Indian Hedgehog and its targets in human endometrium: menstrual cycle expression and response to CDB-2914

Abstract

Context: Progesterone is critical for secretory endometrial differentiation in women, but its downstream mediators are poorly understood.

Objective: Our objective was to investigate endometrial expression of Indian Hedgehog (IHH) and genes involved in its signaling [smoothened (SMO), patched-1 (PTCH1), glioma-associated oncogene homolog 1 (GLI1), and GLI2] during the menstrual cycle and the effects of the selective progesterone receptor modulator CDB-2914 on its expression.

Design and setting: Comparisons between normally cycling volunteers and women with symptomatic fibroids who received CDB-2914 or placebo were made at a clinical research center.

Patients and interventions: Endometrial biopsy was performed on 34 volunteers, 17 additional women with fibroids.

Main outcome measures: Endometrial expression of IHH, SMO, PTCH1, GLI1, and GLI2 by in situ hybridization and/or RT-PCR and IHH, GLI1, and PTCH1 immunohistochemistry were evaluated.

Results: RT-PCR showed expression of IHH, SMO, PTCH1, GLI1, and GLI2, with significant increases in IHH (5.2-fold) and GLI1 (3.6-fold) in endometrium exposed to CDB-2914 compared with placebo. In situ hybridization showed IHH mRNA expression in glands and stroma that was stronger in secretory samples. Among volunteers, IHH and GLI1 immunohistochemistry scores were higher in the secretory than proliferative phase in the nuclei and cytoplasm of glands and stroma (P=0.0002-0.04). Compared with follicular-phase controls, women exposed to CDB-2914 showed increased IHH expression in all compartments except stromal cytoplasm (P=0.0199-0.0423); GLI1 was up-regulated in glandular nuclei and cytoplasm compared with both volunteers and women receiving placebo (P≤0.0416).

Conclusions: The temporal increase in endometrial IHH and GLI1 during the secretory phase, and their modulation by CDB-2914, suggests progestin regulation and a potential role in endometrial differentiation and implantation.

Trial registration: ClinicalTrials.gov NCT00044876.

Figures

Figure 1

Relative expression of IHH, PTCH1, SMO, GLI1, GLI2, PR-A&B, and PR-B in proliferative-phase endometrium as determined by RT-PCR. Samples were obtained after 3 months administration of placebo (black bars) or CDB-2914 (white bars). *, Administration of CDB-2914 resulted in differential gene expression of IHH and GLI1 (P < 0.035) compared with placebo.

Figure 2

In situ hybridization for IHH mRNA and IHC for IHH in representative endometrium. A, Purple staining in proliferative-phase glands and stroma with antisense probe; B, lack of staining with sense probe; C, purple staining in secretory glands and stroma with antisense probe; D, IHC for IHH; brown indicates positive staining.

Figure 3

IHC scores for IHH (A) and GLI1 (B) during the menstrual cycle in normally cycling volunteers. Histological dating included early (EP), mid (MP), and late (LP) proliferative phase and early (ES), mid (MS), and late (LS) secretory phase. Numbers represent mean ± se H-scores for nuclei and average stain intensity for cytoplasm. Asterisks indicate a significant difference between one or more proliferative-phase results compared with the late secretory phase (for IHH: glandular nuclei all P < 0.02, glandular cytoplasm P < 0.001 for mid- and late-proliferative and 0.032 for early proliferative; for GLI1: glandular nuclei P ≤ 0.02, glandular cytoplasm P = 0.0018, stromal cytoplasm P < 0.02).

Figure 4

Representative photomicrographs of endometrial IHC results for IHH (A) and GLI1 (B) in a woman treated with CDB-2914 (CDB), in normal cervix (NEG1, negative control), in endometrium without primary antibody (NEG2), and in normally cycling women in the early (EP), mid- (MP), and late (LP) proliferative phase and the early (ES), mid- (MS), and late (LS) secretory phase. Brown indicates positive staining. Magnification, ×100 for all images.

Figure 5

IHC scores for glandular and stroma cytoplasm and nuclei for IHH (A) and GLI1 (B) in normally cycling volunteers (normal) and women with fibroids who received CDB-2914 (CDB) or placebo (PLC) for 3 months. All biopsies had proliferative-phase (Pro) histological dating. Numbers represent mean ± se H-scores for nuclei (nuc) and average stain intensity for cytoplasm (cyto). Asterisks indicate a significant difference between the CDB-exposed specimens compared with either the normal or PLC group.