Role of human chorionic gonadotropin (HCG) on embryo attachment onto oviductal epithelial cells through downregulation of olfactomedin-1 (OLFM1)

Abstract

Tubal ectopic pregnancy (TEP) occurs in 2% of pregnancy and is potentially fatal to pregnant women because of the risk of Fallopian tube/oviduct rupture. The causes of ectopic pregnancy are associated with pelvic infection, tobacco smoking and the use of assisted reproductive technology. However, the underlying mechanism leading to TEP remains largely unknown. Results from recent studies suggested that human chorionic gonadotrophin (hCG) stimulates miRNA expression in female reproductive tract and miRNAs play important roles on various biological processes. In silico analysis identified miRNA-212 may regulate Olfactomedin-1 (Olfm1) expression which was found to be down-regulated in endometrial epithelium during embryo implantation. Therefore, we hypothesized that embryo-derived hCG stimulates miRNA-212 expression, down-regulated Olfm1 expression in the Fallopian epithelium and favor embryo attachment leading to ectopic pregnancy. Furthermore, the effect of hCG binding onto LH/CG receptor (LH/CGR) and the downstream protein kinase and Wnt signaling pathways was investigated.

It was found that Olfm1 was down-regulated and LH/CGR was up-regulated at the luteal phase of the menstrual cycle in the human Fallopian tube. hCG (25IU/ml) down-regulated Olfm1 in human oviductal epithelial cells (OE-E6/E7). Both hCG (25IU/ml) and hCG beta subunit increased JAR spheroids (blastocyst surrogate) attachment rate onto OE-E6/E7 cells. Knockdown of LH/CGR siRNA reduces, while knockdown of Olfm1 siRNA increases spheroids attachment, respectively.

Progesterone (60nM) increased attachment rate and up-regulated the expression of LH/CGR. LH/CGR could activate its downstream signaling pathways through either protein kinase A or protein kinase C. PKA activator (Forskolin) has no effect on spheroid attachment; while PKC activator (PMA) increased attachment rate, suggesting hCG promotes spheroids attachment through PKC signaling pathway. Furthermore, hCG activates Erk and Wnt signaling pathways; while knockdown of LH/CGR abolished the effect of hCG on Erk activation in OE-E6/E7 cells. Similarly, Erk or Wnt activator down-regulated Olfm1, while Erk or Wnt inhibitor up-regulated Olfm1 expression and stimulates spheroid attachment in OE-E6/E7 cells. In line with this, immunohistochemistry of human Fallopian tubes obtained from ectopic pregnancy and normal non-pregnant women showed activation of Erk signaling pathway in the epithelium of TEP samples.

In silico analysis of the promoter region of hsa-miR212 revealed binding site of LEF-1, a Wnt signaling transcriptional factor. Chromatin immunoprecipitaiton showed binding of LEF-1 to the promoter region of hsa-miR212. Hsa-miR212 mimic down-regulated Olfm1 and promoted spheroids attachment; while hsa-miR212 inhibitor up-regulated Olfm1 and suppressed spheroids attachment. To summarize, our results demonstrated that hCG (25IU/ml) act through LH/CGR, activates Erk and Wnt signaling pathways, induces miR-212 expression and down-regulates Olfm1, which favors embryo attached onto oviductal epithelial cells for TEP.