Regulation of endometrial gland secretome sialylation and its implication in the pathogenesis of early-onset preeclampsia

Abstract

Glycosylation is a post-translational modification that involves the enzymatic attachment of glycans to proteins or other organic molecules. Hormonal regulation of endometrial gland secretome glycosylation during early placentation has been suggested to be crucial for trophoblast functions and placental development. However, direct evidence for the involvement of protein glycosylation in regulating placentation is lacking. Previous studies linked various pregnancy anomalies to the defects of glycosylation in the placenta and uterus. Preeclampsia (PE) is one of these complications, and it is characterized by dysregulated invasion and migration of the trophoblast. Therefore, we hypothesize that the glycosylation of endometrial gland secretome is able to modulate trophoblast-regulatory activity in humans and contributes to PE pathogenesis. Human endometrial organoids (EOs) were derived from the endometrial tissues of patients who underwent in vitro fertilization in the LH +7/8 stage. EOs were treated with sex hormones estrogen (E2), progesterone (P4), and human chorionic gonadotropin (hCG) to mimic the menstrual cycle and early pregnancy environment. The PE and normotensive (NT) EO culture were also established from the decidual tissues of women diagnosed or not diagnosed with PE during their cesarean delivery. EOs were cultured in hormonal conditions mimicking the proliferative phase (E2), secretory phase (E2+P4) and early pregnancy state (E2+P4+hCG). EOs were hormone responsive and hormonal treatments can modulate the sialylation of their secretomes. Especially, the level of α2,6-sialic acid was downregulated in the EO secretome mimicking the early pregnancy state compared with that from the proliferative phase. We found the EO secretomes treated with hormones mimics early pregnant state would promote trophoblast differentiation, invasion, migration and MMP activities compared to those from proliferative phase. The PE EO secretome showed an increase in α2,6-sialylation compared with NT EO secretome. The expression of sialyltransferase ST6GAL1 and ST6GALNAC1 was lower in the early pregnancy state EOs but higher in PE EOs. ST6GALNAC1 is an enzyme that catalyzes the formation of sialyl-Tn antigen. Secretomes from early pregnancy state EOs showed a more promotive effect on trophoblast invasion/migration than that of proliferative phase EOs, whereas PE EO secretomes hindered the trophoblast invasion/migration. These effects could be abolished by the knockout of trophoblastic Siglec-6 (a α2,6-sialic acid receptor). Meanwhile, the blockade of sialyl-Tn antigen – a known ligand for Siglec-6 – could also rescue the inhibitory effect of PE EO secretomes on trophoblast invasion/migration, indicating that the trophoblast-regulatory activity of EO secretome via Siglec-6 was sialyl-Tn antigen dependent. Three sialylated proteins with reported regulatory roles on trophoblast functions were subsequently identified in the secretome of EOs by sialo-proteomic analysis. They were AHSG, S100A9 and MIF. All these glycoproteins have also been shown to be associated with the onset of PE. The sialylated AHSG and S100A9/sialylated S100A9 ratio was also identified as the potential first trimester maternal serum biomarker for early prediction of PE. In conclusion, this project demonstrated that changes in the sialylation of endometrial gland secretome is associated with the alterations in trophoblast functions, which further indicated the importance of secretome glycoprotein sialylation in the PE pathophysiology.