Thrombin and interleukin-1beta decrease HOX gene expression in human first trimester decidual cells: implications for pregnancy loss

Abstract

Bleeding or inflammation in early pregnancy may result in pregnancy loss or defective implantation. Their effect on HOX gene expression in first trimester decidua is unknown. Bleeding results in thrombin generation, although infection or inflammation results in production of cytokines typified by Interleukin-1beta (IL-1beta). First trimester decidual cells were pretreated with 17beta estradiol (E(2)), medroxyprogesterone acetate (MPA) or both and subsequently treated with thrombin or IL-1beta. Affymetrix microarray analysis was used to assess the expression of all HOX genes and confirmed using real-time RT-PCR. E(2) or MPA treatment resulted in significant increases in HOXA10 and HOXA11. Subsequent treatment with thrombin resulted in diminished expression of HOXA10 and HOXA9. Treatment with IL-1beta resulted in decreased expression of HOXA1, 3, 9, 10 and 11. HOXA10 expression was reduced by 70% after thrombin treatment (P = 0.018) and by 90% after IL-1beta treatment (P = 0.004). HOXA11 mRNA expression was decreased by 88% after IL-1beta treatment (P < 0.001), but not by thrombin treatment. Decidua was collected at the time of elective termination of pregnancy (n = 10) or surgical treatment of spontaneous pregnancy loss (n = 10). Real-time PCR and western analysis demonstrated decreased HOXA10 and HOXA11 RNA and protein expression in the decidua of spontaneous pregnancy loss compared with that of viable pregnancies. In conclusion, multiple HOX genes are expressed in decidual cells and inhibited by thrombin and IL-1beta. Since HOXA10 and HOXA11 are known to be necessary for successful pregnancy, these findings suggest a molecular mechanism by which bleeding or inflammation may affect pregnancy outcome.

Figure 1

Sex steroid regulation of HOX genes in decidual cells. (A) Microarray analysis showed HOXA10, and HOXA11 expression was increased in first trimester decidual cells treated with E₂ and MPA. Microarray analysis revealed a statistically significant increase in HOX gene expression after treatment with E₂+MPA (EM) compared with vehicle treated controls. Raw microarray data was normalized using the median value of each gene throughout different chips in the same experimental condition. Fold-ratios were derived from comparing normalized data between control and treatment groups. Genes up- or down-regulated more than 2.0-fold by E₂+MPA (EM) were filtered. (B) Real-time RT–PCR analysis. Each experiment was repeated three times in triplicate. Each replicate consisted of cells obtained from a different subject. Shown are HOX mRNA levels normalized to actin. Fold changes from vehicle treated control are shown. (*) designates P < 0.01 compared with control using t test. The error bars are SEM.

Figure 2

IL-1β regulation of HOX genes in decidual cells. (A) Microarray analysis showed HOXA1, 3, 9, 10 and 11 expression were decreased in decidual cells treated with IL-1β. Microarray analysis revealed a statistically significant decrease in HOX gene expression in first trimester decidual cells treated with IL-1β (I) compared with E₂+MPA (EM) vehicle treated controls. Raw microarray data was normalized using the median value of each gene throughout different chips in the same experimental condition. Fold-ratios were derived from comparing normalized data between control and treatment groups. Genes up- or down-regulated more than 2.0-fold by IL-1β are shown. All changes shown are statistically significant in comparison to EM treated controls. (B) Real-time RT–PCR analysis. IL-1β significantly decreased HOXA10 expression in first trimester decidual cells as determined by real-time RT–PCR. In E₂+MPA (EM) vehicle control pretreated cells, 1 mg/ml IL-1β decreased HOXA1, HOXA3, HOXA9, HOXA10 and HOXA11 expression by 100, 84, 88, 90 and 88%, respectively. * = P < 0.001 compared with EM treated controls. The error bars are SEM.

Figure 3

Thrombin regulation of HOX genes in decidual cells. (A) Microarray analysis showed HOXA9 and HOXA10 expression was decreased in decidual cells treated with thrombin. Microarray analysis revealed a statistically significant decrease in HOX gene expression in first trimester decidual cells treated with thrombin compared with E₂+MPA (EM) vehicle treated controls. Raw microarray data was normalized using the median value of each gene throughout different chips in the same experimental condition. Fold-ratios were derived by comparing normalized data between control and treatment groups. Genes up- or down-regulated more than 2.0-fold by 2.5 U/ml thrombin compared with EM controls are shown. (*) indicates P < 0.01 for each comparison. (B) Real-time RT–PCR analysis. Thrombin significantly decreased HOXA9 and HOXA10 expression in decidual cells in vitro as determined by real-time RT–PCR. (A) In E₂+MPA (EM) pretreated cells, thrombin decreased HOXA9 and HOXA10 expression by 53 and 70%, respectively. * = P < 0.001 compared with EM vehicle treated controls. The error bars are SEM.

Figure 4

Decidual HOX gene expression in spontaneous pregnancy loss. (A) Real-time RT–PCR analysis showed that decidual HOXA10 and HOXA11 expression was decreased in spontaneous miscarriage compared with that of elective termination of pregnancy. Decidua obtained from women undergoing surgical treatment for either elective termination of pregnancy (here considered the control group) or spontaneous miscarriage was analyzed for HOXA10 and HOXA11 expression by real-time PCR. Expression of HOXA10 and HOXA11 in the decidua associated with spontaneous pregnancy loss was decreased to 12 and 27%, respectively, compared the expression of each gene in the decidua of a viable pregnancy (each assigned a value of 100%). * = P < 0.001 expression in decidua of spontaneous loss compared with viable pregnancy controls. (B) Western blot analysis demonstrates decreased HOXA10 protein expression in the decidua obtained from spontaneous pregnancy loss compared with that obtained at the time of termination of pregnancy. Representative data is shown. SAB = spontaneous miscarriage.