Effects of pasteurization on osteopontin concentrations in human breastmilk.

BACKGROUND: Osteopontin (OPN) is an important breastmilk protein involved in infant intestinal, immunological, and brain development. However, little is known about how common milk pasteurization and storage techniques affect this important bioactive protein. METHODS: Human milk osteopontin concentration was measured in single-donor fresh (n = 1) or frozen (n = 20) breastmilk, pooled Holder-pasteurized donor breastmilk (n = 11), and a shelf-stable (retort pasteurized) breastmilk product (n = 2) by ELISA. Single-donor breastmilk samples were subjected to pasteurization and/or freezing before measuring osteopontin concentrations. RESULTS: Holder pasteurization of breastmilk resulted in an ∼50% decrease in osteopontin concentration within single-donor samples. Breastmilk from mothers of preterm infants trended toward higher osteopontin concentration than mothers of term infants; however, samples from preterm mothers experienced greater osteopontin degradation upon pasteurization. A commercial breastmilk product that underwent retort pasteurization had lower osteopontin concentration than a Holder-pasteurized pooled breastmilk product. Finally, freezing breastmilk prior to Holder pasteurization resulted in less osteopontin degradation than Holder pasteurization prior to freezing. CONCLUSIONS: Commonly used breastmilk pasteurization and storage techniques, including freezing and Holder pasteurization, decrease the concentration of the bioactive protein osteopontin in human breastmilk. Holder pasteurization reduced osteopontin concentration by an average of 63%, while freezing resulted in an 8-12% decrease. IMPACT: Pasteurization of human breastmilk significantly decreases the concentration of the bioactive protein osteopontin. Use of both pasteurization and freezing techniques for breastmilk preservation results in greater loss of osteopontin. This study presents for the first time an analysis of osteopontin concentrations in single-donor pasteurized milk samples.

A novel role for PGE2-EP4 in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function.

The ductus arteriosus (DA) is a vascular shunt that allows oxygenated blood to bypass the developing lungs in utero. Fetal DA patency requires vasodilatory signaling via the prostaglandin E2 (PGE2) receptor EP4. However, in humans and mice, disrupted PGE2-EP4 signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP4 during development. We used EP4-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP4 signaling in DA development and function. Expression analyses identified EP4 as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP4 as the primary dilator during this period. Chronic antagonism recapitulated the EP4 KO phenotype and revealed a narrow developmental window when EP4 stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP4 KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP4 KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP4 KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP4 KO DA phenotype. Together, our data suggest that EP4 signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP4 KO mice, the postnatal DA exhibits signaling and contractile properties characteristic of an immature DA, including impairments in the first, muscular phase of DA closure, in addition to known abnormalities in the second permanent remodeling phase.NEW & NOTEWORTHY EP4 is the primary EP receptor in the ductus arteriosus (DA) and is critical during late gestation for its development and eventual closure. The "paradoxical" patent DA (PDA) phenotype of EP4-knockout mice arises from a combination of impaired contractile potential, altered signaling properties, and a failure to remodel associated with an underdeveloped immature vessel. These findings provide new mechanistic insights into women who receive NSAIDs to treat preterm labor, whose infants have unexplained PDA.

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.

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

Currently, there is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and its analog mundulone acetate (MA) as inhibitors of in vitro intracellular Ca 2+ -regulated myometrial contractility. In this study, we probed the tocolytic and therapeutic potential of these small molecules using myometrial cells and tissues obtained from patients receiving cesarean deliveries, as well as a mouse model of PL resulting in preterm birth. In a phenotypic assay, mundulone displayed greater efficacy in the inhibition of intracellular-Ca 2+ from myometrial cells; however, MA showed greater potency and uterine-selectivity, based IC 50 and E max values between myometrial cells compared to aorta vascular smooth muscle cells, a major maternal off-target site of current tocolytics. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted concentration-dependent inhibition of ex vivo myometrial contractions and that neither mundulone or MA affected vasoreactivity of ductus arteriosus, a major fetal off-target of current tocolytics. A high-throughput combination screen of in vitro intracellular Ca 2+ -mobilization identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these synergistic combinations, mundulone + atosiban demonstrated a favorable in vitro therapeutic index (TI)=10, a substantial improvement compared to TI=0.8 for mundulone alone. The ex vivo and in vivo synergism of mundulone and atosiban was substantiated, yielding greater tocolytic efficacy and potency on isolated mouse and human myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone 5hrs after mifepristone administration (and PL induction) dose-dependently delayed the timing of delivery. Importantly, mundulone in combination with atosiban (FR 3.7:1, 6.5mg/kg + 1.75mg/kg) permitted long-term management of PL after induction with 30 µg mifepristone, allowing 71% dams to deliver viable pups at term (> day 19, 4-5 days post-mifepristone exposure) without any visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the future development of mundulone as a stand-alone single- and/or combination-tocolytic therapy for management of PL.

Combination pharmacotherapy for patent ductus arteriosus: Rationale and evidence.

While cyclooxygenase inhibitors have been the most common medications used to facilitate earlier closure of patent ductus arteriosus in preterm infants, adverse effects and low efficacy in extremely low gestational age neonates (ELGANs) have highlighted a need for alternative options. Combination therapy with acetaminophen and ibuprofen is a novel strategy for PDA treatment in ELGANs, as it may facilitate higher ductal closure rates via additive action on two separate pathways inhibiting prostaglandin production. Initial small observational studies and pilot randomized clinical trials indicate potentially higher efficacy of the combination regime to induce ductal closure in comparison to treatment with ibuprofen alone. In this review, we examine the potential clinical impact of treatment failure in ELGANs with significant PDA, highlight the biological rationale in support of studying combination therapy, and review the randomized and non-randomized studies to date. With the rising number of ELGANs receiving neonatal intensive care, who are vulnerable to PDA-related morbidities, there is an urgent need for adequately powered clinical trials to systematically investigate the efficacy and safety of combination therapy for PDA treatment.

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

Mildred T. Stahlman: a centenarian who pioneered modern scientific neonatal intensive care.

Neonatology pioneer Mildred (Millie) T. Stahlman celebrated her 100th birthday on July 31, 2022. Her distinguished career at Vanderbilt University Medical Center in Nashville, TN, is reviewed to commemorate this milestone. Stahlman was arguably the first to establish a modern neonatal intensive care unit in 1961, successfully utilizing negative pressure ventilation and umbilical arterial and venous catheters to monitor blood gasses and pH levels. She received early invaluable training in newborn physiology at the Karolinska Institute in Stockholm, Sweden, under John Lind and Petter Karlberg, and at Vanderbilt under Elliot V. Newman. Stahlman also consulted with luminaries Geoffrey Dawes, Donald Barron, and L. Stanley James. As director of the Vanderbilt NICU, she trained 80 fellows from more than 20 countries. The latter 20 years of her career were highlighted by collaborations with Jeff Whitsett. She was the recipient of the AAP Virginia Apgar Award, the APS John Howland Medal, and served as a member of the Institute of Medicine.

In vivo Raman spectroscopy monitors cervical change during labor.

BACKGROUND: Biochemical cervical change during labor is not well understood, in part, because of a dearth of technologies capable of safely probing the pregnant cervix in vivo. The need for such a technology is 2-fold: (1) to gain a mechanistic understanding of the cervical ripening and dilation process and (2) to provide an objective method for evaluating the cervical state to guide clinical decision-making. Raman spectroscopy demonstrates the potential to meet this need, as it is a noninvasive optical technique that can sensitively detect alterations in tissue components, such as extracellular matrix proteins, lipids, nucleic acids, and blood, which have been previously established to change during the cervical remodeling process. OBJECTIVE: We sought to demonstrate that Raman spectroscopy can longitudinally monitor biochemical changes in the laboring cervix to identify spectral markers of impending parturition. STUDY DESIGN: Overall, 30 pregnant participants undergoing either spontaneous or induced labor were recruited. The Raman spectra were acquired in vivo at 4-hour intervals throughout labor until rupture of membranes using a Raman system with a fiber-optic probe. Linear mixed-effects models were used to determine significant (P<.05) changes in peak intensities or peak ratios as a function of time to delivery in the study population. A nonnegative least-squares biochemical model was used to extract the changing contributions of specific molecule classes over time. RESULTS: We detected multiple biochemical changes during labor, including (1) significant decreases in Raman spectral features associated with collagen and other extracellular matrix proteins (P=.0054) attributed to collagen dispersion, (2) an increase in spectral features associated with blood (P=.0372), and (3) an increase in features indicative of lipid-based molecules (P=.0273). The nonnegative least-squares model revealed a decrease in collagen contribution with time to delivery, an increase in blood contribution, and a change in lipid contribution. CONCLUSION: Our findings have demonstrated that in vivo Raman spectroscopy is sensitive to multiple biochemical remodeling changes in the cervix during labor. Furthermore, in vivo Raman spectroscopy may be a valuable noninvasive tool for objectively evaluating the cervix to potentially guide clinical management of labor.

Cyclooxygenase-2 deficiency attenuates lipopolysaccharide-induced inflammation, apoptosis, and acute lung injury in adult mice.

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation.

Mouse models of patent ductus arteriosus (PDA) and their relevance for human PDA.

The ductus arteriosus (DA) is a unique fetal vascular shunt, which allows blood to bypass the developing lungs in utero. After birth, changes in complex signaling pathways lead to constriction and permanent closure of the DA. The persistent patency of the DA (PDA) is a common disorder in preterm infants, yet the underlying causes of PDA are not fully defined. Although limits on the availability of human DA tissues prevent comprehensive studies on the mechanisms of DA function, mouse models have been developed that reveal critical pathways in DA regulation. Over 20 different transgenic models of PDA in mice have been described, with implications for human DA biology. Similarly, we enumerate 224 human single-gene syndromes that are associated with PDA, including a small subset that consistently feature PDA as a prominent phenotype. Comparison and functional analyses of these genes provide insight into DA development and identify key regulatory pathways that may serve as potential therapeutic targets for the management of PDA.

Diagnosis of an Umbilical Vein Aneurysm at 30 Weeks Gestation.

BACKGROUND: Fetal umbilical vein aneurysm is an uncommon anomaly without clear guidelines regarding the management of these pregnancies. Case Presentation. We describe an ultrasound diagnosis of this condition involving a 38-year-old multigravid woman who presented at 30 weeks and 3 days gestation with severe fetal growth restriction, reverse end-diastolic flow on umbilical artery dopplers, elevated ductus venosus doppler, and an umbilical vein aneurysm. Due to nonreassuring fetal assessment in the setting of an umbilical vein aneurysm, she underwent a cesarean delivery with a favorable neonatal outcome. CONCLUSIONS: There are currently no guidelines for the management of an umbilical vein aneurysm. This case demonstrates a successful multidisciplinary approach for creating a plan of care focused on achieving a favorable outcome for a fetus with a large umbilical vein aneurysm. The approach took into account timing of delivery given the potential for fetal morbidity and mortality, while factoring in the risk of prematurity.

Outcomes in infants < 29 weeks of gestation following single-dose prophylactic indomethacin.

BACKGROUND: Prophylactic indomethacin (3 doses) decreases patent ductus arteriosus (PDA) and intraventricular hemorrhage (IVH) in preterm infants. The study aim was to determine whether single-dose indomethacin (SD-INDO) decreases PDA, IVH, and improves motor function. METHODS: A retrospective cohort (2007-2014) compared infants born < 29 weeks who did (n = 299) or did not (n = 85) receive SD-INDO and estimated outcomes association with ordinal logistic regression, adjusting for multiple variables using propensity scores. RESULTS: Infants who received SD-INDO were more premature (p < 0.001) but had lower odds of PDA (OR 0.26 [0.15, 0.44], p < 0.005), PDA receiving treatment (OR 0.12 [0.03, 0.47], p < 0.005), death (OR 0.41 [0.20, 0.86], p = 0.02), and CP severity (OR 0.33 [0.12, 0.89], p = 0.03). There was less IVH (OR 0.58 [0.36, 0.94], p = 0.03) when adjusted for gestational age. CONCLUSIONS: SD-INDO is associated with decreased PDA and CP severity and improved survival.

Association of chorioamnionitis and patent ductus arteriosus in a national U.S. cohort.

OBJECTIVE: To estimate the effect of clinical chorioamnionitis on the risk of patent ductus arteriosus (PDA). STUDY DESIGN: A secondary analysis of all deliveries >23 gestational weeks from the U.S. Consortium on Safe Labor (CSL) study. The primary exposure was a clinical diagnosis of chorioamnionitis, and the outcome was a diagnosis of PDA. Generalized estimating equations with estimated error variance for women with multiple deliveries were utilized. Models adjusted for age, race, region, delivery year, body mass index, infant sex, multiple gestation, mode of delivery, and antenatal corticosteroid exposure. RESULTS: Among 228,438 deliveries, a diagnosis of PDA was more frequent with chorioamnionitis exposure versus without (9.2% vs. 3.0%; OR: 3.25; 95% CI: 2.92-3.62). Chorioamnionitis was associated with higher adjusted odds of PDA (AOR: 2.18; 95% CI: 1.93-2.45). In sensitivity analyses, the association between chorioamnionitis and PDA held after adjustment for gestational age at delivery (AOR: 1.28; 95% CI: 1.13-1.44). CONCLUSIONS: Chorioamnionitis was associated with increased odds of PDA. Robust exposure and outcome ascertainment with careful assessment of confounding is needed to further investigate this epidemiologic association.

Patent Ductus Arteriosus of the Preterm Infant.

Postnatal ductal closure is stimulated by rising oxygen tension and withdrawal of vasodilatory mediators (prostaglandins, nitric oxide, adenosine) and by vasoconstrictors (endothelin-1, catecholamines, contractile prostanoids), ion channels, calcium flux, platelets, morphologic maturity, and a favorable genetic predisposition. A persistently patent ductus arteriosus (PDA) in preterm infants can have clinical consequences. Decreasing pulmonary vascular resistance, especially in extremely low gestational age newborns, increases left-to-right shunting through the ductus and increases pulmonary blood flow further, leading to interstitial pulmonary edema and volume load to the left heart. Potential consequences of left-to-right shunting via a hemodynamically significant patent ductus arteriosus (hsPDA) include increased risk for prolonged ventilation, bronchopulmonary dysplasia, necrotizing enterocolitis or focal intestinal perforation, intraventricular hemorrhage, and death. In the last decade, there has been a trend toward less aggressive treatment of PDA in preterm infants. However, there is a subgroup of infants who will likely benefit from intervention, be it pharmacologic, interventional, or surgical: (1) prophylactic intravenous indomethacin in highly selected extremely low gestational age newborns with PDA (<26 + 0/7 weeks' gestation, <750 g birth weight), (2) early targeted therapy of PDA in selected preterm infants at particular high risk for PDA-associated complications, and (3) PDA ligation, catheter intervention, or oral paracetamol may be considered as rescue options for hsPDA closure. The impact of catheter-based closure of hsPDA on clinical outcomes should be determined in future prospective studies. Finally, we provide a novel treatment algorithm for PDA in preterm infants that integrates the several treatment modalities in a staged approach.

Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness.

BACKGROUND: Indomethacin treatment for patent ductus arteriosus (PDA) is associated with acute kidney injury (AKI). Fenoldopam, a dopamine (DA) DA1-like receptor agonist dilates the renal vasculature and may preserve renal function during indomethacin treatment. However, limited information exists on DA receptor-mediated signaling in the ductus and fenoldopam may prevent ductus closure given its vasodilatory nature. METHODS: DA receptor expression in CD-1 mouse vessels was analyzed by qPCR and immunohistochemistry. Concentration-response curves were established using pressure myography. Pretreatment with SCH23390 (DA1-like receptor antagonist), phentolamine (α -adrenergic receptor antagonist) or indomethacin addressed mechanisms for DA-induced changes. Fenoldopam's effects on postnatal ductus closure were evaluated in vivo. RESULTS: DA1 receptors were expressed equally in ductus and aorta. High-dose DA induced modest vasoconstriction under newborn O2 conditions. Phentolamine inhibited DA-induced constriction, while SCH23390 augmented constriction, consistent with a vasodilatory role for DA1 receptors. Despite this, fenoldopam had little effect on ductus tone nor indomethacin- or O2-induced constriction and did not impair postnatal closure in vivo. CONCLUSION(S): DA receptors are present in the ductus but have limited physiologic effects. DA-induced ductus vasoconstriction is mediated via α-adrenergic pathways. The absence of DA1-mediated impairment of ductus closure supports the study of potential role for fenoldopam during PDA treatment.

CYP2C9*2 is associated with indomethacin treatment failure for patent ductus arteriosus.

Aims: To identify clinical andgenetic factors associated with indomethacin treatment failure in preterm neonates with patent ductus arteriosus (PDA). Patients & Methods: This is a multicenter cohort study of 144 preterm infants (22-32 weeks gestational age) at three centers who received at least one treatment course of indomethacin for PDA. Indomethacin failure was defined as requiring subsequent surgical intervention. Results: In multivariate analysis, gestational age (AOR 0.76, 95% CI 0.60-0.96), surfactant use (AOR 9.77, 95% CI 1.15-83.26), and CYP2C9*2 (AOR 3.74; 95% CI 1.34-10.44) were each associated with indomethacin failure. Conclusion: Age, surfactant use, and CYP2C9*2 influence indomethacin treatment outcome in preterm infants with PDA. This combination of clinical and genetic factors may facilitate targeted indomethacin use for PDA.