Progesterone resistance in PCOS endometrium: a microarray analysis in clomiphene citrate-treated and artificial menstrual cycles

Abstract

Context: Polycystic ovary syndrome (PCOS), the most common endocrinopathy of reproductive-aged women, is characterized by ovulatory dysfunction and hyperandrogenism.

Objective: The aim was to compare gene expression between endometrial samples of normal fertile controls and women with PCOS.

Design and setting: We conducted a case control study at university teaching hospitals.

Patients: Normal fertile controls and women with PCOS participated in the study.

Interventions: Endometrial samples were obtained from normal fertile controls and from women with PCOS, either induced to ovulate with clomiphene citrate or from a modeled secretory phase using daily administration of progesterone.

Main outcome measure: Total RNA was isolated from samples and processed for array hybridization with Affymetrix HG U133 Plus 2 arrays. Data were analyzed using GeneSpring GX11 and Ingenuity Pathways Analysis. Selected gene expression differences were validated using RT-PCR and/or immunohistochemistry in separately obtained PCOS and normal endometrium.

Results: ANOVA analysis revealed 5160 significantly different genes among the three conditions. Of these, 466 were differentially regulated between fertile controls and PCOS. Progesterone-regulated genes, including mitogen-inducible gene 6 (MIG6), leukemia inhibitory factor (LIF), GRB2-associated binding protein 1 (GAB1), S100P, and claudin-4 were significantly lower in PCOS endometrium; whereas cell proliferation genes, such as Anillin and cyclin B1, were up-regulated.

Conclusions: Differences in gene expression provide evidence of progesterone resistance in midsecretory PCOS endometrium, independent of clomiphene citrate and corresponding to the observed phenotypes of hyperplasia, cancer, and poor reproductive outcomes in this group of women.

Figures

Fig. 1.

A, Venn diagram showing gene expression differences within each group of patients tested, including PCOScc, PCOSp, and MSE samples. B and C, Relationships between groups of subjects is apparent by hierarchical cluster analysis (B) and PCA (C). In the former, unsupervised grouping of each sample based on gene expression differences correctly sorts the nine samples into their appropriate groups. Using PCA, applied to nine endometrium samples that were characterized by the gene expression of all probes on the Affymetrix chip HG U133 Plus 2.0, there are three groupings, including normal controls (blue), PCOScc (yellow), and anovulatory PCOSp (red).

Fig. 2.

A–C, Immunohistochemistry of MIG6 in proliferative endometrium (A) and secretory endometrium from PCOS (B) and normal (C) subjects. D, HSCORE, as described in Patients and Methods, was used as a semiquantitative measure of tissue expression. Comparison by ANOVA revealed significant difference in MIG6 expression between normal and PCOS endometrium (P < 0.03).

Fig. 3.

GAB1 expression in secretory phase endometrium from controls (A) and PCOScc (B). C, HSCORE comparisons in cycling and PCOS women demonstrated increased expression between the proliferative and normal midsecretory phase and significantly more GAB1 in normal midsecretory samples compared with PCOS endometrium in the midsecretory phase.

Fig. 4.

Regulation of the estrogen signaling pathway inside the nucleus. The figure displays gene expression of cofactors that modulate the ER + estrogen complex inside the nucleus. The first bar, from left to right, represents the fluorescent expression of a particular gene in MSEn, and the second bar in the graph represents PCOScc. Genes represented in red are up-regulated, and genes represented in green are down-regulated in PCOScc relative to MSEn. These values were obtained from the microarray analysis and exported to IPA. Note that each gene represented in the MSEn has the opposite expression in the PCOScc. The genes represented with a graph bar are those that reached a P < 0.05. Among the coactivators are: glucocorticoid receptor-interacting protein 1 (GRIP1); cAMP-response element binding protein (CBP/p300); thyroid hormone receptor-associated protein complex component (TRAP220); peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1); nuclear receptor coactivator 3 (ACTR); SWItch/Sucrose NonFermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (BRG1); E1A binding protein p300 (p300) and K (lysine) acetyltransferase 2B (PCAF); proline glutamate and leucine rich protein 1 (PELP1); and steroid receptor RNA activator (SRA). Several corepressors regulate the activity of ERα either directly or via inhibiting a coactivator. Examples of direct inhibition include receptor-interacting protein (RIP140), silencing mediator of retinoid and thyroid receptors (SMRT), and nuclear receptor subfamily 0, group B, member 1 (DAX-1). Corepressors of ER that inhibit indirectly include SMRT/HDAC1 associated repressor protein (SHARP) and C-terminal binding protein 2 (CTBP2).