Chitosan Nanoparticles Cause Pre-and Post-Implantation Embryo Complications in Mice 1 Running Title : Chitosan nanoparticle toxicity in mouse embryos

Embryo development is a complex and tightly controlled process. Nanoparticle injury can affect normal development and lead to malformation or miscarriage of the embryo. However, the risk of these nanoparticles may pose to reproduction are not clear. In this study, chitosan nanoparticles (CSNP) of near-uniform size, in the range of 100 nm, were synthesized and confirmed by particle size analyzer and transmission electron microscopy. Morulae stage embryos exposure to CSNP during in vitro culture caused blastocyst complications, which had either no cavity or a small cavity. Furthermore, CSNP-treated embryos not only showed lower expression of trophectoderm associated genes, but also the pluripotent marker genes. When blastocysts developed in both media without/with CSNP were transferred to recipients, the percentage of blastocysts resulting in viable pups was significantly reduced. These detrimental effects are linked to the reduction of total cell numbers, enhanced apoptosis and abnormal blastocoels forming at the blastocyst stage, indicating that CSNP treatment might have long-term adverse biological effects, in view of pregnancy outcome. Summary: CSNP-treated embryos impairs the mitochondrial function, down regulation of genes responsible for development and eventually it causes structural and functional abnormalities in the placenta.