Circulating Markers in Preterm Infants With Perinatal and Neonatal Inflammation: An Observational Study
Conditions: Sepsis, Necrotizing Enterocolitis, Chorioamnionitis, Feeding Intolerance
Sponsor: Per Torp Sangild

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NEOINFLAM: 
Circulating markers in preterm infants with perinatal and 
neonatal inflammation: An observational study 
 
 
 
 
 

 
 
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Table of Contents 
PROTOCOL TITLE: .................................................................................................................................................. 1 
STUDY SUMMARY ................................................................................................................................................... 2 
1 
INTRODUCTION .............................................................................................................................................. 3 
1.1 
BACKGROUND .................................................................................................................................................. 3 
1.2 
INVESTIGATIONAL AGENT................................................................................................................................ 4 
2 
STUDY OBJECTIVES ...................................................................................................................................... 5 
3 
STUDY DESIGN ................................................................................................................................................ 6 
3.1 
GENERAL DESIGN ............................................................................................................................................ 6 
3.2 
PRIMARY STUDY ENDPOINTS ........................................................................................................................... 6 
3.3 
SECONDARY STUDY ENDPOINTS ...................................................................................................................... 7 
3.4 
SAFETY ENDPOINTS ......................................................................................................................................... 7 
4 
SUBJECT SELECTION AND WITHDRAWAL............................................................................................ 8 
4.1 
INCLUSION CRITERIA ....................................................................................................................................... 8 
4.2 
EXCLUSION CRITERIA ...................................................................................................................................... 8 
4.3 
SUBJECT RECRUITMENT AND SCREENING ........................................................................................................ 8 
4.4 
WITHDRAWAL OF SUBJECTS ............................................................................................................................ 8 
5 
STUDY PROCEDURES .................................................................................................................................... 9 
5.1 
DIAGNOSIS OF CHORIOAMNIONITIS, LOS AND NEC ......................................................................................... 9 
5.2 
INFORMATION IN CRF ...................................................................................................................................... 9 
5.3 
SAMPLE SELECTION, ANALYSIS AND COMPARISON ......................................................................................... 10 
6 
STATISTICAL PLAN ..................................................................................................................................... 11 
6.1 
SAMPLE SIZE DETERMINATION ...................................................................................................................... 11 
6.2 
STATISTICAL METHODS ................................................................................................................................. 11 
7 
SAFETY AND ADVERSE EVENTS ............................................................................................................. 12 
7.1 
DEFINITIONS .................................................................................................................................................. 12 
7.2 
RECORDING OF ADVERSE EVENTS ................................................................................................................. 12 
7.3 
REPORTING OF SERIOUS ADVERSE EVENTS.................................................................................................... 12 
7.3.1 
Study Sponsor Notification by Investigator ......................................................................................... 12 
7.3.2 
Notification to Research Institute by Investigator ............................................................................... 12 
7.4 
UNBLINDING PROCEDURES ............................................................................................................................ 12 
7.5 
STOPPING RULES ............................................................................................................................................ 12 
7.6 
MEDICAL MONITORING.................................................................................................................................. 12 
7.6.1 
Internal Data and Safety Monitoring Board ........................................................................................ 12 
7.6.2 
Independent Data and Safety Monitoring Board ................................................................................. 12 
8 
DATA HANDLING AND RECORD KEEPING .......................................................................................... 13 
8.1 
CONFIDENTIALITY .......................................................................................................................................... 13 
8.2 
HANDLING AND STORAGE OF DATA AND DOCUMENTS.................................................................................... 13 
8.3 
RECORDS RETENTION .................................................................................................................................... 13 
9 
ETHICAL CONSIDERATIONS .................................................................................................................... 14 
10 
STUDY FINANCES ......................................................................................................................................... 15 
10.1 
FUNDING SOURCE ..................................................................................................................................... 15 
10.2 
CONFLICT OF INTEREST ............................................................................................................................. 15 
10.3 
SUBJECT STIPENDS OR PAYMENTS ............................................................................................................ 15 
11 
PUBLICATION PLAN .................................................................................................................................... 16 
12 
REFERENCES ................................................................................................................................................. 17 
13 
APPENDICES .................................................................................................................................................. 18 

 
 
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List of Abbreviations 
 
 
 
 
BPD  
Bronchopulmonary Dysplasia 
 
CRF 
 
Case Report Form 
 
CRP 
 
C-Reactive Protein 
 
LC-MS 
Liquid Chromatography-Mass Spectrometry 
 
LOS 
 
Late-Onset Sepsis 
 
PHI 
 
Protected Health Information 
 
NEC  
Necrotizing Enterocolitis 
 
NETs  
Neutrophil Extracellular Traps 
 
NICU  
Neonatal Intensive Care Unit 
 
PROM  
Premature rupture of membranes 
 
RDS  
Respiratory Distress Syndrome 
 
ROC  
Receiver Operating Characteristic 

Clinical Research Protocol Template 
 
Page 1 
Version 18SEP08 
Protocol title: Circulating markers in preterm infants with perinatal 
and neonatal inflammation: An observational study (NEOINFLAM) 
 
Version 
2 
Date 
September 26th 2017 
Principal investigator 
Neonatal department, Bao’an Maternal and Child Health Hospital  
Ping Zhou, MD, PhD student 
56 Yu Lv Road, Bao’an District, Shenzhen, Guangdong 518102, China 
 
 
Collaborators 
University of Copenhagen, Department of Veterinary Clinical and 
Animal Sciences 
Prof. Per T. Sangild, PhD, DMSc, DVSc 
Duc Ninh Nguyen, PhD 
Dyrlægevej 68, DK-1958 Frederiksberg C, Denmark 
 
 
 
 
Name 
Signature 
Date 
Principal investigators: 
Ping Zhou 
 
 
26.09.2017 
Collaborators: 
Prof. Per T. Sangild 
 
 
 
Duc Ninh Nguyen 
 
 
 
 
 
 
 
26.09.2017 
 
 
 
26.09.2017 
 

 
 
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Study Summary 
 
Title 
Circulating markers in preterm infants with perinatal and neonatal 
inflammation: an observational study (NEOINFLAM) 
Short Title 
Markers for perinatal and neonatal inflammation 
Study type 
Observational study 
Study Duration 
18 months (October 2017-June 2019) 
Study Center(s) 
Single Center- Neonatal department, Bao’an Maternal and Child Health 
Hospital, Shenzhen, Guangdong, China 
Objectives 
The main objective of the study is to identify new circulating markers 
associated with early life inflammatory conditions in preterm infants 
including chorioamnionitis, late-onset sepsis and necrotizing 
enterocolitis in preterm infants during the first four weeks of life. The 
aim is to better understand these conditions, and more long-term, to 
provide earlier and better diagnosis. 
Number of Subjects
200 
Diagnosis and Main 
Inclusion Criteria 
Inclusion criteria: preterm infants (< 32 weeks of gestation) 
Main diagnosis before and during the study: chorioamnionitis, late 
onset-sepsis and necrotizing enterocolitis 
 

 
 
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1 Introduction 
This document is a protocol for a human research study. This study is to be conducted 
according to Chinese medical standards, applicable government regulations and Institutional 
research policies and procedures.  
1.1 Background 
Every year, approximately 15 million infants are born preterm (< 37 weeks of gestation) and 1 
million die (1). Preterm infants, especially those  are born < 32 weeks of gestation, have 
immature gastrointestinal tract and innate immune system, which lead to high risks of 
development of necrotizing enterocolitis (NEC, a devastating intestinal disease affecting 7% 
preterm infants) and early life systemic infection (late-onset sepsis, LOS, up to 20-40% all 
hospitalized infants) (1,2). Those infectious diseases may lead to further long-term complications 
such as neurodevelopmental delay (1). NEC is very often related to LOS, possibly because an 
abnormal gut bacterial colonization and high gut permeability allow bacterial translocation (3). 
Conversely, a systemic inflammatory insult may predispose to NEC via impaired gut motility 
and immunity. As a result, in clinical trials, diagnostic markers are often used for combined LOS 
and/or NEC. Despite tremendous efforts to develop diagnostic markers for LOS and NEC, none 
of them are specific enough for clinical use (4). The current “gold standard” of blood culturing 
for diagnosis of LOS has many highly false negatives and is time-consuming whereas patients 
may develop septic shock and die shortly after clinical presentation. Ng et al performed a 
proteomic study to characterize protein biomarkers for LOS and NEC, but their work was 
exploratory without detailed focus on the mechanism based on an conventional LC-MS protocols 
(4). Recently, new mechanisms have been suggested to play role in the pathogenesis of adult 
sepsis such as circulating neutrophil extracellular traps (NETs), microparticles and miRNA but 
they have not been characterized in neonatal diseases (5,6). 
 
During pregnancy and labor, 20% of term and up to 60% of preterm deliveries show signs of 
maternal/prenatal inflammation (7), chorioamnionitis, which is the most important risk factor for 
preterm birth. Prenatal inflammation is associated with postnatal gut and systemic inflammation 
in newborn infants (8) and this may affect immune development in several ways. However, no 
study has yet investigated whether parameters related to postnatal immune development in 
preterm infants are affected by chorioamnionitis, and how immune parameters in infants with 
chorioamnionitis are associated with postnatal infections. Further, it is unclear how important the 
progression of enteral feeding with milk (mother´s own milk, donor milk or formula) is for the 
postnatal NEC and LOS sensitivity. We speculate that early feeding with an optimal milk diet 
may help to support both gut and systemic immunity development, and thereby increase the 
resistance against NEC and LOS.     
 
To improve the understanding of NEC and LOS, and their diagnostic criteria, and the 
postnatal immune development after chorioamnionitis, we investigate temporal blood samples in 
n=200 preterm infants. We apply proteomics and several immune assays to plasma, as well as 
whole blood transcriptomics/epigenetics to blood collected from preterm infants with and 
without NEC, LOS and/or CA. The aim is to identify new proteins and transcriptomic/epigenetic 
parameters that are related to the mechanisms of NEC, LOS and CA. These biological markers 
may help to provide possible new diagnostic markers for neonatal LOS and NEC, with and 

 
 
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without maternal chorioamnionitis and with/without difficulties in establishing normal enteral 
feeding. 
1.2 Investigational Agent 
Not applicable 

 
 
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2 Study Objectives 
Primary objective: To characterize circulating markers associated with the early life 
complications including chorioamnionitis, LOS and NEC in preterm infants during the first four 
weeks of life. 
 
Secondary objective: To characterize how different feeding regimes and diets may affect 
these markers during the first four weeks of life in preterm infants 
 

 
 
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3 Study Design 
3.1 General Design 
 This observational prospective cohort study includes 200 preterm infants, born < 32 weeks 
of gestation, at Neonatal Department, Bao’an Maternal and Child Health Hospital, 
Shenzhen, Guangdong, China. 
 Patients, who fulfill inclusion criteria, will be recruited following the parental consent. The 
recruitment stops when the sample size reaches 200. Expected study duration: January 2017-
June 2018. 
 Patients will follow usual clinical procedures at the hospital. Patient groups will be 
categorized based on clinical records: infants born without and with clinical 
chorioamnionitis, infants without or with at least one episode of late-onset sepsis (LOS) 
and/or necrotizing enterocolitis (NEC). 
 Enrollment and sampling are performed as indicated in the diagram below. Blood sampling 
is performed every week from birth until 4 weeks of age, or until discharge  
 
 
Prior to  
Enrollment 
 
 
 
 
Visit 1: Day 1 ± 2 
 
 
 
Visit 2: Day 7±3 
Visit 3: Day 14±3 
Visit 4: Day 21±3 
Visit 5: Day 28±3 
 
 
 
 
 
 
 
 
 
 
 
 
3.2 Primary Study Endpoints 
 Circulating markers in temporal postnatal blood samples associated with chorioamniotis. 
200 preterm infants: obtain parental consents. Screen potential participants by inclusion 
and exclusion criteria 
Initial assessments: Examination of clinical sign of sepsis or NEC. EDTA-blood 
collection: 450 µl. 200 µl blood is stored at -70oC, 250 µl is centrifuged at 2000g, 10 min 
for supernatant storage at -70oC.   
Follow-up assessments: Examination of clinical sign of LOS or NEC. EDTA-blood 
collection: 450 µl. 200 µl blood is stored at -70oC, 250 µl is centrifuged at 2000g, 10 min 
for supernatant storage at -70oC.    
Analyses to be performed: whole 
blood transcriptomics/epigenetics, 
plasma proteomics and immune assays 

 
 
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 Circulating markers in temporal postnatal blood samples associated with prognosis and/or 
diagnosis of neonatal LOS and/or NEC 
3.3 Secondary Study Endpoints 
 Circulating markers in temporal blood samples associated with feeding regimes and diets: 
major amount of mother’s own milk (>=50% of feeding times during the first two weeks 
of life as mother’s own milk) or minor amount of mother’s own milk (< 50% of feeding 
boluses during the first two weeks of life as mother’s own milk). 
3.4 Safety Endpoints 
This is an observational study so safety endpoints are not applicable 

 
 
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4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
In order to be eligible to participate in the study, a subject must meet the following criteria: 
 Born at neonatal department, Bao’an Maternal and Child Health Hospital 
 Gestational age < complete 32 weeks 
 Parenteral consent is signed by at least one of the two parents 
4.2 Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the participation of the 
study: 
 Major congenital abnormalities or birth defects 
 Serious perinatal complications such as serious asphyxia or meconium aspiration 
syndrome. 
 Life expectancy < 48 hours 
4.3 Subject Recruitment and Screening 
When infants are admitted to the Neonatal department, parents are informed about this 
observational study and the possibility to participate by the principal investigator. Signed 
parenteral consent is required to enter the study. Subjects should be recruited and the first blood 
sampling will be performed within the first 48 h of life. 
4.4 Withdrawal of Subjects 
A subject will be withdrawn from the study in one of the following conditions: 
 The consent is withdrawn by parents prior to the expected completion of the patient 
 The principal investigator reserves the right during the study to withdraw a patient from the 
study if the patient’s conditions are evaluated to be not suitable for blood sampling. In this 
occasion, another patient needs to be recruited to replace the drop-out patient. 
 Any infants that die before the completion of 4 postnatal weeks and are not diagnosed with 
LOS, NEC or chorioamnionitis beforehand will be withdrawn from the study. In this 
occasion, another patient needs to be recruited to replace the drop-out patient. 

 
 
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5 Study Procedures 
Care of patients follows standard procedures established by the local hospital. Blood samples 
will be taken according to the study design. At each blood sampling period, the study 
investigator will evaluate the suitable blood volume to be taken, based on body weight, health 
status and other guidelines from the hospitals.  
Feeding regime follows the practice at the local NICU: mother’s own milk is the first priority, 
followed by donor milk and infant formula. For each time of feeding, the following details are 
noted in the feeding CRF: time, type of feeding (mother’s milk, donor milk, formula), feeding 
volume, gastric residual from previous feeding. For feeding intolerance, any interventions such 
as aspiration, decreased the amount of feeding or stop feeding are noted in the comments section 
of the CRF. 
5.1 Diagnosis of chorioamnionitis, LOS and NEC 
Once the patient is recruited, information about diagnosis of chorioamnionitis is collected and 
filled in the CRF. Clinical chorioamnionitis is defined in pregnant women with fever (≥38oC), 
high heart rate (≥100/min for mothers and ≥160/min for fetuses), as well as when plasma CRP 
and blood leukocyte counts ≥ 20 mg/L and 12,000 leukocytes/mm3. Histological 
chorioamnionitis is confirmed after histologic examination of the fetal membranes. 
LOS is defined as sepsis occurring after 72 hours after birth. Clinical diagnosis of LOS is 
defined when the patient shows two or more of the following criteria: white blood cell count < 
5x109 cells/L or >20x109/L, total platelet count < 100x109cells/L, CRP > 10 mg/L, procalcitonin 
> 2 ng/mL.  Culture-proven sepsis is defined as clinical sepsis with positive blood bacterial 
culture result. 
Diagnosis of NEC is based on the conventional Bell system (IA, IB, IIA, IIB, IIIA and IIIB). 
Medical NEC is defined when an infant is diagnosed at stage IA to IIB. When an infants is 
classified with Bell stage III A or B (advanced NEC and bowel perforation), surgical 
interventions is required (surgical NEC). 
5.2 Information in CRF 
All collected data from clinical records will be recorded in the study CRF and feeding CRF by 
the study investigator at the hospital, including: 
 Patient ID number 
 Gestational age at birth 
 Birth weight and gender 
 Type of delivery: C-section or vaginal birth 
 Diagnosis of chorioamnionitis:  
o Clinical or histological,  
o Gestational age at diagnosis,  
o Temperature, heart rate, plasma CRP and blood leukocyte counts of mothers at diagnosis 
 Diagnosis of LOS and/or NEC:  
o Day of life at diagnosis,  
o Plasma CRP, total leukocyte counts 
o Clinical or culture-proven LOS 
o For culture-proven LOS: identified bacteria 
o Medical or surgical NEC 

 
 
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 Time of blood sampling and volume of blood samples 
 Feeding details (in the Feeding CRF) 
 Other notes: incidence of RDS, BPD, pneumonia 
5.3 Sample selection, analysis and comparison 
At the end of the study, the selection of appropriate samples for analysis will be conducted by 
all investigators of the study. Samples included in the analysis are those collected from birth until 
the disease diagnosis or completion of 4 postnatal weeks. In case of clinical diagnosis of LOS 
and/or NEC before completion of 4 weeks, further routine sample collection after diagnosis is 
continued if feasible.  
 
Temporal samples will be analyzed to perform three separate comparisons: 
 Infants without versus with LOS and/or NEC 
 Infants without versus with clinical/histological chorioamnionitis 
 Infants with major or minor amount of mother’s own milk during the first two weeks of life (≥ 
or < 50% of feeding times as complete mother’s own milk).  
 
Plasma proteomic study will be prioritized and will include as many samples as possible, 
followed by validation with absolute quantification of potential proteins using immune assays. 
The type of other -omic analysis for plasma (miRNA profiling) whole blood (transcriptomics or 
epigenetics) as well as sample size will be decided depending on the results of proteomic study. 

 
 
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6 Statistical Plan 
6.1 Sample Size Determination 
Based on the practical issues of admission rate of preterm infants and capacity of temporal blood 
sampling at the Neonatal Department, we anticipate to include 200 preterm infants (< 32 weeks 
of gestation) with estimated rate of clinical chorioamnionitis of 15-20% (for overall preterm 
cases, 40-70% of premature PROM cases), and clinical LOS and/or NEC of 20-30%. 
6.2 Statistical Methods 
Infant grouping will be categorized based on the defined endpoints to compare: 1. Infants with 
vs. without chorioamnionitis; 2. Infants with vs. without LOS and/or NEC; 3. Infants with vs. 
without major mother’s own milk feeding. Temporal data obtained from –omic analyses of blood 
and plasma samples collected every week will be analyzed by multivariate analysis with control 
for false discovery rates. Other data obtained from immune assays will be analyzed by univariate 
analysis. A panel of potential biomarkers will be identified by regression algorithm together with 
ROC analysis.  

 
 
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7 Safety and Adverse Events 
This is an observational study so safety reporting will not be applicable 
7.1 Definitions 
7.2 Recording of Adverse Events 
7.3 Reporting of Serious Adverse Events  
7.3.1 Study Sponsor Notification by Investigator 
7.3.2 Notification to Research Institute by Investigator 
7.4 Unblinding Procedures 
7.5 Stopping Rules  
7.6 Medical Monitoring 
7.6.1 Internal Data and Safety Monitoring Board 
7.6.2 Independent Data and Safety Monitoring Board 

 
 
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8 Data Handling and Record Keeping 
8.1 Confidentiality 
Information about study subjects will be kept confidential. In the event that a subject revokes 
authorization to collect or use protected health information (PHI), the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of subject authorization.  
For subjects that have revoked authorization to collect or use PHI, attempts should be made to 
obtain permission to collect at least vital status (i.e. that the subject is alive) at the end of their 
scheduled study period. 
8.2 Handling and storage of data and documents 
All personal data will be coded by serial numbers. Only the investigators will have the access to 
the key. Statistics will be performed with the coded numbers. Published data from this study 
cannot be traced to a specific infant.  
 
All data requested on the case report form (CRF) must be recorded.  All missing data must be 
explained.  If a space on the CRF is left blank because the procedure was not done, write “N/D”.  
If the item is not applicable to the individual case, write “N/A”.  If any entry error has been 
made, to correct such an error, draw a single straight line through the incorrect entry and enter 
the correct data above it.  All such changes must be initialed and dated.  
8.3 Records Retention 
All coded data will be stored in a security place for a minimum of ten years. 

 
 
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9 Ethical Considerations 
This study is to be conducted according to applicable government guidelines and regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to the Ethical Committee at Bao’an 
Maternal and Child Hospital, Shenzhen for formal approval of the study conduct. The decision of 
the Ethical Committee concerning the conduct of the study will be made in writing to the 
investigators. 
 
All subjects for this study will be provided a consent form in Chinese describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study. The consent form will be submitted with the protocol for review and 
approval by the Ethical Committee for the study.  The formal consent of a subject must be 
obtained before that subject is submitted to any study procedure. This consent form must be 
signed by the subject or legally acceptable surrogate, and the investigator-designated research 
professional obtaining the consent.  
 
Blood samples obtained for this cohort are left-over sample materials from routine clinical blood 
sampling for screening and diagnosis in preterm infants at the local hospital (Neonatal 
Department, Bao’an Maternal and Child Health Hospital, Shenzhen). Therefore, there are no 
potential harmful effects of  blood sampling to the participating infants. 
 

 
 
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10 Study Finances 
10.1 Funding Source 
The analytical part of the study will be partly funded by NEOCOL research grant by Innovation 
Fund Denmark (Prof. Per T. Sangild as the grant holder). Additional funding will be searched 
locally in Shenzhen by MD Ping Zhou. 
10.2 Conflict of Interest 
All investigators declare no potential conflicts of interest 
10.3 Subject Stipends or Payments 
Not applicable 

 
 
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11 Publication Plan 
The data are considered joint ownership between the study collaborators and Bao’an Maternal 
and Child Health Hospital, Shenzhen. All results derived from this study will be published 
jointly between these two institutions in pediatrics, nutrition or immunology journals. 
 
Neither the complete nor any part of the results of the study carried out under this protocol will 
be published or passed on to any third party without the consent of all the study investigators.  
Any investigator involved with this study is obliged to provide the principal investigator with 
complete test results and all data derived from the study. 

 
 
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12 References 
 
1.  Strunk T, Inder T, Wang X, Burgner D, Mallard C, Levy O. Infection-induced inflammation 
and cerebral injury in preterm infants. Lancet Infect Dis. 2014 Aug;14(8):751–62.  
2.  Sangild PT, Thymann T, Schmidt M, Stoll B, Burrin DG, Buddington RK. Invited review: 
the preterm pig as a model in pediatric gastroenterology. J Anim Sci. 2013 Oct;91(10):4713–
29.  
3.  Berrington JE, Stewart CJ, Cummings SP, Embleton ND. The neonatal bowel microbiome in 
health and infection. Curr Opin Infect Dis. 2014 Jun;27(3):236–43.  
4.  Ng PC, Ang IL, Chiu RWK, Li K, Lam HS, Wong RPO, et al. Host-response biomarkers for 
diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants. J Clin 
Invest. 2010 Aug;120(8):2989–3000.  
5.  Camicia G, Pozner R, de Larrañaga G. Neutrophil extracellular traps in sepsis. Shock. 2014 
Oct;42(4):286–94.  
6.  Delabranche X, Boisramé-Helms J, Asfar P, Berger A, Mootien Y, Lavigne T, et al. 
Microparticles are new biomarkers of septic shock-induced disseminated intravascular 
coagulopathy. Intensive Care Med. 2013 Oct;39(10):1695–703.  
7.  Garzoni L, Faure C, Frasch MG. Fetal cholinergic anti-inflammatory pathway and 
necrotizing enterocolitis: the brain-gut connection begins in utero. Front Integr Neurosci. 
2013;7:57.  
8.  Tita ATN, Andrews WW. Diagnosis and Management of Clinical Chorioamnionitis. Clin 
Perinatol. 2010 Jun;37(2):339–54.  
 

 
 
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13 Appendices 
• Parental Consent Form (in Chinese) 
• CRF (both English and Chinese)