Abstract
Preterm birth (PTB) is the leading cause of morbidity and mortality in infants <1 year of age. Intrauterine inflammation is a hallmark of preterm and term parturition; however, this alone cannot fully explain the pathobiology of PTB. For example, the cervix undergoes a prolonged series of biochemical and biomechanical events, including extracellular matrix (ECM) remodeling and mechanochemical changes, culminating in ripening. Vaginal progesterone (P4) prophylaxis demonstrates great promise in preventing PTB in women with a short cervix (<25 mm). We used a primary culture model of human cervical stromal fibroblasts to investigate gene expression signatures in cells treated with interleukin-1β (IL-1β) in the presence or absence of P4 following 17β-estradiol (17β-E2) priming for 7–10 days. Microarrays were used to measure global gene expression in cells treated with cytokine or P4 alone or in combination, followed by validation of select transcripts by semiquantitative polymerase chain reactions (qRT-PCR). Primary/precursor (MIR) and mature microRNAs (miR) were quantified by microarray and NanoString® platforms, respectively, and validated by qRT-PCR. Differential gene expression was computed after data normalization followed by pathway analysis using Kyoto Encyclopedia Genes and Genomes (KEGG), Panther, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) upstream regulator algorithm tools. Treatment of fibroblasts with IL-1β alone resulted in the differential expression of 1432 transcripts (protein coding and non-coding), while P4 alone led to the expression of only 43 transcripts compared to untreated controls. Cytokines, chemokines, and their cognate receptors and prostaglandin endoperoxide synthase-2 (PTGS-2) were among the most highly upregulated transcripts following either IL-1β or IL-1β + P4. Other prominent differentially expressed transcripts were those encoding ECM proteins, ECM-degrading enzymes, and enzymes involved in glycosaminoglycan (GAG) biosynthesis. We also detected differential expression of bradykinin receptor-1 and -2 transcripts, suggesting (prominent in tissue injury/remodeling) a role for the kallikrein–kinin system in cervical responses to cytokine and/or P4 challenge. Collectively, this global gene expression study provides a rich database to interrogate stromal fibroblasts in the setting of a proinflammatory and endocrine milieu that is relevant to cervical remodeling/ripening during preparation for parturition.
Highlights
Cervical integrity is crucial for a successful human pregnancy
Cervical stromal fibroblasts were stimulated to elicit the expression of progesterone receptors (Figure 1B)
We conducted follow-up experiments using the identical design with biological replicates to measure by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcripts induced by either IL-1β, P4, or both agents incubated simultaneously
Summary
Throughout most of an uncomplicated gestation, the cervix provides a physical and immune barrier between the interior of the uterus and the vaginal microbiome (Word et al, 2007; Akgul et al, 2014; Vink and Feltovich, 2016). Human and animal studies have yielded a working model for the biomolecular underpinnings of cervical remodeling/ripening (Elovitz and Mrinalini, 2004; Word et al, 2007; Timmons et al, 2010; Yellon, 2017). Progesterone receptor (PR, NR3C3) signaling underpins many of the physiological processes that oppose untimely cervical dilation (Word et al, 2007). Pharmacologic antagonism of the PR by mifepristone (RU-486) promotes cervical ripening in animal models (Chwalisz et al, 1994). Administration of vaginal P4 reduces the incidence of preterm birth in women with a sonographically validated short cervix (Hassan et al, 2011)
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