Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy

Abstract

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic location of the trophoblast.

Figure 1

Expression of CRISP-3 mRNA in uterine decidua; CRISP-3 is differentially expressed. It is increased in women with tubal ectopic pregnancy (Ectopic) when compared with that of women undergoing surgical management of miscarriage (Misc) (P < 0.01; Kruskal–Wallis) and surgical termination of pregnancy (TOP) (P < 0.01; Kruskal–Wallis).

Figure 2

Immunohistochemistry for CRISP-3 in a representative biopsy of decidualized endometrium from a woman with a tubal ectopic pregnancy. Specific immuno-staining for CRISP-3 in all 25 samples was similar and could be detected in the glandular epithelium (A–C), secreted within the glands (A–C) and in the granulocytes of the decidualized endometrium (D). Scale bar = 50 µm.

Figure 3

Representative western blot analysis of CRISP-3 protein in uterine decidua. Western blot analysis revealed bands of 29 and 27 kDa corresponding to N-glycosylated and non-glycoslyated CRISP-3. Miscarriage and TOP groups were combined as they showed a similar pattern. Glycosylated CRISP-3 was increased in the decidualized endometrium from tubal ectopic pregnancy when compared with that from intrauterine pregnancies (P < 0.05; t-test).

Figure 4

Serum CRISP-3 concentrations. Serum samples from patients with tubal ectopic pregnancies (n = 11), miscarriages (n = 6) and viable pregnancies (n = 8) were assayed for CRISP-3 protein. There were no significant differences in measured CRISP-3 concentrations (P > 0.05; ANOVA).

Figure 5

Association between the degree of morphological decidualization, serum progesterone and hCG concentrations and the decidual CRISP-3 mRNA expression. There was no significant association between the degree of morphological decidualization and CRISP-3 mRNA expression (A) (P > 0.05; Kruskal–Wallis). There was no clear association between CRISP-3 mRNA expression and serum progesterone (P > 0.05; Spearman Rank Correlation) (B). Expression of CRISP-3 mRNA in decidualized endometrium was inversely proportional to serum hCG (P = 0.0006; r= −0.64) (C).

Figure 6

Regulation of endometrial epithelial CRISP-3 expression in vitro. At both 6 and 24 h, hCG treatment (100 IU/ml) was associated with a dose dependent decrease in endometrial CRISP-3 mRNA expression (n = 3) (P < 0.01; ANOVA).