Effect of glucose concentration during in vitro culture of mouse embryos on development to blastocyst, success of embryo transfer, and litter sex ratio

Abstract

A high-glucose concentration in the reproductive tract during early development may result in aberrant embryo or fetal development, with effects that could have a greater impact on one sex than the other. Here, we determine if a high-glucose concentration impacts embryo development and pregnancy outcomes in a sex-specific manner in the mouse. Zygotes were cultured in potassium simple optimized medium, which typically contains 0.2 mM D-glucose, with and without additional glucose supplementation to a concentration of 28 mM. Zygote cleavage and blastocyst rate did not differ between treatments, but total and trophectoderm cell counts were reduced in blastocysts cultured in a high glucose. No differences between sexes nor inner cell mass cell number were observed within each treatment. Blastocysts developed in both media were transferred to recipients. The percentage of blastocysts resulting in viable pups was significantly reduced when the blastocysts were cultured in 28 mM glucose (74 ± 4%, controls vs. 55.8 ± 7.1%, 28 mM glucose), but conceptus loss affected both sexes equally as litter sex ratio did not differ between treatments (52.7% and 52.2% males for controls and high glucose, respectively). Pup body weight at birth was higher for males than females, but was not affected by earlier culture in high glucose. In conclusion, in vitro culture in medium with a glucose concentration approximating that of diabetic serum reduces total and trophectoderm cell numbers at the blastocyst stage and conceptus development to term, but these detrimental effects are not sex-specific.

Figure 1

Representative images of blastocyst cell count and sexing. A: 10 μm Z-stack section of a blastocyst. Total cell number were determined based on DAPI nuclei staining (right image), TE cells were detected by antiCDX2 (middle image) and DAPI positive CDX2 negative cells correspond to ICM (left image). B: Result of the PCR sexing of analyzed blastocysts after agarose gel electrophoretic separation. Female embryos exhibited only one band corresponding to Rn18S sequence, whereas males also amplify a Y chromosome-specific product (DYzEms3).