Up-regulation of Cyclooxygenase-2 (COX-2) and Prostaglandin E2 receptor 3 (EP3) in the decidua of patients with unexplained recurrent pregnancy losses (RPL)

Subject:

Abnormal inflammation and unbalanced extra cellular matrix (ECM) remodeling in the fetal-maternal interface lead to impaired trophoblasts invasion and therefore failed blastocyst growth, which are a big part of reasons for unexplained recurrent pregnancy losses (RPL). Prostaglandin E2 (PGE2), the main prostanoid synthesized by the placenta, is involved in inflammation and angiogenesis during early pregnancy. In the history of PGE2, the focus has always been on its application in abortion and labor inducing. Few research testified that PGE2 high expression is associated with RPL. Among the four PGE2 receptors, EP3 receptor was demonstrated to participate in inflammatory swelling. It remains unknown as to whether EP3 receptor signaling is involved in unexplained RPL. Aim of our investigation was to analyze whether Cyclooxygenase-2-Prostaglandin E2 receptor 3 (COX-2-EP3) pathway is associated with unexplained RPL.

Methodology:

Immunohistochemistry was applied to analyze the expression of COX-2 and EP3 receptor in placental tissue of normal pregnancies (n = 19) and unexplained RPL patients (n = 19). Double immunofluorescence with specific markers for stroma and trophoblast cells identified COX-2 and EP3 expressing cells in the placenta. Furthermore, the choriocarcinoma cell line JEG-3 was used as the trophoblasts model in vitro and was stimulated with different concentrations of PGE2, sulprostone (EP1/EP3 agonist) and L798,106 (EP3 antagonist)) for 48 hours, 0.1nM, 1nM and 10nM, respectively. The expression of human chorionic gonadotropin (hCG) and progesterone of JEG-3 cells was evaluated by a two-site sandwich immunoassay. Expression of COX-2 and EP3 of JEG-3 cells was additionally determined by Western blotting and expression of PAI-1 was examined by the enzyme-linked immunosorbent assay (ELISA).

Results:

Both COX-2 and EP3 receptor expressions were up-regulated in decidua of unexplained RPL patients compared to the controls, and there was no correlation between them. It is attributed to that COX-2 was predominantly expressed in stromal cells while EP3 receptor was expressed in extravillous trophoblasts. The production of hCG was suppressed by 10nM PGE2 from 16.33 ± 3.30 mIU/mL in the control to 12.20 ± 1.76 mIU/mL (p = 0.046). Progesterone expression was down-regulated by PGE2, sulprostone and L-798,106, at three different concentrations (0.1nM, 1nM or 10nM) after 48 hours' in JEG-3 cells (p = 0.028, each). COX-2 expression increased nearly 7% after treatment with 10nM PGE2 and EP3 expression decreased 32% after treatment with 10nM L-798,106 compared with control group. PAI-1 level was significantly stimulated by sulprostone to 34% higher than in the control group, while no significant change was observed in PGE2 and L798,106 group.

Conclusion:

These results demonstrate that PGE2 combines with EP3 receptor, which induces high expression of plasminogen activator inhibitor type 1 (PAI-1) and low expression of hCG and progesterone. PAI-1 is the main inhibitor of ECM degradation and its high expression hinders trophoblasts invasion through the decidua and maternal uterine spiral arteries, thus contributing to RPL.

Key Words:

unexplained recurrent pregnancy loss, prostaglandin E2, cyclooxygenase-2, prostaglandin E2 receptor 3, plasminogen activator inhibitor type 1.