Effect of hydroxychloroquine and chloroquine on syncytial differentiation and autophagy in primary human trophoblasts

During placentation, cytotrophoblasts differentiate and fuse to form multinucleated cells (syncytiotrophoblasts) in a process that involves autophagy. Appropriate syncytial differentiation is essential for establishing a healthy pregnancy. In this study, we evaluated the effect of two chloroquine compounds, hydroxychloroquine (HCQ) and chloroquine (CQ), on syncytial differentiation and autophagy in cultured primary human trophoblasts (PHTs). PHT cells were isolated from the human term placenta. Bafilomycin, a well-known autophagy inhibitor, was used as a positive control. Biochemical and morphological differentiation was assessed in syncytiotrophoblasts, and autophagy-related proteins and genes were evaluated. Affymetrix Human Gene 2.0 ST Array profiling was used to identify genes affected by HCQ during syncytial differentiation. Chloroquine compounds lowered the production of beta-human chorionic gonadotropin (β-hCG) and the fusion index in PHTs. Syncytial differentiation in PHT was associated with the increased expression of ATG4C mRNA (autophagy-related gene), and this expression was affected by CQ but not by HCQ. Microarray analysis revealed that HCQ or CQ affected several genes (MMP15, GPC3, CXCL10, TET-1, and S100A7) during syncytial differentiation, which were different from that of the syncytial differentiation suppression (Ham’s/Waymouth media) or autophagy inhibition (bafilomycin treatment). Using Kyoto Encyclopedia of Genes and Genomes analysis we identified that HCQ might affect JAK2 signaling in the syncytial differentiation of PHT. In conclusion, chloroquine compounds could mitigate biochemical and morphological syncytial trophoblast differentiation in cultured PHT cells through the JAK signaling pathway rather than the inhibition of autophagic activity.