Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02696343 |
| Other study ID # |
UNL IRB Project ID #15446 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
July 13, 2016 |
| Est. completion date |
March 31, 2023 |
Study information
| Verified date |
July 2023 |
| Source |
University of Nebraska Lincoln |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Two innovative approaches, pulsatile orocutaneous entrainment of non-nutritive suck via
orosensory entrainment (NTrainer) device technology and serial salivary gene expression
analyses, will be merged to examine the relation between gene expression, oral somatosensory
stimulation, feeding behavior, and neurodevelopmental outcomes at 18 months corrected age
(CA) on 180 extremely preterm infants [EPIs] (24 0/7-26 6/7 GA and 27 0/7 - 28 6/7 GA)
enrolled at three neonatal intensive care units: Catholic Health Initiative (CHI) Health St.
Elizabeth (Lincoln, NE), Tufts Medical Center (Boston, MA), and Santa Clara Valley Medical
Center (San Jose, CA). EPIs will be randomized to a blind pacifier (SHAM) or PULSED NTrainer
treatment groups, and stratified by GA, sex, and bronchopulmonary dysplasia status (BPD vs
non-BPD). We hypothesize that the combination of the NTrainer® intervention for improved oral
feeding skills, along with objective salivary gene expression data to monitor response to
treatment and feeding development, will result in a novel, objective, and personalized
approach to neonatal oral feeding and reduce the duration of time to attain oral feeds while
improving feeding, growth and neurodevelopmental outcomes at 18 months' CA.
Description:
The purpose of this study is to combine molecular and behavioral methods in an experimental
conceptualization approach to map the effects of trigeminal (PULSED orocutaneous)
somatosensory stimulation on salivary gene expression in the context of the acquisition of an
exquisitely complex oromotor skill, namely oral feeding, in high-risk human neonates. The
aims of this study represent the first attempt to combine non-invasive treatment strategies
and transcriptomic assessments of high-risk EPIs to (1) improve oral feeding behavior and
skills, (2) further our understanding of the gene ontology of biologically diverse pathways
related to oral feeding, (3) use gene expression data to personalize neonatal care and
individualize treatment strategies and timing interventions, and (4) improve long-term
developmental outcomes. This integrative approach to oral feeding is a paradigm shift in
neonatal care where rapidly emerging technological advances and personalized transcriptomics
can safely and non-invasively be brought to the neonatal bedside to inform medical care
decisions and improve care and outcomes.
Methods Participants: Only EPIs born between 24 0/7 and 28 6/7 weeks' gestation, as
determined by obstetric ultrasound at < 15 weeks or last menstrual period, are eligible to
participate in this study. We will actively enroll EPIs once they have a corrected PCA of ≥
29 weeks to limit the number of infants who develop serious sequelae of prematurity and would
not be eligible for this study based upon the criteria listed below. Parents of enrolled
subjects will receive a Babies R Us $50 gift card for their participation. Subjects will be
recruited from four neonatal intensive care units including 1) CHI Health St. Elizabeth
[Lincoln, NE], 2) Tufts Medical Center NICU [Boston, MA], 3) Santa Clara Valley Medical
Center [San Jose, CA], and 4) Childrens Hospital Orange Country [Los Angeles, CA] by a study
site PI/Co-Investigator or the neonatal study coordinator. Informed consent will be obtained
prior to participants' entry into the study, following consultation with the attending
physician and nurse(s). A total of 180 EPIs (approximately equal numbers of males and
females, no exclusion based on race/ethnicity) will participate in the study (power >.85,
Type I error <.05).
Exclusion criteria: EPIs will not be recruited for this study if they have any of the
following: 1) chromosomal and congenital anomalies including craniofacial malformation,
nervous system anomalies, cyanotic congenital heart disease, gastroschisis, omphalocele,
diaphragmatic hernia and/or other major gastrointestinal anomalies; 2) congenital infection,
3) no documented GA, 4) severe IUGR (3%), 5) abnormal neurological status including head
circumference < 10th or > 90th percentile, intracranial hemorrhage grades III and IV,
seizures, meningitis, neurological examination showing abnormal tone or movements of all
extremities for PCA; 6) history of necrotizing enterocolitis (stage II and III); and, 7)
culture-positive sepsis at the time of study enrollment.
Protocol: EPIs will be stratified among two gestational age groups (24 0/7 - 26 6/7 weeks,
and 27 0/7 - 28 6/7 weeks). Each infant will be randomized to receive either the PULSED
NTrainer or SHAM intervention using a software random integer function in Minitab v.17
(www.minitab.com). Infants assigned to the PULSED NTrainer group will receive a progressive
dose of the pulsatile orocutaneous stimulation. Beginning at 30 weeks' PCA, these infants
will receive 2 weeks of low-dose PULSED NTrainer stimulation (2 x 3-minute blocks) with a
1-minute stimulus 'off-period' between the stimulation blocks. This form of stimulation will
be given simultaneous with tube feedings 3 times/day. The stimulus dose will then be
increased over the next 2 weeks (3 x 3-minute blocks of PULSED NTrainer stimulation) with a
1-minute stimulus 'off-period' between the stimulation blocks, also given simultaneously with
tube feedings 3 times/day. EPIs randomized to the SHAM condition will be given a regular
silicone pacifier during tube feedings over the same time period.
A 1-minute rest period (no stimulation) occurs between stimulation blocks. Criteria for
initiation of orocutaneous therapy include the following: 1) stable vital signs and not on
continuous vasopressor medications; 2) tolerating enteral feeds in previous 48 hours; and 3)
not intubated and mechanically ventilated. If the infant is on nasal intermittent positive
pressure ventilation, continuous positive airway pressure or nasal cannula >2 liters per
minute, then the FiO2 must be <40%.
NNS Assessment and Automated NNS Digital Signal Processing and Feature Extraction: In
addition to the oral stimulation interventions (PULSED NTrainer vs. SHAM pacifier), all study
infants will be assessed 3 times/week (e.g., M/W/F) for NNS performance. The NTrainer System
will be used in 'assessment mode' to record the compression dynamics of NNS during a 3-minute
session to immediately precede a tube feeding that is not associated with an intervention
condition.
The most active 2-minute period of NNS behavior based on suck cycle count is automatically
extracted from each suck assessment data file using an automated waveform feature extraction
algorithm on the NTrainer System. Pressure peaks > 1.6 cm H2O are subjected to feature
extraction criteria, including suck cycle symmetry, cycle duration, and burst identification
defined as two or more NNS events occurring within 1200 ms. This algorithm permits objective
identification of NNS burst activity distinct from non-NNS mouthing compressions or tongue
thrusts against the pacifier. Four measures will be objectively extracted from the indexed
records of suck compression, including minute-rates for (1) NNS Bursts where an individual
burst includes 2 or more suck cycles, (2) NNS Cycle Events defined as suck compression cycles
with cycle periods less than 1200 ms, (3) Total Oral Compressions defined as the sum of all
pressure events, and (4) NNS compression pressure expressed in cm H2O.
PO Feeding Advancement: EPIs will advance on a standardized cue-based feeding schedule
utilized by each site NICU, known as Infant Driven Feeding® that will lead to full nipple
feeds. This standardization of oral feeding advancements across sites will limit confounders
that may ultimately skew the data.
Saliva Collection, Processing, and Gene Expression Analysis: Saliva Collection: Saliva
samples will be collected from all enrolled infants on the first day prior to receiving
either the SHAM or PULSED NTrainer intervention. This sample will serve as our baseline gene
expression profile. Samples will then be obtained twice per week (~ 3 day intervals) up to
the time of achievement of full oral feeds or discharge home from the hospital with either a
nasogastric tube (NG) or gastronomy tube.
Saliva samples at each site will be collected with techniques that have been optimized and
validated through the Maron laboratory. Briefly, saliva samples will be collected with a 1 mL
syringe, end caps removed and attached to low wall suction. Saliva will be collected for
approximately 20 seconds and immediately stabilized in 500 μL of Qiagen's RNA Protect Saliva
to halt gene expression changes, inhibit destructive RNases, and limit microbial overgrowth.
Samples will be vortexed briefly and frozen at -20 deg C. Once frozen, samples are stable for
up to 18 months prior to the need for total RNA extraction. Thus, samples from Nebraska and
Santa Clara will be shipped once a month on ice overnight to the Maron laboratory at the
Mother Infant Research Institute at Tufts Medical Center in Boston, MA for processing.
Generation of a Saliva Biobank: One additional saliva sample will be obtained each week from
all enrolled subjects to generate a biobank repository. Banking an additional sample each
week from study subjects will ensure that we will limit loss of data points. We anticipate
that there are informative salivary biomarkers of oral feeding maturation that have yet to be
identified. By generating a biobank of additional saliva samples that may be retrospectively
interrogated on either a microarray gene expression platform or with RNASeq, we will have the
potential to discover novel hypotheses about gene-gene interactions and/or transcriptional
regulatory processes related to oral feeding in the EPI population.
Total RNA Extraction: Salivary samples will extracted for total RNA using established
protocols optimized in the Maron laboratory. RNA extraction will occur with the QIAGEN
RNAProtect Saliva Mini kit per manufacturer's instructions. On column DNase treatment will be
performed for each sample to eliminate genomic DNA contamination. Samples that are part of
the biobank will be frozen at -80°C pending need for future analysis. Samples that will be
used for RT-qPCR experiments will first undergo cDNA conversion with the Life Technologies
SuperScript Vilo kit per manufacturer's instructions. cDNA will be stored at -80°C in the
Mother Infant Research Institute at Tufts Medical Center pending gene expression analysis.
Gene Expression Analysis: For the purpose of this study, we will interrogate each sample for
nine genes, six target genes of interest and three reference genes for quality control and
potential normalization of gene expression data. Genes to be analyzed in this study have been
previously shown to be directly linked to oral feeding in the newborn and include PLXNA1,
PLXNA3, NPY2R, WNT3, AMPK, and NPHP4. The three reference genes include GAPDH, HPRT1, and
YWHAZ, all of which have been shown to maintain stable gene expression across advancing PCA.
All cDNA samples will undergo a targeted pre-amplification with a custom TaqMan® PreAmp
Master Mix for all nine genes. This targeted approach to amplification will ensure that only
those genes of interest will be amplified and not all genes across the transcriptome which
may introduce bias. Preamplified cDNA will then undergo PCR with TaqMan Fast Advance Master
Mix on the Life Technologies Quant Studio 7 Flex Real-Time PCR System. This instrument is
housed with the Mother Infant Research Institute at Tufts Medical Center and is an advanced
state-of-the-art PCR machine and software system. All efforts will be made to adhere to the
Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)
guidelines for this study. All samples will be run in duplicate with appropriate positive and
negative controls.
Initial Gene Expression Analyses: All gene expression data will be normalized on the Quant
Studio 7 Flex Real-Time PCR System. Only samples that have successful amplification of all
three reference genes will be considered in the analysis. Previously, genes have been
informative in a binary fashion (+/- expression). However, for the purpose of this study, we
will be prepared to calculate relative gene expression of each target gene with the delta
delta cycle threshold (ΔΔCt) method. In accordance to the MIQE guidelines, we will use the
three reference genes for normalization and determine a geometric mean of their Ct in each
sample to calculate relative gene expression.
Neurodevelopmental Follow-Up: As part of standard of care, each site will complete the Bayley
Scales of Infant and Toddler Development 3rd Edition, on EPIs at 18 to 24 months' corrected
age (CA). The primary subtests of interest include Fine Motor, Gross Motor, Cognition, and
Language. These data will be recorded, analyzed, and correlated with feeding outcomes in the
neo-and post-natal period, gene expression data, and intervention status. In addition, growth
parameters including head circumference, length and weight will be recorded.
