Mouse models of preterm birth: Suggested assessment and reporting guidelines

Preterm birth affects approximately 1 out of every 10 births in the United States, leading to high rates of mortality and long-term negative health consequences. To investigate the mechanisms leading to preterm birth so as to develop prevention strategies, researchers have developed numerous mouse models of preterm birth. However, the lack of standard definitions for preterm birth in mice limits our field\u27s ability to compare models and make inferences about preterm birth in humans. In this review, we discuss numerous mouse preterm birth models, propose guidelines for experiments and reporting, and suggest markers that can be used to assess whether pups are premature or mature. We argue that adoption of these recommendations will enhance the utility of mice as models for preterm birth

Transcriptional profiling of the ductus arteriosus: Comparison of rodent microarrays and human RNA sequencing

DA closure is crucial for the transition from fetal to neonatal life. This closure is supported by changes to the DA’s signaling and structural properties that distinguish it from neighboring vessels. Examining transcriptional differences between these vessels is key to identifying genes or pathways responsible for DA closure. Several microarray studies have explored the DA transcriptome in animal models but varied experimental designs have led to conflicting results. Thorough transcriptomic analysis of the human DA has yet to be performed. A clear picture of the DA transcriptome is key to guiding future research endeavors, both to allow more targeted treatments in the clinical setting, and to understand the basic biology of DA function. In this review, we use a cross-species cross-platform analysis to consider all available published rodent microarray data and novel human RNAseq data in order to provide high priority candidate genes for consideration in future DA studies

Structural and Functional Evidence for the Interaction of Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins with Vitronectin

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and 125iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells

Dual excitation wavelength system for combined fingerprint and high wavenumber Raman spectroscopy

A fiber optic probe-based Raman spectroscopy system using a single laser module with two excitation wavelengths, at 680 and 785 nm, has been developed for measuring the fingerprint and high wavenumber regions using a single detector. This system is simpler and less expensive than previously reported configurations of combined fingerprint and high wavenumber Raman systems, and its probe-based implementation facilitates numerous in vivo applications. The high wavenumber region of the Raman spectrum ranges from 2800-3800 cm-1 and contains valuable information corresponding to the molecular vibrations of proteins, lipids, and water, which is complimentary to the biochemical signatures found in the fingerprint region (800-1800 cm-1), which probes DNA, lipids, and proteins. The efficacy of the system is demonstrated by tracking changes in water content in tissue-mimicking phantoms, where Voigtian decomposition of the high wavenumber water peak revealed a correlation between the water content and type of water-tissue interactions in the samples. This dual wavelength system was then used for in vivo assessment of cervical remodeling during mouse pregnancy, a physiologic process with known changes in tissue hydration. The system shows that Raman spectroscopy is sensitive to changes in collagen content in the fingerprint region and hydration state in the high wavenumber region, which was verified using an ex vivo comparison of wet and dry weight. Simultaneous fingerprint and high wavenumber Raman spectroscopy will allow precise in vivo quantification of tissue water content in the high wavenumber region, paired with the high biochemical specificity of the fingerprint region

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility

Paracrine Signals from the Mouse Conceptus Are Not Required for the Normal Progression of Decidualization

The purpose of this study was to determine whether the conceptus directs the formation of a tight- and adherens-dependent permeability barrier formed by the primary decidual zone and normal progression of decidual cell differentiation during embryo implantation. Four artificial models of decidualization were used, some apparently more physiological than others. The results show that both the formation of the permeability barrier and decidual cell differentiation of three of the artificial models were quite different from that of pregnant uteri. One artificial model of decidualization, namely pseudopregnant animals receiving concanavalin A-coated Sepharose bead transfers on d 2.5 of pseudopregnancy, better recapitulated the decidual changes that occur in the pregnant uterus undergoing decidualization. This included the formation of a primary decidual zone-like permeability barrier and decidual growth. This model also exhibited similar temporal changes of the expression of genes involved in decidualization that are markers of decidual cell differentiation. Overall, the results of this study indicate that some models of inducing decidualization artificially produce responses that are more similar to those occurring in the pregnant uterus, whereas others are quite different. More importantly, the results suggest that concanavalin A-coated Sepharose beads can provide an equivalent stimulus as the trophectoderm to cause the formation of the primary decidual zone permeability barrier

Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4–5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL