Using stem cell oxygen physiology to optimize blastocyst culture while minimizing hypoxic stress

Abstract

This review is a response to the Fellows Forum on testing 2% oxygen for best culture of human blastocysts (J Ass Reprod Gen 34:303-8, 1; J Ass Reprod Gen 34:309-14, 2) prior to embryo transfer. It is a general analysis in support of the position that an understanding of stem cell physiology and responses to oxygen are necessary for optimization of blastocyst culture in IVF and to enhance reproductive success in fertile women.

Keywords: Blastocyst; Hypoxic stress; IVF; Oxygen optimization; Stem cells.

Fig. 1

The mouse blastocyst has two initial lineages, embryonic and placental, with Oct4-dependent FGF4 synthesized in the ICM sustaining the polar trophectoderm, mural trophectoderm initiating differentiation from polar trophectoderm and primitive endoderm allocating from the ICM. a Mitochondrial charge is highest in mural trophectoderm > polar trophectoderm > ICM as indicated by the width of white lines. Lactate is made by mouse and human blastocyst stem cells. b Hypoxic stress diminishes the velocity of the stem cell pipeline by forcing decreases in anabolism, proliferation and potency and by increasing compensatory differentiation and epigenetic changes

Fig. 2

a–d TSCs cultured with FGF4 for 24 h show increased differentiation (Hand1, GCM, TPBPa proteins) and stress (SAPK activity) and decreased potency (Cdx2, Id2, and ErrB nuclear TF proteins) and growth below the optimum O₂ level of 2%. O₂ levels below 2% override FGF4 and cause hypoxic stress (modified from [34]). Note the broken line in the x axis chart frame which indicates that changes from 20 to 2% O₂ are small and large changes from 2 to 0% O₂ occur over small changes in O₂ levels. e TSCs cultured with FGF4 for 48 h larger increases in SAPK-dependent increase in first lineage (Hand1) and suppression of a later lineage (GCM1) at levels of O₂ above (20%) and below (0.5%) the optimum at 2% O₂, where SAPK activity is lowest (Fig. 1c). f Mouse preimplantation embryos were cultured from two cell embryo to blastocyst stage (which is ~80% TSC lineage) for 72 h in seven media and assayed by immunoblot and immunofluorescence for level of SAPK activity. In conclusion, stress/SAPK activity is inversely proportional to cell number and development rates and directly proportional to differentiation rates

Fig. 3

a Although outcomes that depend on long-term cumulative effects like growth are ambiguous in human postcompaction and postthaw culture, instantaneous stress levels indicated by rapid turnover stress genes encoding transcripts are low and similar between 2 and 5% O₂ stimulated blastocysts. Heat shock protein 70.1 (HSP70.1) is a cytoplasmic stress factor, glucose 6 phosphate dehydrogenase (G6PD) and glucose transporter (Glut1) enable energy increase in response to stress, manganese superoxide dismutase (MnSOD) detoxifies reactive oxidative stress species, and Bax mediates stress-induced apoptotic responses (modified after [55]). We anticipate that TSCs at 2% O₂ might be lower than 5% for these stress marker mRNA levels if microfluidics reduced lactate that arises due to high anabolism of stem cells and cause second stress after 6–8 h (b) (modified after [6, 46]). TSCs at 2% O₂ grow most rapidly, but to obtain highest growth media, must be changed every 12 h or TSCs become morbid instead. b indicates that stress enzyme AMPK (which mediates hyperosmotic and genotoxic stress, drug-/diet supplement-induced AMPK antagonist-sensitive potency, growth and anabolism loss in two-cell embryos, blastocysts, TSCs, and ESCs [11, 46, 48]) is highly activated between 6 and 8 h after culture of TSCs in media preequilibrated at optimal 2% O₂