Objectives To evaluate salivary biomarkers that elucidate the molecular mechanisms by which in utero opioid exposure exerts sex-specific effects on select hypothalamic and reward genes driving hyperphagia, a hallmark symptom of infants suffering from neonatal opioid withdrawal syndrome (NOWS). Study design We prospectively collected saliva from 50 newborns born at ³34 weeks of gestational age with prenatal opioid exposure and 50 sex-and gestational age-matched infants without exposure. Saliva underwent transcriptomic analysis for 4 select genes involved in homeostatic and hedonic feeding regulation (neuropeptide Y2 receptor [NPY2R], proopiomelanocortin [POMC], leptin receptor [LEPR], dopamine type 2 receptor [DRD2]). Normalized gene expression data were stratified based on sex and correlated with feeding volume on day of life 7 and length of stay in infants with NOWS requiring pharmacotherapy. Results Expression of DRD2, a hedonistic/reward regulator, was significantly higher in male newborns compared with female newborns with NOWS (D threshold cycle 10.8 AE 3.8 vs 13.9 AE 3.7, P = .01). In NOWS requiring pharmacotherapy expression of leptin receptor, an appetite suppressor, was higher in male subjects than female subjects (D threshold cycle 8.4 AE 2.5 vs 12.4 AE 5.1, P = .05), DRD2 expression significantly correlated with intake volume on day of life 7 (r = 0.58, P = .02), and expression of NPY2R, an appetite regulator, negatively correlated with length of stay (r = À0.24, P = .05). Conclusions Prenatal opioid exposure exerts sex-dependent effects on hypothalamic feeding regulatory genes with clinical correlations. Neonatal salivary gene expression analyses may predict hyperphagia, severity of withdrawal state, and length of stay in infants with NOWS.
Introduction The BCRP/ABCG2 efflux transporter protects the developing fetus by limiting the transplacental transfer of drugs and chemicals and prevents the apoptosis of trophoblasts. The purpose of this study was to determine whether hypoxia-related signaling alters placental BCRP expression and function in vitro and in human pregnancies. Methods Human BeWo choriocarcinoma cells were treated with the hypoxia mimetic, cobalt chloride (CoCl2), or 3% oxygen for 24-48 h. Activation of HIF-1α signaling and regulation of BCRP was assessed using qPCR, ELISA, western blotting and a fluorescent substrate transport assay. In addition, healthy term placentas from high altitude pregnancies with chronic hypoxia were assessed for BCRP expression. Results CoCl2 and 3% oxygen increased HIF-1α protein signaling and decreased the mRNA and protein expression of BCRP by 30-75% in BeWo cells. Reduced BCRP expression corresponded with impaired efflux activity during hypoxia as evidenced by accumulation of the substrate Hoechst 33342. A number of transcription factors known to regulate BCRP, including AHR, NRF2 and PPARγ, were also coordinately down-regulated by 3% oxygen in BeWo cells. Moreover, women who gave birth at a high altitude (3100 m) exhibited signs of chronic placental hypoxia, including enhanced protein expression of the HIF-1α target GLUT1, and had reduced BCRP levels in microvillous membranes compared to women at a moderate altitude (1600 m). Discussion This study provides novel insight into the regulation of the placental BCRP transporter by hypoxia, which may be important for exposure of the fetus to chemicals during early development and in hypoxia-related pregnancy disorders.
Non-invasive techniques to monitor and diagnose neonates, particularly those born prematurely, are a long-sought out goal of Newborn Medicine. In recent years, technical advances, combined with increased assay sensitivity, have permitted the high-throughput analysis of multiple biomarkers simultaneously from a single sample source. Multiplexed transcriptomic and proteomic platforms, along with more comprehensive assays such as RNASeq, allow for interrogation of ongoing physiology and pathology in unprecedented ways. In the fragile neonatal population, saliva is an ideal biofluid to assess clinical status serially and offers many advantages over more invasively obtained blood samples. Importantly, saliva samples are amenable to analysis on emerging proteomic and transcriptomic platforms, even at quantitatively limited volumes. However, biomarker targets are often degraded in human saliva, and as a mixed source biofluid containing both human and microbial targets, saliva presents unique challenges for the investigator. Here, we provide insight into technical considerations and protocol optimizations developed in our laboratory to quantify and discover neonatal salivary biomarkers with improved reproducibility and reliability. We will detail insights learned from years of experimentation on neonatal saliva within our laboratory ranging from salivary collection techniques to processing to downstream analyses, highlighting the need for consistency in approach and a global understanding of both the potential benefits and limitations of neonatal salivary biomarker analyses. Importantly, we will highlight the need for robust and stringent research in this population to provide the field with standardized approaches and workflows to impact neonatal care successfully.
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