the vast majority of infants admitted to the neonatal intensive care unit ( nicu ) must acquire the skills for successful oral feeding prior to discharge . for the preterm infant , successful oral feeding involves the maturation and integration of the nervous , sensory , muscular , and digestive systems . failed oral feeding trials can result in an array of morbidities including choking , aspiration , bradycardia , desaturations , feeding aversions , and both short- and long - term impaired neurological outcomes [ 14 ] . further , there is a subset of term infants who have either delayed or unsafe oral feeding skills , resulting in prolonged hospitalization and/or surgical placement of a gastric tube for administration of enteral nutrition . despite the fact that the vast majority of infants in the nicu are at risk for these morbidities , research on neonatal feeding is relatively scarce compared to other complications of prematurity , such as bronchopulmonary dysplasia   and necrotizing enterocolitis , that almost exclusively affect a much smaller percentage of infants born at < 32 weeks ' gestation and/or at extremely low or very low birthweights . currently , newborns rely on the interpretation of subjective feeding cues by their caregivers to determine when it is safe to orally feed [ 8 , 9 ] . our understanding of the complexities of oral feeding maturation in the developing infant at the molecular level is largely unknown . thus , there is an opportunity to incorporate emerging molecular techniques with conventional clinical approaches to improve our understanding of the complexities of oral feeding , and to develop objective diagnostic assays to accurately assess neonatal oral feeding skills . previously , our laboratory described the enormous amount of real - time global developmental information available from premature infants through noninvasive salivary gene expression analyses . although in the initial study we did not specifically target gene transcripts involved in oral feeding , this original discovery - driven research identified key regulatory genes , as well as novel pathways , associated with oral feeding in the preterm infant .   in the two years since this initial work was published , our understanding of the physiological functions of the genes initially identified continues to improve . this has prompted a targeted reexamination of the data to identify previously unrecognized genes in neonatal saliva that may correlate to oral feeding success . identifying these genes and their associated biological and physiological pathways may not only lead to objective , noninvasive salivary biomarkers that accurately predict oral feeding readiness , but may also highlight aberrant developmental pathways that correlate with pathological feeding behavior in the newborn . pertinent clinical information , including medical complications , of each subject can be found in table 1 . salivary samples were collected from each subject from the following time points : ( 1 ) no feeds ; ( 2 ) partial gastric feeds ; ( 3 ) full gastric feeds ; ( 4 ) some oral feeds ; ( 5 ) advanced oral feeds . salivary rna was extracted , amplified , and hybridized onto the affymetrix hg u133 2.0 plus gene expression arrays . all arrays ( n = 25 ) underwent normalization and bioinformatic analysis as previously described . genes that were shown to statistically significantly alter their gene expression over time at a false discovery rate p value of < 0.05 were identified ( n = 9,286 ) . there were 5,764 up - regulated and 3,522 down - regulated genes that met statistical criteria and altered their expression profile as the subjects matured through the feeding stages . for the purpose of this study , the up- and down - regulated gene lists were uploaded into the newest version of ingenuity pathway analysis ( ipa content version : 11904312 , release date 12 - 15 - 2011 ) . ipa then performed a functional analysis of the gene lists with a right - tailed fisher 's exact test to calculate a p value determining the probability that each biological function assigned to that data set was due to chance alone . this analysis provided a comprehensive biological assessment of the gene - gene interactions , gene functions and gene regulation in our data sets . in order to provide a targeted analysis of genes most likely to be related to oral feeding skills , genes that clustered into statistically significantly biological pathways by ipa were only considered if they met the following criteria : ( a ) they were in the five most statistically significantly up- or down - regulated physiological pathways ; ( b ) they were associated with the key terms  feeding ,   digestion ,  and/or  development  in the ipa analysis . thus , genes had to statistically significantly alter their gene expression over time , and be identified by ipa as significantly clustering into pathways believed to be associated with oral feeding skills to be considered in this analysis . genes that met these criteria were further reviewed by the author with the use of ipa , pubmed , and entrezgene to better understand their functions and possible roles in neonatal oral feeding . from 2010 when the data was initially published until present , the functions of 4% of the genes in the original data sets have been modified . as such , those genes are no longer considered valid for previously identified biological functions , canonical pathways , or networks . this recategorization of genes is a direct result of emerging literature and an improved understanding of gene function , gene - gene interaction , and gene regulation . although it is common for gene expression analyses to change over time , the slight difference in gene analysis from this data set has allowed for the identification of genes not previously known to be associated with neonatal oral feeding . the most significant physiologic pathways identified in this analysis highlight the complexity of oral feeding and involve not only tissue and nervous system development , but key pathways involved in feeding behavior . the five most statistically significantly up - regulated pathways were  behavior  ( 10 < p < 10 ) ,  nervous system development the five most statistically significantly down - regulated pathways were  hematological system development and function  ( 10 < p < 10 ) ,  hematopoiesis ( 10 < p < 10 ) , and  cell - mediated immune response for example , within nervous system development , the subcategories  development of cranial nerve there were 1,807 up - regulated genes that met the search criteria ; 379 down - regulated genes were also considered . one of the most novel aspects of both the current and previous analysis is the prominent role of  behavior  in neonatal oral feeding . a subcategory entitled  feeding  was highlighted ( p < 10 ) . genes within this pathway were associated with hyperphagia ,   satiety ,   obesity ,  and  weight gain  ( table 2 ) . this novel pathway suggests that oral feeding in the newborn is neither merely reflexive nor solely dependent upon oral musculature and nervous system development . rather , newborns rely , in part , on complex neurological signaling related to hunger , satiety , and energy expenditure for successful oral feeding . the enormous amount of caloric intake required for such exponential growth is unique to the newborn period of the human lifespan . thus , it is not surprising that the most statistically significant pathway in our analysis is related to biological mechanisms driving feeding behavior .   in the five most significantly up - regulated physiological pathways , new gene functions have also emerged . for example , in the prior study , the development of only the trigeminal nerve ( cranial nerve [ cn ] v ) was highlighted . at the time , we speculated that the development of this nerve , which provides motor innervation to the muscles of mastication , was essential for proper coordination of the suck- and swallow - reflex . in this updated analysis , we not only see upregulation of genes involved in the development of the trigeminal nerve , but also have identified genes involved in the developing facial ( cn vii ) and glossopharyngeal ( cn ix ) nerves ( table 3 ) . each of these nerves is known to be essential for safe coordination of swallowing with respiration , with cn vii innervating the sensory component of the facial mask , and cn ix providing sensory taste fibers to the posterior tongue . in addition , the subcategory  development of cranial nerve  was new to this analysis . here , genes associated with the oculomotor ( cn iii ) and vestibulocochlear ( cn viii ) nerves were identified . there is an upregulation of genes involved in the developing eye ( p < 0.01 ) and ear ( p < 0.02 ) , as well as the olfactory system ( p < 0.01 ) . genes involved in olfactory receptor development were significantly upregulated as infants matured and learned to orally feed . this , too , is of great interest as emerging literature supports the role of the infant 's olfactory system in the establishment of successful breastfeeding . genes involved in the developing nervous , skeletal and muscle systems were also highlighted in this analysis . there was a wide range of nervous system functions including the development of the brain , spine , central nervous system , neurons , neurites , and ganglions . development of striated muscle ( p = 0.009 ) was significantly upregulated over time , as was developing bone ( p = 0.006 ) and cartilage ( p = 0.005 ) . one of the limitations of this study is that neonatal subjects were followed over several weeks while in the nicu . during that time however , by limiting salivary collection to five predefined feeding stages , there was an opportunity to capture , in real - time , gene expression changes directly related to the attainment of safe oral feeding skills . these data support the complexity of oral feeding and further demonstrate how multiple processes involved in oral feeding may be monitored simultaneously in the newborn at the molecular level . of note , statistically significant down - regulated genes were related only to the developing hematological and lymphoid system . there was no obvious link between lymphoid and/or hematological development and oral feeding success that appeared when reviewing the down - regulated pathways and their associated subcategories . while it is possible that the development of the gastrointestinal immune system plays an important role in readiness to feed , our current understanding of these genes prohibits further speculation on their role , if any , in neonatal oral feeding skills . this research demonstrates that with the advent of recent technical advances and high - throughput screening methods , we are able to monitor , in real - time , normal and aberrant developmental processes occurring in the newborn from mere drops of saliva . utilizing this information for the development of noninvasive salivary diagnostic panels is a novel and exciting aspect of translational medicine . indeed , salivary diagnostic platforms are currently in development for adult patients with oral cancer , breast cancer , sjgren 's disease , pancreatic cancer , melanoma , nonsmall cell lung cancer , acute myocardial infarction , diabetes , and ovarian cancer [ 1217 ] . these platforms are designed to be utilized at the bedside in order to give an accurate diagnosis within minutes with the use of point - of - care technology currently in development . while these platforms are clinically important , none targets a neonatal or even a pediatric patient population . yet neonates , with their limited blood volumes and clinical fragility , are the ideal patient population on which to apply this technology . compared to our prior analysis which examined global developmental processes in the newborn , the present study focused solely on genes believed to be involved in the attainment of successful oral feeding skills . this targeted analysis of the data is laying the foundation for the development of a neonatal oral readiness to feed salivary diagnostic platform . for example , our laboratory recently determined that salivary detection of one of the genes identified in this data set , neuropeptide y2 receptor ( npy2r ) , a known hypothalamic regulator of feeding behavior , has a 95% positive predictive value for immature oral feeding skills . a limitation to this biomarker is that if undetected in saliva , it is only 27% accurate in determining a safe and effective oral feeding pattern . however , as this work moves forward , other genes identified in this targeted analysis could be incorporated into a comprehensive platform , not only for the development of an accurate and objective diagnostic assay , but also to improve our understanding of aberrant feeding patterns in the newborn ( figure 1 ) . neonatal salivary transcriptomic analysis provides noninvasive and objective information about the learning process of oral feeding in the newborn at the molecular level . these data further confirm the complexities of oral feeding and suggest that the development of feeding behavior is a novel and essential biological component to successful oral feeding . this research lays the foundation for the development of an objective , noninvasive assay for the determination of readiness to feed in the neonatal population in order to reduce morbidities and improve care and outcomes .
background . 
 the development of safe and effective oral feeding skills in the newborn is complex and may be associated with significant morbidities . 
 our understanding of neonatal oral feeding maturation at the molecular level is limited , providing an opportunity to utilize emerging molecular techniques to accurately assess neonatal oral feeding skills . 
 objective . to identify key regulatory genes in neonatal saliva involved in successful oral feeding . methods . 
 previously , our laboratory identified 9,286 genes in saliva that statistically significantly altered their gene expression as premature newborns gained advanced oral feeding skills . in this report 
 , genes previously identified underwent an updated and targeted pathway analysis with ingenuity pathway analysis ( ipa ) to identify potential candidate genes involved in successful oral feeding . 
 genes were considered if they were in the five most significantly up- and down - regulated physiological pathways and were associated with the keywords  feeding  ,  digestion  and  development  . results . 
 there were 2,186 genes that met criteria . 
 pathways associated with feeding behavior , cranial nerve development , and the development of the nervous , skeletal , and muscular systems were highlighted . 
 discussion . 
 these data provide important insights into the biological processes involved in oral feeding in the newborn at a molecular level and identify novel pathways associated with successful oral feeding .