Randomized controlled trial of low (5%) versus ultralow (2%) oxygen for extended culture using bipronucleate and tripronucleate human preimplantation embryos

Extended culture in ultralow (2%) oxygen significantly improved the blastocyst yield among sibling bipronucleate and tripronucleate human embryos and altered utilization of key anabolic amino acids and metabolites involved in redox homeostasis.

Volume 109, Issue 6, Pages 1030–1037.e2


Daniel J. Kaser, M.D., Berhan Bogale, B.S., Vishnudas Sarda, M.B.B.S., M.P.H., Leslie V. Farland, Sc.D., Paige L. Williams, Ph.D., Catherine Racowsky, Ph.D.



To determine whether [1] exposure of embryos to 5% oxygen (O2) from day 1 (D1) to D3, and then to 2% O2from D3 to D5, improves total blastocyst yield, as compared with continuous exposure to 5% O2; and [2] extended culture in 2% O2 alters key metabolic processes and O2-regulated gene expression in human preimplantation embryos.


Randomized controlled trial.


Academic medical center.


Bipronucleate and tripronucleate embryos donated for research.


On D1, sibling zygotes were randomized to culture in 5% O2 from D1 to D5 (n = 102; “5% group”) or 5% O2from D1 to D3, then 2% O2 from D3 to D5 (n = 101, “2% group”).

Main Outcome Measure(s)

Developmental stage and grade; D5 total cell counts; mass spectrometry of spent media; quantitative polymerase chain reaction of 21 genes in inner cell mass and trophectoderm.


Among cleaved embryos (n = 176, 87%), those in the 2% group were less likely to arrest at the cleavage stage on D5 (34 of 87, 39.1%) compared with the 5% group (52 of 89, 58.4%) (adjusted odds ratio 0.38, 95% confidence interval 0.18–0.80). Those in the 2% group were more likely to blastulate (35 of 87, 40.2%) than those in the 5% group (20 of 89, 22.5%) (adjusted odds ratio 2.55, 95% confidence interval 1.27–5.12). Culture in 2% O2 was associated with significantly fewer cells in early and advanced blastocysts, alteration in relative abundances of anabolic amino acids and metabolites involved in redox homeostasis, and differential expression of MUC1 in trophectoderm.


These findings provide foundational evidence for future investigation of 2% O2 as the preferred O2 tension for extended culture of human embryos.

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