Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen

We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions. Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated beta-galactosidase (SA-beta-gal) and phosphorylated H2A.X (gamma-H2A.X), the latter being a mark of DNA oxidative damage. Expression of senescence-associated genes p21, p16, and interleukin 6 (IL6) were also assessed. Compared with in vivo-derived blastocysts, in vitro embryos had high levels of SA-beta-gal, nuclear gamma-H2A.X, and p21 mRNA expression, indicating that a senescence-like phenotype is induced by in vitro culture. To determine the role of culture conditions, we studied the effect of oxygen (5 % vs 20 %) and protein supplementation on senescence markers. Blastocysts in reduced oxygen (5 %) had low levels of both SA-beta-gal and gamma-H2A.X compared with blastocysts cultured in ambient oxygen. Senescence markers also were reduced in the presence of protein, suggesting that antioxidant properties of protein reduce oxidative DNA damage in vitro. Elevated SA-beta-gal, gamma-H2A.X, and p21 suggest that in vitro stress can induce a senescence-like phenotype. Reduced oxygen during embryo culture minimizes these effects, providing further evidence for potential adverse effects of culturing embryos at ambient oxygen concentrations.