Abstract
Background: The cardiovascular dysfunction in children born after in vitro fertilization (IVF) has been of great concern, in our study, we aim to explore potential molecular mechanisms for such long-term outcomes.Methods:Real-time qPCR was used to test long non-coding RNA MEG3 and endothelium-derived factors such as endothelial nitric oxide synthase (eNOS), endothelin-1(ET1), and vascular endothelial growth factor (VEGF). ELISA was used to determinate levels of the first and second oxidation products of NO (nitrite, nitrate), ET1 and VEGF. Primary HUVECs collected after caesarean section were treated with different estradiol concentrations in vitro. Additionally, knockdown of MEG3 on HUVEC provided further evidence between MEG3 expression and alteration of NO, ET1, VEGF. Then, by using pyrosequencing, we uncovered the methylation status of the MEG3 region.Results: We found that the expression level of MEG3 was higher in human umbilical vein endothelial cells (HUVECs) of IVF offspring than that in spontaneously born offspring. Furthermore, we found decreased expression of eNOS and VEGF along with elevated expression of ET1 in HUVECs from IVF offspring compared to spontaneously born offspring, accompanied by lower secretion of nitrite, VEGF, and higher secretion of ET1 in the umbilical cord serum of IVF offspring. We confirmed the results from in vivo experiments by demonstrating that high estradiol intrauterine environments lead to abnormal expression of MEG3 and endothelium derived factors. Meanwhile, silencing MEG3 expression decreased ET1 expression, and increased nitrite, nitrate, and VEGF secretion, which could account for the effects we observed in vivo. With pyrosequencing technology, we found that elevated expression of MEG3 in IVF offspring derived HUVECs was the result of hypomethylation of the MEG3 promoter.Conclusions: Our results demonstrated that increased expression of MEG3 in IVF-born HUVECs, accompanied by lower secretion of eNOS and VEGF along with higher secretion of ET1, which is closely related with endothelial dysfunction, together provide a potential mechanism addressing high risk of hypertension in IVF offspring.