The effects of insulin–transferrin–selenium (ITS) and CHIR99021 on the development of pre-implantation human arrested embryos in vitro

Pre-implantation development arrest poses a significant challenge in infertility treatment cycles. This study aims to evaluate the effect of Insulin, Transferrin, Selenium (ITS), and CHIR99021 on arrested human embryos. Arrested human embryos were obtained from the Embryology Department of the Royan Institute. After determining optimal concentrations, the embryos were assigned to control, CHIR99021, and ITS groups and cultured for 48-72 h. The arrest rate significantly decreased in the ITS and CHIR99021 groups compared to the control group (P < 0.05). The developmental rate up to the pre-morula stage significantly increased in the CHIR99021 group compared to the control group (P < 0.05). Additionally, there were significant increases in the expression of SOX2 in the CHIR99021 group and CCNA2 in the ITS group compared to the control group (P < 0.05). Immunofluorescent staining confirmed the expression of NANOG protein in the experimental groups. GSK3 inhibition by CHIR99021 and the application of ITS can alleviate arrest in human embryos, promote cell cycle induction, and enable progression to the blastocyst stage. Comprehensive characterization of these blastocysts in future studies is crucial to support ITS and CHIR99021 probable application in culture systems, particularly for women of advanced maternal age and those experiencing severe male factor infertility.