Increased ammonium in culture medium may promote cellular apoptosis and negatively affect pluripotency of human blastocysts.

Abstract

To determine the association between ammonium concentration in culture medium and blastocyst development and to assess the influence of increased ammonium concentration on the expression of Bax, Bcl-2 and Oct4. A total of 254 cleavage-stage embryos were individually cultured in 30μL G2-plus medium on Day 3, and then culture media samples were collected on Day 5 for ammonium concentration determination immediately after evaluating the embryos morphology. Poor-quality blastocysts (combined score of CC) were used for gene expression analysis. The blastocyst formation rate, good-quality blastocyst rate and relative expression levels of Bax, Bcl-2 and Oct4 were analyzed. Based on receiver operating characteristic curve, the cutoff value of ammonium concentration produced by embryos was 16.07μmol/L (AUC = 0.722, 95% CI 0.637-0.807; P = 0.000), so all embryos were assigned to two groups according to the cutoff value: normal group (< 16.07μmol/L) and increased group (≥ 16.07μmol/L). There was a significant difference in blastocyst formation rate (80.5% vs 59.0%, P < 0.01) between normal group and increased group, as well as for good-quality blastocyst rate (21.0% vs 3.4%, P < 0.01). A significantly higher expression level of Bax (P < 0.05) and considerably lower expression level of Oct4 (P < 0.01) were observed in increased group compared to normal group. Our data demonstrated for the first time that increased ammonium concentration in culture medium may promote cellular apoptosis and negatively affect pluripotency of human blastocyst.