There is a growing appreciation for the symbiotic relationship between humans and their microbiome to support healthy development. Early microbial colonizers of the gastrointestinal tract play a pivotal role in the type of microbial community structure that forms during infancy and childhood. However, this process can be interrupted by several pre-, peri-, and postnatal factors that commonly occur in early life, such as cesarean section (C-section) delivery, antibiotic exposure, and formula feeding [1,2]. This is especially true for infants born preterm (<37 weeks gestation) who may experience a number of aberrant prenatal and postnatal exposures, combined with their structurally and functionally immature gut [3-5]. Alterations to the gut microbiota in preterm infants are common and associated with an elevated risk of numerous disorders, including necrotizing enterocolitis, late-onset sepsis, and impaired neurodevelopment [3-7]. However, the specific microbiome features involved in acute and long-term disease development, and the underlying mechanisms that lead to disease progression and poor neurodevelopment, represent one of the least understood frontiers in pediatric care.

Being born preterm is one of the leading causes of pediatric morbidity and mortality worldwide [8,9]. In Canada alone, 8% of infants are born preterm (approximately 29,000 infants every year), with the earliest gestational ages having the highest risks of health complications [10,11]. Prematurity is a spectrum, with the earliest born infants classified as extremely preterm (<28 weeks gestation), followed by very (28‐32 weeks), moderately (32‐34 weeks), and late (34‐37 weeks) preterm. Unsurprisingly, infants born extremely and very preterm are more likely to experience greater clinical interventions than those born at later gestational ages, such as greater antibiotic administration, respiratory support (eg, mechanical ventilation), intravenous and enteral tube feeding, stress-inducing procedures (eg, surgery and frequent blood draws), reduced contact with parents, and extended hospital stays [12]. Furthermore, being born preterm predisposes individuals to developing numerous health complications into childhood and adulthood, including asthma, metabolic syndrome, and impaired neurodevelopment [13-17]. The earlier an infant is born, the greater the risk of these health complications along with poorer rates of employment, lower income, and reduced enrollment in higher education [13-18]. Therefore, interventions aimed at targeting microbiome development and the health of preterm-born children will depend upon their gestational age at birth and associated early-life exposures, with important implications for their overall health and well-being throughout life.

The term “microbiome” encompasses microbial communities, along with their functions (eg, metabolites) in the surrounding environment [19]. Microbiomes reside in numerous body sites including, but not limited to, nasal, oral, vaginal, and skin microbiomes; however, the gut is the most widely studied microbiome to date.

During the first months of life, the patterns of gut microbiome development in preterm infants are altered compared to term-born infants, which becomes more pronounced with earlier gestational ages at birth [20,21]. There are numerous reasons for this, including immaturity of the gastrointestinal and immune systems, higher likelihood of antibiotic exposure pre- and postnatally, greater likelihood of C-section delivery, increased risk of exposure to pathogenic microbes pre- and postbirth, reduced contact with parents, and reduced mother’s milk feeding—all of which are associated with altered early colonization patterns of the infant gut [3-5]. In order to gain a thorough understanding of these altered microbial patterns, a comprehensive evaluation of perinatal factors (eg, maternal microbiome, pregnancy complications, delivery mode, nutrition, and antibiotics) and their effects on the preterm microbiome, metabolome, and immune development over time is needed.

Microbiomes established in early life have the potential to shape the trajectory of microbial development and sculpt the immune system to thereby influence health outcomes into childhood [2,22]. For instance, the nasal microbiome during the first few weeks postnatally has shown links to bronchopulmonary dysplasia risk in preterm infants later in hospitalization [23]. For the gut microbiome, it is thought to stabilize by approximately 3 years of age, offering an early window of opportunity to modify [1,2]. Although the gut microbiome of preterm- versus term-born children generally converges over the first 1‐3 years of life, there are often still microbial and metabolite signals that can differentiate these gestational age groups, which are thought to be driven by pre- and postnatal exposures (eg, delivery mode, human milk feeding, and pharmacological treatments) [24-28]. This is especially true among preterm infants who experience serious morbidity during the neonatal period, such as necrotizing enterocolitis [29]. Furthermore, the early-life gut microbiome in preterm infants has been associated with later health outcomes into childhood. For instance, Rozé et al [30] surveyed 577 very preterm infants across France and found that gut microbiome patterns dominated by Enterococcus, Staphylococcus, or low bacterial loads at 4 weeks postnatally were associated with a greater risk of suboptimal neurodevelopment (as measured by the Ages & Stages Questionnaires, Second Edition [ASQ-2]), cerebral palsy, and/or death by 2 years corrected age (CA).

Asthma is a leading cause of school absenteeism, pediatric hospitalizations, and adult work loss [31-33]. This debilitating disease affects approximately 1 in 9 Canadian children with a total economic burden of CAD $2.20 billion/year in 2010 (CAD $1=US $1.02 as of November 1, 2010)—an amount that is expected to grow to CAD $4.20 billion by 2030 [34,35]. In Canada and globally, preterm infants are at 1.7 times greater risk of developing childhood respiratory disorders, including asthma, with those born extremely or very preterm having a 3 times greater risk than infants born at term, likely due to several mechanisms that are unique to the preterm population [36]. Lung immaturity and immune dysregulation, combined with the need for respiratory support, can induce inflammation and long-term structural lung damage, subsequently leading to bronchopulmonary dysplasia [37]. Preterm infants also experience immune system immaturity, rendering them susceptible to respiratory infections. In fact, experimental animal models that use antibiotics to shift maternal and neonatal microbial communities suggest that, mechanistically, these microbial shifts can have a profound effect on the stereotypic development of lymphocyte and innate immune cell subsets, with long-term consequences in adult responses and susceptibility to disease [22]. Lastly, exposure to stress (eg, medical procedures and enhanced exposure to sensory stimuli in the neonatal intensive care unit) can alter microbiota-to-brain signaling via dysregulation of the hypothalamic-pituitary-adrenal stress axis. Together, their immature immune system and external stress exposures can alter asthma susceptibility [38-41]. Recent studies have provided evidence of a causal link between early-life gut microbiome alterations and the development of asthma; however, these have largely excluded preterm infants, despite them being at the highest risk of developing asthma [42-46].

Although preterm infants are at an elevated risk of immune dysregulation and asthma, there is little data exploring their risk of developing a food or environmental allergy. Findings from observational cohort studies have been mixed, although generally showing a lower risk of IgE sensitization with earlier gestational ages [47-50]. However, from a microbiome perspective, recent findings from the Canadian Health Infant Longitudinal Development (CHILD) cohort (>1000 children across Canada) have shown that delays in microbiome development at 1 year strongly predict allergy diagnoses by the age of 5 in term-born children [51]. Given the unique postnatal exposures and altered patterns of microbial colonization observed among preterm infants, the crosstalk between microbial species, their metabolic byproducts, and the mechanisms relevant to asthma and allergy development in preterm infants may differ from those born at term.

Preterm birth is a significant determinant for impaired neurodevelopment in infancy and into adulthood [52]. Similar to asthma, infants born preterm are significantly more likely to develop neurodevelopmental issues than their term-born counterparts, including cerebral palsy, developmental disabilities (eg, intellectual/cognitive delays, speech and language disorders, and neuro-motor disorders), attention-deficit/hyperactivity disorder, autism spectrum disorder, and psychiatric disorders in adulthood [53-60]. Head circumference growth is often used as a proxy for brain development, with some evidence showing slower growth being linked to lower total cerebral volumes and poorer neurodevelopment in childhood [61,62]. Although impaired neurodevelopment in preterm infants is multifactorial, the gut microbiome and its actions via the gut-brain axis are thought to play an important role. In a recent narrative review by Beghetti et al [7], specific microbial signatures (eg, pathogenic microbes and lower Bifidobacterium abundance) were linked to head circumference growth, performance on developmental scales, and clinical measures (eg, electroencephalogram) across different preterm infant cohorts in early life and into childhood. This is likely due to mechanisms including microbially derived metabolites and hormones, and the induction of proinflammatory cytokines (particularly from pathogenic microbes and infections), which can all mediate brain maturation [7].

The overarching goals of this cohort are to comprehensively study microbiome development in preterm infants and understand how microbial patterns during this critical early-life window contribute to development and disease risk in childhood. The primary objective for this cohort is to profile microbiome development from birth to 3.5 years CA in children born preterm (≤36^+6/7 days gestation) and compare it to children born at term.

The secondary objectives are (1) to determine how perinatal factors are associated with microbiome development in children born preterm versus at term and (2) to determine the links between microbiome development and infant outcomes at 1- and 3-years CA, including the Asthma Predictive Index and other atopy such as food and environmental allergies.

Exploratory objectives are (1) to examine the role of environmental, nutritional, and pharmacological exposures in the development of the preterm and term infant microbiome, metabolome, and immunobiome; (2) to explore correlations between the gut microbiome, early-life stress, and fronto-amygdalar brain connectivity at 1 month CA; and (3) to establish how microbiome development in preterm infants is associated with head circumference at term CA and with neurodevelopment at ages 1 to 5.5 years.

We hypothesize that specific microbial patterns and/or metabolites will be significantly associated with prenatal, maternal, and/or infant factors (or combinations of factors). As well, we hypothesize that microbial alterations resulting from preterm birth causally contribute to the higher rates of allergies and asthma observed in preterm infants through immune mechanisms, which differ from those in infants born at term. Lastly, we hypothesize that the microbiome in infants born preterm is associated with head circumference growth at term age and improved neurodevelopment at ages 1 to 5.5 years.

The Alberta BLOOM (Begin a Life of Health With Observation and Optimization of the Microbiome) study was approved by the University of Calgary Conjoint Health Research Ethics Board under protocols REB17-1877 (BLOOM–Preterm Neonate Study), REB19-1780 (BLOOM–Long-Term Follow-Up Study), REB20-1442 (BLOOM–Premature Child Study), and REB19-0613 (BLOOM–Brain Magnetic Resonance Imaging substudy). All study procedures were conducted in accordance with the Declaration of Helsinki and applicable institutional and national ethical guidelines.

Written or electronic informed consent was obtained from the parent or legal guardian of each participating child prior to study enrollment. Participants were informed of the study objectives, procedures, potential risks and benefits, the voluntary nature of participation, and their right to withdraw at any time without affecting their medical care. A copy of the signed informed consent form was provided to participating families.

Participant privacy and confidentiality were protected by assigning each participant a unique study identification number used for all study records and biological samples. Personal identifiers were stored separately from research data in secure, access-restricted systems, with access limited to authorized study personnel. Electronic data were stored on secure University of Calgary servers, hard-copy records were maintained in locked facilities, and all biological samples were deidentified prior to analysis and any future sharing for ethically approved research.

Participants received modest compensation to acknowledge their time and participation. Families enrolled shortly after birth received gift cards upon enrollment and completion of the 2-week and 2-month questionnaires, participating children received a small gift at the 3-month corrected age visit, and families attending the 1-year and 3-year follow-up visits received CAD $50 to offset expenses such as parking, transportation, and time. Participants in the neurodevelopment arm also received a CAD $15 gift card following completion of each developmental assessment, CAD $25 gift cards upon enrollment and completion of the 2-week and 2-month questionnaires, participating children received a small gift at the 3-month corrected age visit, and families attending the 1-year and 3-year follow-up visits received CAD $50 to offset expenses such as parking, transportation, and time.

The neonatal intensive care unit (NICU) at Foothills Medical Centre is a Level III NICU, where infants needing the highest level of intensive care are admitted. Pregnant individuals who go into labor at <32 weeks of gestation in the Calgary region are sent to Foothills Medical Centre (if possible) to deliver and infants born <32 weeks gestation are generally admitted to the Foothills Medical Centre NICU immediately following birth. Pregnant individuals who go into preterm labor at >32 weeks gestation may deliver at one of the Calgary hospitals with Level II NICUs, including Peter Lougheed Centre, South Health Campus, or Rockyview General Hospital. For the BLOOM studies, preterm-born participants will therefore be recruited from the NICU at Foothills Medical Centre, in addition to NICUs at the Peter Lougheed Centre (as well as their Pediatrics Unit), South Health Campus, and Rockyview General Hospital.

Study team members will screen admissions to participating hospitals to identify eligible infants born preterm. For all potential study participants, the first approach will be made by a member of the infant’s primary care team with an existing relationship with the child’s caregiver, who will request permission to be approached by a member of the study team. If the caregiver agrees, the research study team member will then approach the family. For BLOOM-LTFU and/or PCS, previous participants of PROBIO and PTN who consented to be contacted for future research will be invited via email and/or telephone. For previous participants of the PROBIO and PdP studies, the first approach will be conducted by a member of the PROBIO or PdP study team, respectively, to ask if they are willing to be contacted by a member of the BLOOM study team to discuss the study.

For the BLOOM-PCS Neurodevelopment arm, a member of the study team will invite previous BLOOM participants (PTN, LTFU, PCS, and/or PROBIO) who provided consent to contact for future research and meet the eligibility criteria. Families will be contacted via email, mail, text, and/or phone for participation invites.

For the BLOOM-MRI substudy, a member of the BLOOM study team will contact the families of potential BLOOM participants (PTN and PCS) between 36 and 40 weeks CA via phone, email, and/or mail to introduce the study and provide an informational pamphlet. Caregivers of potential participants will be subsequently contacted between term CA and up to 6 weeks CA to be invited to join the study and screen for eligibility. Only participants who agreed to be contacted for future studies will be approached.

Written or electronic informed consent will be obtained from caregivers to participate in the BLOOM studies. Generally, this consent will be obtained from the birthing parent to access their medical chart and their child’s. The nature of the research study will be explained to caregivers, and a copy of the signed informed consent form and contact information for the investigators will be provided. Caregivers will be advised that their participation is voluntary, and they may withdraw from the study at any time. Once consent is obtained, a study identification number will be assigned, and all study data and subsequent specimen collection will commence. In cases where in-person written consent cannot be obtained at that time (eg, caregiver illness preventing them from visiting the unit, caregiver requires additional time to consider the study and would prefer to be contacted later), a consent to contact form will be completed to allow the research team to contact the participant in-person or via telephone, text, or a secure virtual communication tool, depending on the caregiver’s preference. Informed consent will be obtained thereafter.

Participants will be advised that their involvement is voluntary and they may withdraw from the BLOOM studies at any time upon request. Participants who choose to stop participating in this study will have no new data collected or linked to other data that becomes available after their withdrawal date. The study may access, collect, and use data and samples that are available up to the withdrawal date, unless the participating family requests otherwise. Upon withdrawal, participants will be contacted to determine whether they still consent to the completion of all required data extractions from medical databases up to the withdrawal date, and/or permit the study to continue using the data and samples collected during their participation. If participants request that their collected data or samples be removed from the study, any data or samples that have not been processed to remove identifying information will be destroyed. Participants will be made aware that any data or samples that have already been analyzed, processed to remove identifying information, published, or linked with other data cannot be removed.

Investigators may also terminate a study participant’s involvement in the study if a medical condition, event, or situation occurs such that continued participation in the study will not be in the best interest of the participant, or if the participant meets an exclusion criterion (either newly developed or not previously recognized) that precludes further study participation.

Clinical data are extensively recorded in paper and/or electronic medical records and the Alberta Health Services databases. Data extraction from these electronic resources will be used to ascertain necessary data including, but not limited to, birth characteristics (eg, gestational age at birth, delivery mode, premature rupture of membranes, and small for gestational age at birth), infant acuity scores (eg, Score for Neonatal Acute Physiology-II), infant clinical testing parameters (eg, heart rate, blood pressure, and urine output), maternal history and characteristics (eg, pregnancy complications and socioeconomic status), infant/maternal medications (eg, antibiotics, probiotics, and corticosteroids), infant feeding (eg, daily enteral nutrition, vitamin/mineral supplementation, and breast/chest/bottle feeding), infant diagnoses (eg, necrotizing enterocolitis, sepsis, bronchopulmonary dysplasia, and brain injury), infant procedures (eg, surgery and hospitalizations), child health care visits (eg, vaccinations and weight gain), and laboratory results (eg, bilirubin and complete blood count with differential). A summary of key data collected from BLOOM participants and their families is shown in Table 2.

REDCap will also be used to collect and store study data, especially when families have been discharged home and their information is no longer entered into electronic medical charts for data extraction [67,68]. This includes data tracking consent, continued participation in BLOOM, samples collected, study questionnaires, and descriptive data (eg, demographics, medications, and feeding).

All hard copy data will be stored in a locked, secure location with access limited to authorized study personnel. Electronic data are securely stored on university servers with authenticated access for restricted and confidential access. All data (hard copy and electronic) will be retained for a minimum of 15 years after the project is closed and in accordance with the University of Calgary’s Data Retention Policy.

At 1‐2 years CA and 3‐3.5 years CA, participating children in LTFU and PCS attend follow-up visits at the Alberta Children’s Hospital Pediatric Infectious Diseases Clinic to undergo multiple tests.

This study arm involves the completion of the Ages & Stages Questionnaires, Third Edition (ASQ-3) and the Ages & Stages Questionnaires: Social-Emotional, Second Edition (ASQ:SE-2) by participating parents and their children during the first years of life [70,71]. The ASQ-3 is a developmental screening tool that asks parents a set of questions about their child’s development, including communication, gross motor, fine motor, problem-solving, and personal-social development [70]. The ASQ:SE-2 focuses on screening social and emotional behaviors in young children by asking questions specific to self-regulation, compliance, social-communication, adaptive functioning, autonomy, affect, and interaction with people [71].

Parents will complete the online questionnaires using the study’s secure and validated electronic data capture tool, REDCap. Each questionnaire will take 15‐45 minutes to complete. Accommodations will be made for parents who prefer to complete paper questionnaires. These developmental screening tools are not diagnostic and are intended to answer research questions; however, the results may identify areas that may benefit from a formal assessment by a qualified professional. After the completion of each questionnaire, results will be calculated immediately by REDCap, and caregivers will be presented with the option to view and save their child’s results to learn how they compare to the cutoffs. The number of ASQ-3 and ASQ:SE-2 administered to participants will depend on the child’s CA at enrollment (Figure 2B). The five administration intervals include 1-year (<13 months CA), 2-years (13‐25.5 months CA), 3-years (25.5 to <39 months CA), 4-years (39 to <51 months CA), and 5-years (51 to <66 months CA).

To support participant engagement and retention, participating families will receive study reminders about upcoming clinic visits, and required sample collection and questionnaire completion, when applicable. Reminders will be sent via telephone, text, email, and/or the secure REDCap database. A description about the studies is available on the ClinicalTrials.gov and AlbertaBLOOM.ca websites, which participants will be able to access at any time. These websites do not include information that can identify participants; instead, they include resources such as a study overview and sample collection videos, in addition to links to published results.

Participating children (from PTN and PCS-Arm A) will remain in the study when discharged from the hospital, and samples will continue to be collected at home per the study schedule (Figure 3). A courier will transport home-collected samples from the participant’s home to the Arrieta Lab. Weekly phone calls (or via paper or electronic formats, if preferred) will also be made to the participants by the research team to ask about infants’ anthropometrics, feeding, supplements, and medications up to 60 days after birth.

Caregivers will be contacted by the research team to schedule 3-month (PTN/PCS), 1-year (LTFU/PCS), and 3-year (PCS) CA clinic visits, when applicable, as well as complete the ASQ-3 and ASQ:SE-2 (PCS-neurodevelopment arm) at each 1-year timepoint until 5.5 years CA.

This cohort is also focused on setting up a biobank and data repository. Therefore, as described in the informed consent form for the study, data and samples will be stored for at least 15 years. The cohort will facilitate storage and reuse of study data through a secure data repository to facilitate future research, in addition to allowing linkage of study data to other datasets. Personal information (eg, name, address, and telephone number) will be removed prior to sharing with other researchers. Researchers who wish to use study data must (1) have their new study approved by an ethics board and (2) sign an agreement that ensures confidentiality and restricts data use to only the approved study. Study data may then be linked with other cohort data to enable further research. This information will be stored on a secure, isolated server, and use of this information will only link data to other datasets.

All samples and any related documentation in the BLOOM studies will be labeled with a study identification number. All biological samples will be stored in freezers in a secure research facility. The labeling of the samples will be done with a study identification number without identifiers such as name, health care number, or initials. The master list, which matches names to identification numbers, will be kept in a separate and secure location that can be accessed only by authorized study personnel. Only study team members will have access to the personal information collected in this study.