Differential regulation of osteopontin and CD44 correlates with infertility status in PCOS patients.

Endometrial receptivity is mediated by adhesion molecules at the endometrium-trophoblast interface where osteopontin (OPN) and CD44 form a protein complex that plays an important role in embryo recognition. Here, we undertook a prospective study investigating the expression and regulation of OPN and CD44 in 50 fertile and 31 infertile ovulatory polycystic ovarian syndrome (PCOS) patients in the proliferative and secretory phases of the natural menstrual cycle and in 12 infertile anovulatory PCOS patients. Endometrial biopsies and blood samples were evaluated for expression of OPN and CD44 using RT-PCR, immunohistochemistry and ELISA analysis to determine circulating levels of OPN, CD44, TNF-α, IFN-γ and OPN and CD44 levels in biopsy media. Our findings highlighted an increased level of circulating OPN and CD44 in serum from infertile patients that inversely correlated with expression levels in endometrial tissue and positively correlated with levels secreted into biopsy media. OPN and CD44 levels positively correlated to each other in serum and media from fertile and PCOS patients, as well as to circulating TNF-α and IFN-γ. In vitro analysis revealed that hormone treatment induced recruitment of ERα to the OPN and CD44 promoters with a concomitant increase in the expression of these genes. In infertile patients, inflammatory cytokines led to recruitment of NF-κB and STAT1 proteins to the OPN and CD44 promoters, resulting in their overexpression. These observations suggest that the endometrial epithelial OPN-CD44 adhesion complex is deficient in ovulatory PCOS patients and displays an altered stoichiometry in anovulatory patients, which in both cases may perturb apposition. This, together with elevated circulating and local secreted levels of these proteins, may hinder endometrium-trophoblast interactions by saturating OPN and CD44 receptors on the surface of the blastocyst, thereby contributing to the infertility associated with ovulating PCOS patients. KEY MESSAGES: • Endometrial epithelial OPN-CD44 adhesion complex levels are deficient in ovulatory PCOS patients contributing to the endometrial infertility associated with ovulating PCOS patients. • Circulating levels of OPN, CD44 and inflammatory cytokines TNF-α and IFN-γ are altered in infertile PCOS patients. • Increased levels of both OPN and CD44 in biopsy media and serum inversely correlate with endometrial expression of these markers in endometrial tissue. • In infertile PCOS patients, high levels of oestrogens and inflammatory cytokines stimulate the recruitment of transcription factors to the OPN and CD44 promoters to enhance gene transcription. • Our study identifies a novel crosstalk between the CD44-OPN adhesion complex, ERα, STAT1 and NF-κB pathways modulating endometrial receptivity.

Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors.

The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways, that yield a cytokine output in response to pathogenic stimuli, have been postulated to link infection at the maternal-fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll-like receptors (TLR-3, -7, -8 and -9), and retinoic acid-inducible gene 1 (RIG-I)-like receptors [RIG-I, melanoma differentiation-associated protein 5 (MDA5) and Laboratory of Genetics and Physiology 2(LGP2)] in human term gestation-associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)-6 and/or IL-8 production in response to specific agonists for TLR-3 (Poly(I:C); low and high molecular weight), TLR-7 (imiquimod), TLR-8 (ssRNA40) and RIG-I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR-9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL-8 response, while the choriodecidua and amnion generate a more similar IL-6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR-3, TLR-7, TLR-8 and RIG-1/MDA5 is a broad feature of human term gestation-associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes.

Reflux composition influences the level of NF-κB activation and upstream kinase preference in oesophageal adenocarcinoma cells.

Oesophageal adenocarcinoma (OA) incidence is rising and prognosis is poor. Understanding the molecular basis of this malignancy is key to finding new prevention and treatment strategies. Gastroesophageal reflux disease is the primary cause of OA, usually managed with acid suppression therapy. However, this often does little to control carcinogenic bile acid reflux. The transcription factor nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of OA and its activity is associated with a poor response to chemotherapy, making it an attractive therapeutic target. We sought to decipher the role of different bile acids in NF-κB activation in oesophageal cell lines using short, physiologically relevant exposure times. The effect of an acidic or neutral extracellular pH was investigated concurrently, to mimic in vivo conditions associated with or without acid suppression. We found that some bile acids activated NF-κB to a greater extent when combined with acid, whereas others did so in its absence, at neutral pH. The precise composition of an individual's reflux, coupled with whether they are taking acid suppressants may therefore dictate the extent of NF-κB activation in the oesophagus, and hence the likelihood of histological progression and chemotherapy success. Regardless of pH, the kinase inhibitor of κB kinase was pivotal in mediating reflux induced NF-κB activation. Its importance was confirmed further as its increased activation was associated with histological progression in patient samples. We identified further kinases important in acid or bile induced NF-κB signalling in oesophageal cells, which may provide suitable targets for therapeutic intervention.

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are important mediators of reflux-induced cell signalling in esophageal cells.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has been implicated in both DNA damage induction and aberrant cell signalling in various tissue and cell backgrounds. We investigated here the role of iNOS and NO in DNA damage induction and nuclear factor-kappa B (NF-κB) signalling in esophageal cells in vitro. As esophageal adenocarcinoma develops in a background of Barrett's esophagus secondary to reflux disease, it is possible that inflammatory mediators like NO may be important in esophageal cancer development. We show that reflux components like stomach acid and bile acids [deoxycholic acid (DCA)] can induce iNOS gene and protein expression and produce NO generation in esophageal cells, using real-time PCR, western blotting and NO sensitive fluorescent probes, respectively. This up-regulation of iNOS expression was not dependent on NF-κB activity. DCA-induced DNA damage was independent of NF-κB and only partially dependent on iNOS and NO, as measured by the micronucleus assay. These same reflux constituents also activated the oncogenic transcription factor NF-κB, as measured by transcription factor enzyme-linked immunosorbent assay and gene expression studies with NF-κB linked genes (e.g. interleukin-8). Importantly, we show here for the first time that basal levels of NF-κB activity (and possibly acid and DCA-induced NF-κB) are dependent on iNOS/NO and this may lead to a positive feedback loop whereby induced iNOS is upstream of NF-κB, hence prolonging and potentially amplifying this signalling, presumably through NO activation of NF-κB. Furthermore, we confirm increased protein levels of iNOS in esophageal adenocarcinoma and, therefore, in neoplastic development in the esophagus.