Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast

Abstract

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy.

Figure 1. Heat map showing hierarchical clustering of individual arrays by gene expression.

Hierachical clustering was performed on the decidua from women with ectopic pregnancy (EP) and intrauterine pregnancy (IUP) on entities (genes) using the Pearson Centroid method in Array Assist. The genes used in this comparison (∼14,000) had passed an initial filtering stage to filter out genes with very low absolute levels of expression (<64) in 50% of the arrays. Samples are clustered into 3 groups based on degree of decidualization. The most decidualized (++) IUP cluster together as do the non-decidualized (−) EP. Between these is a mixture of partially decidualized (+) IUP and EP samples. The numbers are the codes for the samples (IUP; 1- is from viable intrauterine [termination of pregnancy] and 2- is from non-viable intrauterine pregnancy [miscarriage]. EP; 3- is from tubal ectopic pregnancy).

Figure 2. Expression of markers of decidualization.

PRL and IGFBP1 are known markers of endometrial decidualization. A) Both PRL (P<0.001) and IGFBP1 (P<0.001) were higher in the decidualized samples (++) (n = 7) than the non-decidualized samples (−) (n = 5). B) In tubal ectopic pregnancy (EP) both PRL (P<0.001) and IGFBP1 (P<0.001) were higher in the partially decidualized samples (+) (n = 6) than the non-decidualized samples (−) (n = 5). C) When standardized for decidualization (+) there was no difference in the expression of PRL or IGFBP1 in samples from EP (n = 6) and intrauterine pregnancy (IUP) (n = 6).

Figure 3. Expression of decidualization-related genes.

Expression of genes in partially decidualized (+) (n = 6) and non-decidualized (−) (n = 5) endometrium from ectopic pregnancy (EP) and in the partially decidualized (+) (n = 6) and decidualized (++) (n = 7) endometrium from intrauterine pregnancy (IUP). A) SCARA5 was higher in partially decidualized EP than non-decidualized EP samples (P<0.01). B) DKK1 was higher in partially decidualized EP than non-decidualized EP samples (P<0.001) and in decidualized IUP than in partially decidualized IUP (P<0.05). In contrast, C) MMP7 (P<0.001) and D) INHBA (P<0.01) were increased in the non-decidualized EP samples when compared to the partially decidualized EP endometrium.

Figure 4. Expression of trophoblast-regulated endometrial genes.

Expression of genes in partially decidualized (+) (n = 6) and non-decidualized (−) (n = 5) endometrium from ectopic pregnancy (EP) and in the partially decidualized (+) (n = 6) and decidualized (++) (n = 7) endometrium from intrauterine pregnancy (IUP). A) CRISP3 was lower (P<0.05) in endometrium from IUP when compared to that from decidualization matched EP as is B) GAST (P<0.01). However both A) CRISP3 (P<0.05) and B) GAST (P<0.001) were increased by partial decidualization in EP. In contrast, C) CSH1 (P<0.05) and D) CGA (P<0.01) were increased in the partially-decidualized IUP samples when compared to the partially decidualized EP endometrium.

Figure 5. The effect of hCG on trophoblast-regulated genes in an endometrial epithelial cell line.

Expression of both A) CSH1 and B) CGA could be detected in hTERT-EEpC cells but the trend to increase after treatment with 10 IU/ml or 100 IU/ml did not reach statistical significance.