the metabolic activity of human embryos. The developed separation conditions allowed the separation of all AAs, Tau, and Ala‐Gln dipeptide in 24 min [209].…”

Non-Invasive Human Embryo Metabolic Assessment as a Developmental Criterion

“…In the early stages of development, the tricarboxylic acid (TCA) cycle and its central substrates, pyruvate (Pyr) and lactate (Lac), are the main energy sources, which are replaced by glycolysis and increasingly glucose (Glc) consumption upon the transition from the morula to the blastocyst stage [6,7]. However, most knowledge on this subject has been derived mainly through the noninvasive separate assessment of the depletion/appearance of milieu components, such as AAs [8][9][10][11] and carbohydrates (i.e., Glc and its metabolites, Pyr and Lac) [6,12] in the early cleavage-stage embryos. For the establishment of a more reliable predictive biomarker, this study focused on the simultaneous evaluation of all these metabolites in the blastocyst stage of embryo development using a noninvasive, cost-effective, reproducible, rapid, and efficient strategy.…”