Early cleaving embryos result in blastocysts with increased aspartate and glucose consumption, which exhibit different metabolic gene expression that persists in placental and fetal tissues

Abstract

Objectives: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC-MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer.

Methods: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts.

Results: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts.

Conclusions: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

Keywords: Amino acids; Embryo; Gene expression; Metabolism; Time-lapse.

Fig. 1

Effect of aspartate concentration on aspartate uptake by in vivo developed blastocysts. a) Rate of aspartate uptake per blastocyst. b) Rate of aspartate uptake per cell. n = 40 blastocysts in total, 5 biological replicates. Data is expressed as mean ± SEM. ^{a, b, c}P < 0.01. ^{a, b, c}Different letters represent significant differences between groups. The blastocyst cell numbers (mean ± SEM) for each group are as follows: 0.1 mM (54.3 ± 2.4), 0.25 mM (57.3 ± 4.0), 0.5 mM (56.7 ±  3.4), 1.0 mM (58.9 ± 3.9), 2.0 mM (41.1 ±  3.9)

Fig. 2

Effect of aspartate concentration on glucose uptake and lactate production by in vivo developed blastocysts. a) Glucose consumption per blastocyst. b) Glucose consumption per cell. c) Lactate production per blastocyst. d) Lactate production per cell. e) Glycolytic rate. n = 54 blastocysts in total, 7 biological replicates. Data is expressed as mean ± SEM. ^bP < 0.05, ^cP < 0.01. ^{a, b, c}Different letters represent significant differences between groups. White bars represent glucose consumption, dark bars represent lactate production and striped bars represent glycolytic rates of embryos. The blastocyst cell numbers (mean ± SEM) for each group are as follows: 0.0 mM (58.1 ± 3.4), 0.1 mM (62.1 ± 3.4), 1.0 mM (62.2 ± 3.8), 10.0 mM (54.1 ± 3.7)

Fig. 3

Aspartate and glucose consumption of in vitro kinetically different embryos. n = 31 blastocysts in total, 4 biological replicates. Data is expressed as mean ± SEM. ^*P < 0.05, white bars represent ‘fast’ cleaving embryos, dark bars represent ‘slow’ cleaving embryos

Fig. 4

Summary of gene expression levels referenced to gene 18S. a) In vitro ‘slow’ blastocysts relative to ‘fast’ blastocysts, n = 4 biological samples per group, where 1 sample consists of 36 blastocysts in total, collected over 12 culture weeks. b) Placental tissue developed from implanted ‘slow’ blastocysts relative to placental tissue developed from implanted ‘fast’ blastocyst, n = 10 biological samples per group. c) Fetal liver tissue developed from implanted ‘slow’ blastocysts relative to fetal liver tissue developed from implanted ‘fast’ blastocysts, n = 10 biological samples per group. #Significantly different genes (P < 0.05). Data is expressed as mean ± SEM

Fig. 5

Schematic diagram of metabolism within a kinetically faster blastocyst. In kinetically faster blastocysts, it was observed that there was an increase in glucose consumption, as well as higher levels of glucose transporter genes (Slc2a1 and Slc2a3), reflecting the considerable energy required for biosynthetic precursors and formation of the blastocoel cavity. Glucose is converted to pyruvate, which can either be oxidised through the tricarboxylic acid cycle (TCA), or converted to lactate via aerobic glycosis. Uniquely, the blastocyst exhibits high levels of aerobic glycolysis, in order to form lactate that may facilitate several key processes involved in implantation. In support of this, high levels of lactate formation were determined in kinetically faster blastocysts [28]. Although high levels of cytosolic NAD⁺ can be generated via conversion of aerobic glycolysis, this is energetically unfavorable, and a second means of generating cytosolic NAD⁺ is through increased malate aspartate shuttle (MAS) activity. Driven by increased levels of aspartate consumption, kinetically faster blastocysts convert aspartate to oxaloacetate via glutamic oxaloacetic transaminase (Got1), of which enzyme levels have been measured to be higher in these blastocysts compared to kinetically slower blastocysts. Oxaloacetate is subsequently converted by malate dehydrogenase to malate, thereby generating cytosolic NAD⁺. This can then be used to increase conversion of glucose to lactate