Markers of cellular senescence are higher in in-vitro cultured embryos compared to in-vivo embryos

Cellular senescence is characterized by stable cell cycle arrest triggered by a variety of stresses. Senescent cells are viable and metabolically active. The aim of this study was to test if in-vitro embryo culture induces senescence.

Single-cell mouse embryos from superovulated FVB-mice were cultured in-vitro to blastocysts. In-vivo developed mouse blastocysts were collected from superovulated FVB-mice 4 days post coitum. Blastocysts were stained for senescence-associated-beta-galactosidase (SA-beta-gal) and phosphorylated H2A.X (γ-H2A.X). Blastocysts were SA-beta-gal-positive when blue color was evident. γ-H2A.X-foci were assessed by confocal microscopy and positive when > 5 γ-H2A.X-foci were detected. p21, p16, and Interleukin6 were assayed by qRT-PCR.

SA-beta-gal was positive in 76.7% of in-vitro-blastocysts and 3.3% of in-vivo-blastocysts (P < 0.0001). Nuclear γ-H2A.X was positive in 45.2% of cells of in-vitro-blastocysts and 14.2% of cells of in-vivo-blastocysts (P < 0.0001). p21 mRNA expression was 22.1 fold higher in the in-vitro than the in-vivo group (P < 0.05), whereas p16 could not be detected. Interleukin6 mRNA was 1.2 fold higher in the in-vitro compared to the in-vivo group (P = 0.99).

Fig. 1: SA-β-gal & γ-H2A.X in in-vivo/in-vitro blastocysts

Elevated SA-beta-gal, γ-H2AX, and p21 in cultured embryos compared to in-vivo-embryos suggest that in-vitro stress can induce a senescence-like phenotype. Clinically, markers of senescence could be used to enhance embryo culture conditions.