Probiotics and the Neurodevelopment in the Premature Infant <32 Weeks Gestational Age and <1500g
Phase: NA
Conditions: Infant, Newborn, Diseases, Infant Development
Sponsor: Fundacion Clinic per a la Recerca Biomédica

Probio&cs 
and 
the 
Neurodevelopment 
in 
the 
Premature Infant <32 Weeks Gesta&onal Age and 
<1500g 
 
 
Unique Protocol ID: HCB/2021/0454 
 
Date of document: 03/05/2021 
 
Version 2.3 
 
 

1 
General Informa-on 
 
1.1       Iden(ﬁca(on of the Study 
 
Title: Probio(cs and the neurodevelopment of the very premature infant <32 weeks gesta(on and <1500g 
 
1.2       Promotor  
 
Name and address of promotor: Benjamin J Baucells and Giorgia Sebas(ani, Neonatology Service, BCNatal 
Hospital Clinic, ICGON, 08028, Barcelona, Spain 
 
Collaborators: Marta Astals1, Vicente Andreu-Fernández2,3, Elisabet Navarro-Tapia2,3, Òscar García-Algar1, 
Josep Figueras-Aloy1 
 
1. Neonatology services, Hospital Clínic-Maternitat, ICGON, BCNatal, 08028 Barcelona, Spain 
2. Grup de Recerca Infància i Entorn (GRIE), Institut D’investigacions Biomèdiques August Pi i Sunyer 
(IDIBAPS), 08036 Barcelona, Spain 
3. Valencian International University (VIU), 46002 Valencia, Spain 
 2   Jus-ﬁca-on  
 
Introduc(on 
Probio(cs were deﬁned by the WHO as those microorganisms that, when administered in adequate 
amounts, conferred a health beneﬁt for the host1. This declara(on empowered probio(cs and paved the 
way for the research in this ﬁeld, allowing the rise of a variety of studies in diﬀerent ﬁelds, being the 
premature infant the popula(on with great advances2. Some of these studies demonstrated that in the 
premature neonate there is a delay in microbiota acquisi(on, as well as a substan(ve diﬀerence in 
composi(on, presen(ng a decrease in Lactobacillus and Biﬁdobacterium (considered protec(ve 
microorganisms) and a rise in Enterobacteriaceae, that contains pathogens such as Escherichia coli and 
Klebsiella3. Nevertheless, dysbiosis (disbalance between commensal ﬂora and pathogenic ﬂora that leads 
to pathology) could entail a dysregula(on between proinﬂammatory factors and protec(ve factors, puhng 
the extreme premature infant at bigger risk of major complica(ons and increasing the morality4. The study 
of the microbiota in the premature infant and the applica(on of probio(cs have encompassed diﬀerent 
lines of research in dysbiosis (related to obesity, allergy and breathing problems in early stages of 
development5), however the most studied aspect has been the use of probio(cs to prevent necro(sing 
enterocoli(s, one of the most severe disease a newborn infant can confront-10. Moreover, although there 
is s(ll controversy, there has been a clear beneﬁt in the prophylac(c use of diﬀerent strands of probio(cs, 
specially combined, for the preven(on and reduc(on of the incidence of necro(sing enterocoli(s7,8. 
Branching from here, we can also ﬁnd the use of probio(cs in preven(on of late-onset sepsis, considered 
the main side eﬀect of probio(cs, and neonatal mortality, where evidence has also shown probio(cs to 
have poten(al beneﬁt, especially those probio(cs containing genres Biﬁdobacterium and Lactobacillus8. 
For instance, in animal models Biﬁdobacterium spp. has been shown to have a possible an(nﬂammatory 
eﬀect through the produc(on of cytokines and synthesis of Th2 lymphocites, as well as regula(on IgE. 
Addi(onally, produc(on of short chain fajy acids could help maintain intes(nal barrier integrity2. 
Seeing these promising results, one has to ask if the eﬀect of probio(cs could extend beyond necro(sing 
enterocoli(s, late-onset sepsis and mortality, having other eﬀects in the premature infant, specially those 

more immature (<32 weeks gesta(onal age) and very low birthweight (<1500g). A premature neonate is 
daily challenged by many complica(ons and poten(al sequalae that will aﬀect the rest of their lives. 
Between these we have the neurodevelopment with eﬀects that extend beyond cerebral palsy, 
neurosensorial hearing loss and blindness11. 
Regarding neurodevelopment, a gut-brain axis has previously been described, connected through neural, 
endocrine, and immune pathways12-14. Microbiota with its ability to process indispensable metabolites, 
regulate pathogen microbiota and modula(ng the immune response, by for example reducing intes(nal 
permeability to lipopolysaccharide (pro-inﬂammatory factor), could have a key role in the gut-brain axis14. 
For instance, substances such as brain derived neurotrophic factor, neutrophines o interleukin-6, 
implicated in neuroinﬂamma(on and neurodevelopment, could see themselves modiﬁed by changes in 
microbiota16. Nevertheless, the brain derived neurotrophic factor, nerve growth factor and neutrophines 
3 and 4 promote neuronal survival and diminish apoptosis of central and peripheral nervous system 
neurones, being vital for the development of the pre and post-natal brain17-18. Despite everything, 
unfortunately there is a lack of published evidence, with only 4 studies analysing the eﬀect of probio(cs 
on the neurodevelopment of the premature new-born, without ﬁnding a beneﬁt19-22. However, it is 
important to note that the assessment of neurodevelopment in those studies is limited around the 2 years 
of follow-up with the use of a Bayley test. Alas, studies such as the EPIcure11 have shown that behavioural 
changes and impairment in neurodevelopment appear beyond the 2 years of age, even in adulthood, being 
some of these diﬃcul(es in learning, psychiatric disorders o ajen(on deﬁcit and hyperac(vity disorder, 
diﬃcult to detect at the age where the majority of the follow-up is discon(nued23,24. 
 
Hypothesis 
 
1. The prophylac(c use of a combina(on of probio(cs containing Biﬁdobacterium biﬁdum and 
Lactobacillus acidophilus in premature infants <32 weeks gesta(on and <1500g could contribute 
to improving neurodevelopment at 2 years follow-up. Said improvement will be even more evident 
at 6 years of age, as well as entailing changes in biomarker pajerns of neuroplas(city and 
neuroinﬂamma(on at 6 years of age.   
2. The said combina(on of probio(cs will contribute to reducing necro(sing enterocoli(s, late-onset 
sepsis, intraventricular haemorrhage, and neonatal mortality. Addi(onally, there will be a 
reduc(on in allergy and breathing disorders in early infancy.  
 
Task distribu(on 
Neonatology (BCNatal Hospital Clinic) 
• 
Dr Benjamin J Baucells: informa(on of study, recruitment, informed consent. Data collec(on and 
sampling. Coordina(on of neuropsychology team and laboratory. Interviews with families. 
Maintenance of database and support to rest of researchers. Elabora(on of future manuscripts 
and sta(s(cal analysis of data.  
• 
Dr Georgia Sebas(ani: informa(on of study, recruitment, and informed consent. Data collec(on 
and sampling. Coordina(on and supervision of the project and neuropsychology and laboratory 
teams. Maintenance of database. Supervision and wri(ng of future manuscripts.  
• 
Ms Marta Astals: informa(on of the study. Neuropsychological assessment of pa(ents at 2 and 6 
years of age. Performance of semi structured interviews to families. Coordina(on and 
collabora(on with other psychologists ajached to the Neonataology Services of BCNatal Hospital 
Clinic.  
• 
Dr Òscar García-Algar: review of protocols and research. Supervision of project and achievement 
of objec(ves. Supervision and wri(ng future manuscripts.  Coordina(on of research teams. 
Support to other teams and services.  

• 
Dr Josep Figueras-Aloy: review of protocols and research. Supervision of project and achievement 
of objec(ves. Data quality assessment and sta(s(cal data analysis. Supervision and wri(ng of 
future manuscripts.  
Laboratory (IDIBAPS-GRIE): 
• 
Vicente Andreu-Fernández: processing and collec(on of biological samples. Analysis of 
biomarkers of neuroinﬂamma(on and neurodevelopment with kits based on Luminex Mul(plex 
technology of R&D. Analysis of intes(nal permeability with ELISA kit of LifeSpan BioSciences (LS-
F55511) in serum. Database management of recruited cohorts.  
• 
Elisabet Navarro-Tapia: processing and collec(on of biological samples. Analysis of biomarkers of 
neuroinﬂamma(on and neurodevelopment with kits based on Luminex Mul(plex technology of 
R&D. Analysis of intes(nal permeability with ELISA kit of LifeSpan BioSciences (LS-F55511) in 
serum. Database management of recruited cohorts.  
 
2.1     Relevant references 
 
1. Food and Agricultural Organiza3on of the United Na3ons and World Health Organiza3on. Health and 
nutri3onal proper3es of probio3cs in food including powder milk with live lac3c acid bacteria. World Health 
Organiza3on, (2001). 
2. Navarro-Tapia E, Sebas3ani G, Sailer S, Almeida Toledano L, Serra-Delgado M, Garcia-Algar O, Andreu-
Fernández V. Probio3c Supplementa3on during the perinatal and infant period: Eﬀects on gut dysbiosis and 
disease. Nutrients. Aug 2020; 112(8): 2243. 
3. Mai, V.; Young, C.M.; Ukhanova, M.; Wang, X.; Sun, Y.; Casella, G.; Theriaque, D.; Li, N.; Sharma, R.; Hudak, 
M. Fecal microbiota in premature infants prior to necrotizing enterocolitis. PLoS ONE 2011, 6.  
4. Underwood MA, Sohn K. The Microbiota of the Extremely Preterm Infant. Clin Perinatol. 2017 Jun; 44(2): 
407–427. 
5. Sjögren, Y.M.; Jenmalm, M.C.; Böicher, M.F.; Björkstén, B.; Sverremark-Ekström, E. Altered early infant gut 
microbiota in children developing allergy up to 5 years of age. Clin. Exp. Allergy 2009, 39, 518–526  
6. Thomas JP, Raine T, Reddy S, Belteki G. Probio3cs for the preven3on of necrosi3ng enterocoli3s in very low-
birth-weight infants: a meta-analysis and systema3c review. Acta Paediatr. 2017 Nov; 106(11):1729-1741.   
7. Baucells BJ, MercadalHally M, Álvarez Sánchez AT, Figueras-Aloy J. Probio3c associa3ons in the preven3on 
of necrosi3ng enterocoli3s and the reduc3on of late-onset sèpsia and neonatal mortality in preterm infants 
under 1500g: A Systema3c review. An Pediatr (Barc). Nov 2016; 85 (5): 247-255.  
8. Bi LW, Yan B, Yang Q, Li M, Cui H. Which is the best probiò3c treatment strategy to prevent the necro3sing 
enterocoli3s in premature infants. A network meta-analysis revealing the eﬃcacy and safety. Medicine 
(Bal3more). 2019 Oct;98(41):e17521. doi: 10.1097/MD.0000000000017521. PMID: 31593123; PMCID: 
PMC6799420. 
9. van den Akker C, van Goudoever J, Shamir R, Domellöf M, Embleton ND, Hojsak I, et al. Probio3cs and 
Preterm Infants: A Posi3on Paper by the European Society for Paediatric Gastroenterology Hepatology and 
Nutri3on Commiiee on Nutri3on and the European Society for Paediatric Gastroenterology Hepatology and 
Nutri3on Working Group for Probio3cs and Prebio3cs. JPGN. May 2020; 70 (5): 664-680.  
10. Alfaleh K, Anbrees J, Bassler D, Al-Kharﬁ T. Probio3cs for preven3on of necro3sing enterocoli3s in preterm 
infants. Cochrane Database of Systema3c Reviews 2011. Issue 3. Art No: CD005496. 
11. Wood NS, Marlow N, Costeloe K, Gibson AT, Wilkinson AR. Neurologic and Developmental Disability aser 
Extremely Preterm Birth. N Engl J Med 2000; 343:378-384.  
12. Hortensius LM, van Elburg RM, Nijboer CH, Benders MJNL, de Theije CGM. Postnatal Nutri3on to improve 
Brain Development in the Preterm Infant: A Systema3c Review from Bench to Bedside. Front. Physiol. 10:961.   
13. Cryan JF, O'Mahony SM. The microbiome-gut-brain axis: from bowel to behaviour. Neurogastroenterol Mo3l 
2011;23:187–92.  
14. Yang I, Cornwin EJ, Brennan PA, Jordan S, Murphy JR, Dunlop A. The infant Microbiome: Implica3ons for 
Infant Health and Neurocogni3ve Development. Nurs Res. 2016; 65 (1): 76-88.  

15. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and func3on. Annu Rev Neurosci. 
2001;24:677-736. doi: 10.1146/annurev.neuro.24.1.677. PMID: 11520916; PMCID: PMC2758233. 
16. Ranuh R, Athiyyah AF, Darma A, et al. Eﬀect of the probio3c Lactobacillus plantarum IS-10506 on BDNF and 
5HT s3mula3on: role of intes3nal microbiota on the gut-brain axis. Iranian Journal of Microbiology. 2019 
Apr;11(2):145-150. 
17. Nikolaou KE, Malamitsi-Puchner A, Boutsikou T, Economou E, Boutsikou M, Puchner KP, Baka S, Hassiakos D. 
The varying paierns of neurotrophin changes in the perinatal period. Ann N Y Acad Sci. 2006 Dec;1092:426-
33. doi: 10.1196/annals.1365.041. PMID: 17308169.  
18. Liu DY, Shen XM, Yuan FF, Guo OY, Zhong Y, Chen JG, Zhu LQ, Wu J. The Physiology of BDNF and Its 
Rela3onship with ADHD. Mol Neurobiol. 2015 Dec;52(3):1467-1476. doi: 10.1007/s12035-014-8956-6. Epub 
2014 Oct 30. PMID: 25354496. 
19. Jacobs SE, Hickey L, Donath S, Opie GF, Anderson PJ, Garland SM, Cheong JLY. Probio3cs, prematurity and 
neurodevelopment: follow-up of a randomised trial. BMJ Paediatrics Open 217; 1:e0000176.  
20. Chou IC, Kuo HT, Chang JS, et al. Lack of eﬀects of oral probio3cs on growth and neurodevelopmental 
outcomes in preterm very low birth weight infants. J Pediatr2010;156:393–6. 
21. Romeo MG, Romeo DM, Trovato L, et al. Role of probio3cs in the preven3on of the enteric coloniza3on by 
Candida in preterm newborns: incidence of late-onset sepsis and neurological outcome. J 
Perinatol2011;31:63–9. 
22. Sari FN, Eras Z, Dizdar EA, et al. Do oral probio3cs aﬀect growth and neurodevelopmental  outcomes in very 
low-birth-weight preterm infants? Am J Perinatol2012;29:579–86. 
23. Johson S, Hollis C, Kochhar P, Hennessy E, Wolke D, Marlow N., Psychiatric disorders in extremely preterm 
children: longitudinal ﬁnding at age 11 years in the EPICure study. J m Acad Child Adolesc Psychiatry. May 
2010;49 (5): 453-63.e1 
24. O’Reilly H, Johnson S, Ni Y, Wolke D, Marlow N. Neuropsychological Outcomes at 19 Years of Age following 
Extremely Preterm Birth. Pediatrics. Feb 2020; 145 (2): e20192087. 
 
3      Objec-ves and purpose of the study 
 
The objec(ve of the study is to determine the eﬀect of the combina(on of 2 probio(cs (Biﬁdobacterium 
biﬁdum NCDO 2203 y Lactobacillus acidophilus NCDO 1748) in the neurodevelopment and neuroimage of 
extreme premature infants less than 32 weeks gesta(on and under 1500g at 2 years of age, and the 
neurodevelopment and neuronal plas(city biomarkers at 6 years of age.  
 
Secondarily the incidence of necro(zing enterocoli(s, late-onset sepsis, intraventricular haemorrhage and 
mortality of premature infants will also be assessed comparing those neonates that received probio(cs 
from those that did not. Moreover, growth curves will be considered to determine the moment of “catch-
up” (moment where the infant returns to his correspondent growth curves) between the two groups, and 
to elucidate whether there is any eﬀect from the moment of “catch-up” and neurodevelopment. Finally, 
there will be a comparison between obesity, asthma, and respiratory disease at 6 years of age.  
 
On the other hand, at 6 years of age there will be an assessment of neurodevelopment biomarkers and 
neuroplas(city in blood serum to determine changes in expression linked to probio(c administra(on, and 
therefore contribute to the enforcement of the existence of a brain-gut axis and it’s importance in 
neurodevelopment.  
 
3.1 Main and secondary variables 
 
• 
Gesta(onal age, weight, length and head circumference at birth 
• 
Sex 
• 
Mul(ple gesta(on 

• 
Gesta(onal pathology (diabetes, preeclampsia, maternal sepsis, placental disease, in vitro 
fecunda(on) 
• 
IUGR and degree of IUGR 
• 
Inborn vs Outborn 
• 
Apgar at 5 and 10 minutes 
• 
Antenatal steroids 
• 
Type of delivery (vaginal vs caesarean) 
• 
Coriamnioni(s 
• 
Type of feed (breasqeeding, donor milk or formula) 
• 
Dura(on of breasqeeding 
• 
Necro(sing enterocoli(s, early and late-onset sepsis, mortality 
• 
An(bio(c use beyond 48 hours of life 
• 
Days of parenteral nutri(on 
• 
Days of enteral nutri(on 
• 
Days of invasive ven(la(on, days of non-invasive ven(la(on, days of oxygen 
• 
Days of stay in intensive care 
• 
Chronic lung disease 
• 
Intraventricular haemorrhage (I-IV) 
• 
Periventricular leukomalacia 
• 
Cranial ultrasound result 
• 
Re(nopathy of prematurity 
• 
Neurosensorial hearing loss 
• 
Cerebral palsy 
• 
Colic in early infancy 
• 
Bayley score at 2 years of age 
• 
MRI at 2-3 years of age 
• 
Growth chart 
• 
Moment of “catch-up” 
• 
Childhood diseases (asthma, atopic skin, hospital admissions, allergy,…) 
• 
Need for an(bio(cs or probio(cs during childhood >48 hours 
• 
Weight and height at the assessment 
• 
WISC-V at 6 years 
• 
CBCL 6-18 (Child Behaviour Checklist 6-18) 
• 
BRIEF-2 
• 
Mother tongue 
• 
Parental age 
• 
Cultural level of parents 
• 
Ethnical origin 
• 
Parents work 
• 
Kind of family (single parent, …) 
• 
Social services requirements 
• 
Need to ajend early s(mula(on centre 
 
4     Study design 
 

Quasi-experimental, sequen(al, unicentric, cohort study with the performance of neuropsychological 
tests and biomarkers of neurodevelopment, inﬂamma(on, and intes(nal permeability.  
 
We es(mate an ini(al phase of 2 years with a comparison at 6 years of age (4 years aver start of trial). 
 
5    Enrolment of par-cipants   
 
Infants born below 32 weeks gesta(onal age and birth weight under 1500g cared for at BCNatal Hospital 
Clínic (ter(ary neonatal unit) in Barcelona between January 2014 and December 2019. There will be a 12 
month wash out period (year 2017) to alleviate cross contamina(on of probio(cs. 
 
Par(cipants will be contacted via phone (parent/legal guardian) for those that consented to be included 
for a 6-year assessment and blood sampling. In those cases where contact via phone has not been possible, 
contact to reference Paediatrician will be sought.  
 
5.1   Inclusion criteria 
 
Extreme premature infants <32 weeks and <1500g, cared at BCNatal Hospital Clinic during years 2014-
2019. Par(cipants will have to survive beyond 7 days of life (moment where the probio(c interven(on will 
be given). Recruitment will start from January 2014 and will last un(l December 2019. 
 
5.2    Exclusion criteria 
 
All neonates presen(ng with suspected congenital anomalies, inborn errors of metabolism, or gene(c 
defects will be excluded. Infants with a suspected syndrome, or who had suﬀered events beyond the 
neonatal period, not related to prematurity, that could entail impairment in neurodevelopment (severe 
cranioencephalic trauma, oncological process, meningi(s, or exposure to toxic substances) will also be 
excluded.  
 
6   Treatment and study calendar 
 
Interven(on 
 
Daily dose of 6x109 UFC Inﬂoran® -Berne, Switzerland- (Biﬁdobacterium biﬁdum NCDO 2203 and 
Lactobacillus acidophilus NCDO 1748) from 7 days of life un(l reaching a postmenstrual age of 34 weeks 
or discharge. The probio(c mixture will be provided in capsules, opened, dissolved in water and given 
orally or via nasogastric tube, according to the feeding regime of each neonate (breast milk, donor milk or 
formula). Where concerns for NEC or LOS are present, probio(cs will be stopped and reinstated once 
enteral feeds are recommenced. 
 
Evalua(on of eﬀect of interven(on  
 
At 24 months corrected age there will be a Bayley-III scale test in an extensive examina(on performed by 
independent assessors, blinded to group alloca(on. Aver this evalua(on, pa(ents will be divided into four 
categories according to their degree of neurodevelopment: survival without neurodevelopment 
impairment (normal neurodevelopment), mild impairment, moderate impairment, or severe 
impairment26. Mild impairment will be considered if they have muscle tone changes, impaired ﬁne or 
gross motor coordina(on, Bayley scale score between 71-84, moderate behaviour disorders or mild visual 

disability. Moderate impairment will be diagnosed when suﬀering from spas(c diplegia, hemiplegia, 
seizures (non-febrile), Bayley scores between 50-70, severe behaviour disorders, moderate visual disability 
or mild-moderate hypoacusis. Severe impairment will be ajributed to subjects with spas(c quadriplegia, 
choreoathetosis, ataxia, Bayley score <50, blindness or severe hypoacusis.  
 
Posteriorly, at 6 years of age, we will assess the presence of cerebral palsy, learning diﬃcul(es, behavioural 
disorders (au(sm spectrum, ajen(on deﬁciency and hyperac(vity disorder) and scores in the tests WISC-
V, CBCL 6-18 and BRIEF-2. We will also perform a semi-structured interview to parents or legal guardians 
to determine the degree of impairment of the child, that might have not been previously included.  
 
On the other hand, at 6 years of age we will proceed to sampling of blood plasma for the study of diﬀerent 
biomarkers related to neurodevelopment, inﬂamma(on, and intes(nal permeability. We will analyse the 
following: NeuN, Doblecor(na, GFAP, GDNF, Ki67, Nrf2, BDNF, NGF, neurotroﬁna-1 (NT-1), neurotroﬁna-3 
(NT-3), neurotroﬁna-4 (NT-4), DYRK1A, HIF1α, S100B i GSK3B. Regarding inﬂamma(on we will assess IL1B, 
IL6, IL8, IL10, IL12, TNF- α. We will ﬁnally determine presence of lipopolysaccharide in plasma with an 
ELISA.  
 
7   Sta-s-cs 
 
Considering an alpha risk of α = 0.05 and a beta risk of β = 0.2 in a bilateral contrast, a minimum of 90 
subjects in each group were required to detect a sta(s(cally signiﬁcant diﬀerence between groups, where 
for the control group the propor(on of some neurodevelopmental altera(on was expected to be at least 
0.4 and for the group treated with probio(cs at least 0.2. 
 
7.2      Sta(s(cal analysis  
IBM SPSS Sta(s(cs 27.0.1.0 will be used for the sta(s(cal analysis. Non-parametrical analysis with a 
double-sided Mann-Whitney U test will be calculated for all con(nuous variables, whilst for categorical 
variables the chi-square test will be applied. In all cases, 0.05 will be considered the threshold of sta(s(cal 
signiﬁcance. The rela(ve risk (RR) and 95% conﬁdence intervals will be determined for dichotomous 
variables. When encountering signiﬁcant diﬀerences, the number needed to treat (NNT) will be analysed.  
 
8   Ethics 
 
The study will be in accordance with the Declara(on of Helsinki (Fortaleza Version, Brazil October 2013) 
and will follow the legal requirements to fulﬁl the Spanish Biomedical Law of 14/2007. All pa(ents, parents 
or legal guardians will be sought for informed consent.  
 
9   Data processing and conﬁden-ality 
 
Treatment, communica(on and cease of data of personal character will be following the EU Ruling 
2016/679 of the European Parliament and the Counsel of 27th April 2016, referring to protec(on of physical 
people and the treatment of personal data and the free circula(on of data, being legally binding on the 
25th of May 2018. The legal base of use of data will be in accordance to this act, established in the ar(cle 
9 of EU 2016/679 Ruling.  
 
All collected data will be deiden(ﬁed and allocated a speciﬁc code, that will not allow any recogni(on of 
par(cipants. Only the main promotor and collaborators will have access to data and will be allowed to 
enter the clinical history of pa(ents for the purpose of this study.  

 
Only legal requirement of medical emergency will allow the disclosure of any data related to par(cipants 
of the study.  
 
There will, however, be access upon request of non-iden(ﬁed data to Health Authori(es, Ethics Commijee 
and authorised personnel, for the purpose of quality assessment and study evalua(on. Nevertheless, this 
will follow the exis(ng legal frame and always guaranteeing conﬁden(ality.  
 
If data is to leave the EU, whether it is to en((es related to the Hospital where pa(ent was cared for, o 
third healthcare par(es or collaborators, data will be protected via contracts or other established 
mechanisms to comply with EU legal requirements to data protec(on.  
 
As the study promotors we agree to treat data in accordance to EU Ruling 2016/679, and therefore register 
all ac(vi(es undertaken with the data and an assessment of data security and risks of the treatment of 
data.  
 
Par(cipants will be allowed to access, modify, oppose and cancel data (supress in the new ruling) and limit 
collected data as well as its processing. They will also be allowed to request copies or cession to a third 
party. In order to fulﬁl with their rights they will have to contact the Main promotor or the Delegate of 
Data Protec(on at Hospital Clinic I Pronvicial de Barcelona (protecciodades@clinic.cat). If they are not 
sa(sﬁed with the process they may contact the Data Protec(on Agency.  
 
If a pa(ent opts out of the study, their data will not be deleted to guarantee the validity of the research 
and fulﬁl their requirements with the healthcare authori(es and pharmaceu(cal authori(es.  
 
The main promotor is obliged to preserve all collected data for the study for at least 5 years aver the 
ﬁnalisa(on of the study. Posteriorly, personal informa(on will only be stored by the Healthcare centre for 
healthcare purposes and the main researcher only if the pa(ent has authorised such treatment, always in 
accordance to exis(ng law. 
 
10   Biological sampling and processing  
 
We will proceed to collect 2 mL of blood plasma for the study of plas(city, inﬂamma(on and permeability 
biomarkers.  
 
They will be analysed at the laboratory of Ins(tut d’Inves(gacions Biomèdiques Agus€ Pi I Sunyer (IDIBAPS) 
by Dr Vicente Andreu-Fernández and Elisabet Navarro-Tapia.  
 
Aver study comple(on all samples will be destroyed appropriately.  
 
11   Funding 
 
We will proceed to apply to na(onal and interna(onal grants to achieve necessary funding (es(mated 
25,500€). 
 
Researchers will receive no payments and all budget will be des(ned to pay for material and processing of 
samples, with some budget allocated to scien(ﬁc sharing.   
 

12    Publica-on policy 
 
 All results will be published in recognised peer review journals. Addi(onally, all par(cipants will be 
en(tled to dispose of their personal results for their convenience and discre(on.