Effects of Probiotic Mixture Supplementation and Evaluation of Intestinal Mucosal Tolerance and Gut Microbiome in Newborns with Perinatal Asphyxia Receiving Hypothermic Treatment: A Randomized, Multicentric, Blinded, Controlled TRIAL (VISNAT)


 IntroductionNeonatal encephalopathy is still a major cause of mortality and morbidity for newborns, although hypothermia treatment has improved the prognosis of term newborns with hypoxic-ischemic injury.Recent evidence suggests that one of the crucial but understudied mechanisms of secondary neuronal injury after global hypoxic-ischemic injury is inflammation.Hence, blocking the inflammatory reaction promotes neuroprotection and has a potential for use in the clinical treatment of ischemic brain injury.Many preclinical studies have shown bidirectional interactions between the central nervous system, the enteric nervous system, and the gastrointestinal tract, suggesting a prominent role for the gut microbiota in these gut-brain interactions. Early human studies suggest that altering the microbiota with beneficial bacteria, or probiotics, can lead to changes in brain function, as well as subjective reports of mood.Methods and analysisThe VISNAT trial is a PILOT STUDY developed according to SPIRIT checklist. It is a randomized, placebo-controlled, blinded, multicentre superiority trial with two parallel groups and a primary outcome of mortality and/or disability at 18 months of age.The experimental intervention will consist in administrating a high-dose multi-strain probiotic (SIVOMIXXTM) for 30 days in addition to hypothermic therapy.Primary outcome will be to compare mortality and/or disability at 18 month of age between the two groups. Disability will be assessed by means of the Bayley Mental Development Index. Secondary outcomes will be to compare inflammatory cytokines level and brain injury biomarkers (i.e. Tau and neurofilament light protein) between groups.A subgroup analysis will be performed stratifying patients according to both probiotic/placebo treatment and breast/artificial milk.DiscussionProbiotics supplementation represents a simple and reproducible intervention. If proven effective, probiotics supplementation in asphyxiated babies would improve clinical outcomes and reduce health cost. Hence, this study may cast a new light on the management of hypoxic-ischemic encephalopathy. Trial registration numberStudy has been registered on clinicaltrials.org on 23rd October 2019 with number NCT04145713. Trial StatusThe present trial (VISNAT Study Version 1.0 - 23 October 2019) has been approved by the Ethics Committees of the coordinator centre “Azienda Ospedaliera Universitaria Consorziale Policlinico di Bari”. Approval from the Ethics Committees of each participating hospital has also been requested. Enrollment of patients is planned to start in July 2021 and should last approximately 2 years.


Introduction Research Question
In newborns with perinatal asphyxia receiving hypothermic treatment, might the add-on of high-dose of multi-strain probiotic (SIVOMIXX TM ) reduce mortality and/or disability at 18 months of age versus placebo?

Background
Neonatal encephalopathy is still a major cause of mortality and morbidity for newborns, although hypothermia treatment has improved the prognosis of term newborns with hypoxic-ischemic (HI) injury. 1 Long-term neurological sequelae still develop in approximately 65% of all the asphyxiated newborns treated with hypothermia, yet an improvement compared to 78% neurodevelopmental morbidity in the non-treated newborns. 2 Recent evidence suggests that a cascade of cellular and biochemical responses to the initial HI insult can lead to secondary neuronal injury after re-oxygenation. 3 One of the crucial but understudied mechanisms of secondary neuronal injury after global HI is in ammation which is characterized by activation of microglia, the innate immune cells of brain; 4 migration of peripheral macrophages; 5 release of proin ammatory cytokines and chemokines, and phagocytosis of injured and uninjured neurons. 6 Some evidence suggests that blocking the in ammatory reaction promotes neuroprotection and has a potential for use in the clinical treatment of ischemic brain injury. 3 In this regard, it should be stressed that many preclinical studies have shown bidirectional interactions between the central nervous system, the enteric nervous system, and the gastrointestinal tract, suggesting a prominent role for the gut microbiota in these gut-brain interactions. 7 Current evidence suggests that multiple mechanisms, including immune, endocrine and neurocrine pathways, may be involved in gut microbiota-to-brain signalling and that the brain can in turn alter microbial composition and behaviour via the autonomic nervous system. 8 Early human studies suggest that altering the microbiota with probiotics, can lead to short-and long-term health bene ts. In particular, probiotics have being increasingly scrutinized as prophylactic 9 and therapeutical tools 10 to reduce the socio-economic burden of functional gastrointestinal diseases, 11,12 a model of disorders of Gut-Brain Interaction in children. 13 Authors have speculated that probiotics supplementation affects release of immunity-modulating molecules, with consequent health bene ts related to the gut microbiota-to-brain signalling.
There are increased chemokine gene expression and release in the developing brain after HI, some investigators have measured serum cytokines in newborns that received hypothermic treatment, showing a distinctive pattern of chemokines in the acute phase of injury. In particular, increased levels of serum interleukin (IL)-6 and IL-8 have been found in the rst 24-48 hours after birth. 14 Furthermore, recent studies have con rmed that diffusion-weighted imaging (DWI) is the most reliable MRI sequence to assess injury during the rst week after an HI event, specially to estimate the injury to the deep grey matter area that has the most predictive property in prognosticating outcome. Early detection is important for the selection of future additional neuroprotective strategies, which may need to be initiated within the rst week after birth. For these reasons some investigators have developed a novel easily applicable score based on the assessment of all MRI abnormalities of suspected importance for prognostication in infants with HI encephalopathy; this kind of score system has the advantage of including DWI, in contrast to most of the previously published MRI score system, such as Barkovich scoring system, that were designed to be performed using T1-and T2-weighted images only and therefore use scans obtained in the second week of life. 17 Considering that none of these studies have taken into account the effects of probiotics on neonates with asphyxia, our group intends to conduct a pilot study that compares the add-on of SIVOMIXX TM probiotic over the baseline therapy (hypothermia) versus placebo.
According to the Italian national guidelines on hypothermia, babies are kept nil by mouth throughout the duration of the hypothermic treatment. 18 Thus far, there is no evidence available regarding whether the type of milk started right after the discontinuation of the hypothermic treatment might in uence the patient feeding tolerance. Therefore, we also aim to assess the impact of different types of milk (breast vs arti cial milk) on gut microbiome and time to reach full feeds in asphyxiated babies after hypothermic treatment.

Methods And Analysis Study Design
The VISNAT trial is a PILOT STUDY due to absence of previous evidences in literature on using probiotic in newborns with perinatal asphyxia. It has been developed according to SPIRIT checklist (Supplementary 1). It is designed as a randomized, placebo-controlled, blinded, multicentre superiority trial with two parallel groups and a primary outcome of mortality and/or disability at 18 months of age. After informed consent (Supplementary 2) is obtained from both parents, randomization will be performed as block randomization with a 1:1 allocation using a computer-generated allocation sequence, while the allocation concealment will be performed using locked bags. Randomization data and allocation list will be stored in a secure place and will not be available to any of the components of the study apart of data collectors.
Participant les will be stored for a period of 10 years after completion of the study. All the components of the study will be blinded including: participants and their parents, healthcare providers, outcome assessors, data collectors, data analysts.

Patient and Public Involvement
Patient and Public Involvement was not sought in the design of this study.
Participants: inclusion/exclusion criteria Inclusion criteria: Newborns with gestational age >/= 35 weeks and birth weight >/= 1800 grams Intrapartum asphyxia de ned according at least one of the following: -APGAR index at 10 minutes </=5; -Resuscitation with endotracheal tube or mask IPPV for more than 10 minutes; -pH </= 7 or excess of base >/= 12 mmol/l on arterial blood gas (ABG) analysis within the rst 60 minutes of life Moderate/severe Hypoxic-ischemic encephalopathy assessed between 30 and 60 minutes of life according to Sarnat & Sarnat de nition Hypothermic treatment. According to the Italian national guidelines on Perinatal Asphyxia and Therapeutic Hypothermia, the hypothermic therapy will be started in the rst six hours of life and will last 72 hours.

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Exclusion criteria: Inability to obtain the informed consent from both parents Congenital major malformations or syndromes

Surgical diseases
No concomitant care and interventions are prohibited during the trial.

Setting and recruitment
In order to reach the prespeci ed sample size (see below 13. Statistical Issue: hypothesis, sample size and power) the VISNAT trial will involve 9 centres: . Azienda Ospedaliera Universitaria "Federico II" di Napoli (Maria Vendemmia)
The coordinator centre will be the Neonatal Intensive Care Unit "A.Mautone" of Policlinico di Bari. It is an academic department specialized in the care of newborns at risk due to several conditions such as extreme prematurity, surgical and cardiac diseases, metabolic diseases, neurological diseases and perinatal asphyxia treated with systemic hypothermia. Our research team is made of neonatologists with considerable experience in intensive care, neonatal gastroenterology and neurology, supported by a group of residents and young paediatricians constantly involved in research and clinical trials. Our team also actively collaborates with other academic units, as well as, Neuroradiology unit for brain MRI in asphyxiated infants treated with systemic hypothermia and Laboratory unit for all blood chemistry tests. During time we have performed many research protocols with scienti c papers published in peer-reviewed scienti c journal.
Each of the others centres has a Neonatal Intensive Care Unit and has been chosen based on patient recruitment capability.
After obtaining the informed consent, the healthcare providers will enrol the patients that meet the inclusion criteria and proceed to randomization. All the interventions will take place in the Neonatal Intensive Care Units.

Timeframe
The recruitment period will extend over 2 years, while the follow-up will last up to 2 years.

Intervention
The experimental intervention will consist in administrating a high-dose multi-strain probiotic (SIVOMIXX TM ) for 30 days in addition to hypothermic therapy. The administration of probiotics will begin during the rst 24 hours of hypothermia, at the dose of 1 capsule open in 5 millilitre of water. It will be administered daily for 30 days, either via nasogastric tube or orally.

Comparison
The comparison intervention will consist in administrating placebo for 30 days in addition to hypothermic therapy. The administration of placebo will begin during the rst 24 hours of hypothermia, at the dose of 1 capsule open in 5 millilitre of water. It will be administered daily for 30 days, either via nasogastric tube or orally.

Type of milk
Newborns will not be randomized for type of milk. Mothers will be encouraged to give their own breast milk. In case of unavailability of breast milk, all enrolled newborns will be fed with the same arti cial milk.
The type of milk (breast/arti cial) will be registered in clinical notes.
Clinical assessment, laboratory tests and brain imaging (Supplementary 3) 1. Weekly auxological data (weight, height, head circumference) will be recorded during hospitalisation.
Upon discharge, enrolled patients will be followed up in the outpatient service with periodical clinical evaluation. Bayley Mental Development Index (MDI) will be performed at 18 months to assess the degree of disability (no disability, mild, moderate or severe disability). A stool sample will be collected for microbiome analysis in every enrolled newborn at the end of hypothermic treatment (i.e. before the beginning of oral feeding) and at the end of probiotic treatment. The analysis of the samples will be performed by Real-time PCR at the Department of Microbiology -University "La Sapienza", Rome (Prof. Paola Mastromarino).
3. According to the Italian national guidelines on neonatal asphyxia and therapeutic hypothermia, brain MRI will be scheduled at the end of the rst week of life and during the second month of life.
Outcomes Primary outcome will be to compare mortality and/or disability at 18 month of age between the two groups.
Disability will be assessed by means of the Bayley MDI.
Severe disability will be de ned by any of the following: Bayley MDI < 70; Gross Motor Function (GMF) level of 3-5; blindness or profound hearing loss requiring ampli cation.
Moderate disability will be de ned as Bayley MDI between 70-84 and any of the following: GMF level = 2, a seizure disorder or a hearing de cit.
Mild disability will be de ned by either Bayley MDI of 70-84 alone or a MDI > 85 and any of the following: GMF level = 2, seizure disorder or hearing loss not requiring ampli cation.
Infants with Bayley MDI > 85, GMF level = 1, absence of any neurosensory de cits will be de ned as "no disability".
Secondary outcomes will be to compare in ammatory cytokines level and brain injury biomarkers (i.e. Tau and neuro lament light protein) between groups.
A subgroup analysis will be performed stratifying patients according to both probiotic/placebo treatment and breast/arti cial milk. The aim will be to assess changes in gut microbiota and compare oral tolerance, the latter being de ned as the number of days to reach full enteral feeding (140 ml/kg), in each subgroup.
Statistical Issue: hypothesis, sample size and power This is prospective superiority study. The sample size, due to absence of previous evidences in literature on using probiotic in newborns with perinatal asphyxia, was established on the bases of TOBY's results in which 45% of mortality and/or disability in the group with hypothermic therapy was observed. 19 Hypotizing a 30% (with a superiority margin of 10% ) of reduction in the proportion of mortality and/or disability in the group with a high-dose multi-strain probiotic in addition to hypothermic therapy respect to the hypothermic treatment group, a power 1-b=0.80 and a signi cant level a=0.05, a sample of 55 newborns for each group are needed. Considering a 10% of dropout we decided to enrol 120 newborns over a two-year period (60 probiotics, 60 placebo). 20 Statistical analysis will be performed by Prof. Margherita Fanelli.
The primary outcome will be evaluated comparing proportions of mortality and/or disability by means of z test. The effect of probiotics on the primary outcome will be evaluated adjusting for the centre and some clinically relevant variables (gestational age, birth weight) by means of logistic regression model. A survival analysis will be performed by Kaplan Meyer Overall Survival and Free Survival curves that will be compared by Log Rank test. Cox regression models will be built to evaluate the effects of probiotic and other clinically variables on the survival time.
The secondary outcomes will be analysed as follows: binary outcomes will be compared between the two arms using Chi-square test, in ammatory cytokines level and brain injury biomarkers will be compared between the two arms using Student t test or Mann-Whitney test, as appropriate while number of days to reach full feeds will be evaluated by Kaplan Meyer curves compared between the two arms by log rank test. Logistic regression models will be built to evaluate the effect of probiotics on the binary outcomes, adjusting for the centre and some clinically relevant variables (gestational age, birth weight, Sarnat).
A p-value less than 0.05 will be considered statistically signi cant. Statistical analysis will be performed using SPSS vers. 21.

Ethics And Dissemination
Compliance to protocol Compliance will be de ned as full adherence to protocol. Compliance with the protocol will be ensured by the local PI and collaborators in each hospital; they will be responsible for local data collection.
SIVOMIXX ™ packages of each study will be collected be the investigators at the end of treatment to assess adherence. The local PI will weekly monitor the adherence to the study protocol and will input the patients' data in an electronic data sheet. Double data entry will be performed by 2 independent members at each centre to promote data quality.

Missing data
Once an infant is enrolled or randomized, the study site will make every reasonable effort to follow the infant for the entire study period. It is projected that the rate of loss-to-follow-up on an annual basis will be at most 5%. Study site staff are responsible for developing and implementing local standard operating procedures to achieve this level of follow-up.
Investigators and study staff will be trained on the importance of the completion of the study period of enrolled patients. Parents will also be informed about this crucial aspect to reduce dropout, and a local investigator will be available at each site anytime the parents may need further information or clari cation during the study period.

Data Safety and Monitoring Board
Safety measures will include incidence, severity and causality of reported serious adverse events (SAEs), represented by changes in occurrence of the expected common neonatal complications and the development of unexpected SAEs. During systemic hypothermia the protocol to control temperature, metabolic status, haematological status, infectious disease will be applied.
SAE will be de ned as unexpected death, sepsis and enterocolitis. All SAEs will be followed until complete resolution or until the clinician responsible for the care of the recruited patient considers the event to be chronic or the infant to be stable. Any adverse event will be reported to the Principal Investigator Centre.
A monitoring board including an independent assessor (not involved in the study) from the Azienda Ospedaliera Universitaria Consorziale Policlinico di Bari and assessors from each participating hospital will review all the deaths and adverse effects. If there is a reasonable suspected causal relationship with the intervention, SAEs will be reported to the Ethics Committee to guarantee the safety of the participants. The intervention will be discontinued if any of the following occur: parents' consent withdrawal, attending physician decision or occurrence of serious adverse event possibly related to probiotic supplementation.
To maintain the overall quality and legitimacy of the clinical trial, code breaks should occur only in exceptional circumstances when knowledge of the actual treatment is absolutely essential for further management of the patient. Investigators are encouraged to discuss with the Medical Advisor if unblinding is necessary.
If unblinding is deemed to be necessary, the investigator should use the system for emergency unblinding through local emergency number.
The Investigator is encouraged to maintain the blind as far as possible. The actual allocation must NOT be disclosed to the patient and/or other study personnel including other site personnel, monitors, corporate sponsors or project o ce staff; nor should there be any written or verbal disclosure of the code in any of the corresponding patient documents.
The Investigator must report all code breaks (with reason) as they occur on the corresponding CRF (case report form) page.
Unblinding should not necessarily be a reason for study drug discontinuation.
An interim analysis will be performed on the primary endpoint and SAEs from the rst 16 infants enrolled. The interim analysis will be performed by the statistician, blinded for the treatment allocation, who will report to the principal investigator. The principal investigator will discuss the results of the interim analysis with the monitoring board and the trial will be ended in case of harm. Criteria for stopping for harm include: a statistically signi cant difference in the primary outcome between the treatment groups; a reasonable suspected causal relationship between the intervention and SAEs.

Con dentiality
Only local PI will have access to the electronic database with an assigned personal account and password. Subjects will be identi ed by sex, birth date, and assigned trial number, during and after the trial, in accordance with personal data protection law.

Access to data
The principal investigators of each site will have complete access to the nal trial dataset, and no contractual agreement exists to limit such access for the investigators.

Consent/assent
Sub-investigators will introduce the trial to patients. Patients will also receive information sheets. Patients will then be able to have an informed discussion with the sub-investigators, who will obtain written consent from patients willing to participate in the trial. Information sheets and consent forms are provided for all parents involved in the trial.
Other issue (registration, sponsor etc.) The VISNA trial will involve all the parts cooperatively. A preliminary meeting intended to clarify the design, methods and outcomes of the study will be the rst step of the protocol. Teaching materials will be designed, realised and distributed to all the members of the research team. Monthly meetings will be planned to discuss any aspects of the protocol. Training sessions will take place in the teaching room of Neonatal Intensive Care Unit "A. Mautone" of Policlinico di Bari and will be chaired by the Principal Investigator together with the collaboration of co-investigator.
Study has been registered on clinicaltrials.org on 23rd October 2019 with number NCT04145713.
The Investigational Product (probiotic mixture) and placebo will be supplied by Ormendes SA.

Strengths And Limitations Of This Study
This study comprises a well-powered clinical trial investigating the additional effect of probiotics on the neurodevelopmental outcome of asphyxiated neonates treated with hypothermia.
A strength of the trial is the pragmatic nature of the study and applicability in daily clinical practice.
The multicentric nature of the study enables to assess the reproducibility of the intervention. This study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and will be consistent with Good Clinical Practice (GCP) and applicable regulatory requirements.
The study will be conducted in compliance with the protocol. The protocol and any amendments and the informed consent will receive Ethics Committee (EC) approval/favourable opinion prior to initiation.
Freely given written informed consent must be obtained from every participant's parent or legally authorized representative prior to clinical trial participation.
The rights, safety, and well-being of the trial participants are the most important consideration and should prevail over interests of science and society.
Patients that are enrolled into the study are covered by local insurance policies.
Study personnel involved in conducting this trial will be quali ed by education, training, and experience to perform their respective task(s).
The trial has been approved by the Ethics Committees of the coordinator centre "Azienda Ospedaliera Universitaria Consorziale Policlinico di Bari" (Supplementary 4) and will be sent to the Ethics Committees of each participating hospital. We have planned to start enrollment of patients in July 2021.
Any modi cations to the protocol which may impact on the conduct of the study, potential bene t of the patient or may affect patient safety, including changes of study objectives, study design, patient population, sample sizes, study procedures, or signi cant administrative aspects will require a formal amendment to the protocol. Such amendment will be agreed upon by all Principal Investigators, and approved by the Ethics Committee prior to implementation.

Consent for publication
The results of the trial are expected to be published in a scienti c journal and to be presented in medical seminars and conferences. The nal reporting will follow the CONSORT Report guidelines (http://www.consort-statement.org).
The VISNAT Participant Box will list all professionals that have participated in the VISNAT study for a minimum of one year.

Availability of data and materials
The data that support the nding of this study will be available from the PI (M.E.B.) upon reasonable request.

Competing interests
The authors declare no con ict of interest.
Funding statement