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Longitudinal body mass index trajectories at preschool age:

children with rapid growth have differential composition of the
gut microbiota in the first year of life
Myrtha E. Reyna1,12, Charisse Petersen 2,12, Darlene L. Y. Dai2, Ruixue Dai1, Allan B. Becker3,4, Meghan B. Azad 3,4,5, Kozeta Miliku6,
Diana L. Lefebvre6, Theo J. Moraes1, Piushkumar J. Mandhane7, Rozlyn C. T. Boutin 8, B. Brett Finlay8,9,10, Elinor Simons3,
✉
Anita L. Kozyrskyj7, Wendy Lou11, Stuart E. Turvey2,13 and Padmaja Subbarao 1,6,11,13

© The Author(s) 2022

BACKGROUND/OBJECTIVE: The steep rise in childhood obesity has emerged as a worldwide public health problem. The first 4
years of life are a critical window where long-term developmental patterns of body mass index (BMI) are established and a critical
period for microbiota maturation. Understanding how the early-life microbiota relate to preschool growth may be useful for
identifying preventive interventions for childhood obesity. We aim to investigate whether longitudinal shifts within the bacterial
community between 3 months and 1 year of life are associated with preschool BMI z-score trajectories.
1234567890();,:

METHODS: BMI trajectories from birth to 5 years of age were identified using group-based trajectory modeling in 3059 children.
Their association with familial and environmental factors were analyzed. Infant gut microbiota at 3 months and 1 year was defined
by 16S RNA sequencing and changes in diversity and composition within each BMIz trajectory were analyzed.
RESULTS: Four BMIz trajectories were identified: low stable, normative, high stable, and rapid growth. Infants in the rapid growth
trajectory were less likely to have been breastfed, and gained less microbiota diversity in the first year of life. Relative abundance of
Akkermansia increased with age in children with stable growth, but decreased in those with rapid growth, abundance of
Ruminococcus and Clostridium at 1 year were elevated in children with rapid growth. Children who were breastfed at 6 months had
increased levels of Sutterella, and decreased levels of Ruminococcus and Clostridium.
CONCLUSION: This study provides new insights into the relationship between the gut microbiota in infancy and patterns of growth
in a cohort of preschool Canadian children. We highlight that rapid growth since birth is associated with bacteria shown in animal
models to have a causative role in weight gain. Our findings support a novel avenue of research targeted on tangible interventions
to reduce childhood obesity.
International Journal of Obesity (2022) 46:1351–1358; https://doi.org/10.1038/s41366-022-01117-z

INTRODUCTION the microbiota both directly shapes nutrient availability within the
The steep rise in rates of obesity in the last decades has emerged gut as well as regulates host metabolic pathways responsible for
as a worldwide public health problem from which children are not transporting and storing those nutrients [7–10]. A number of
exempt [1]. Obesity as early as 6 months of age has been reported studies have investigated the role of the microbiota in obesity in
to track into school age [2], and has been linked to increased risk adult humans and animal models, even going so far as to develop
of many adverse health conditions in adolescence and adulthood weight loss therapies using microbial candidates such as
including cardiometabolic, pulmonary, and psychological disor- Akkermansia muciniphila [11–14]. However, studies focusing on
ders [3–6]. children are limited to cross-sectional indicators of obesity, and
The collective consortia of resident bacteria and their functional only a few analyzed the relationship between the gut microbiota
genetic capacity making up the microbiota and microbiome, and longitudinal changes in growth [15].
respectively, is a key regulator of host metabolism and obesity. Longitudinal studies of childhood growth are essential due to
With an estimated 1012 bacteria residing along our digestive tract, the individual variations in patterns of weight over time, and

1
Translational Medicine Program, Department of Pediatrics, The Hospital for Sick Children, Toronto, ON, Canada. 2Department of Pediatrics, BC Children’s Hospital, University of
British Columbia, Vancouver, BC, Canada. 3Section of Allergy and Immunology, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada.
4
Developmental Origins of Chronic Diseases in Children Network (DEVOTION), Children’s Hospital, Winnipeg, MB, Canada. 5Department of Food and Human Nutritional Sciences,
University of Manitoba, Winnipeg, MB, Canada. 6Department of Medicine, McMaster University, Hamilton, ON, Canada. 7Department of Pediatrics, University of Alberta,
Edmonton, AB, Canada. 8Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada. 9Michael Smith Laboratories, UBC, Vancouver, BC,
Canada. 10Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada. 11Dalla Lana School of Public Health, University of Toronto,
Toronto, ON, Canada. 12These authors contributed equally: Myrtha E. Reyna, Charisse Petersen. 13These authors jointly supervised this work: Stuart E. Turvey, Padmaja Subbarao.
✉email: [email protected]

Received: 3 August 2021 Revised: 23 March 2022 Accepted: 25 March 2022

Published online: 15 April 2022

Content courtesy of Springer Nature, terms of use apply. Rights reserved

 M.E. Reyna et al.
1352
have been shown to have better predictive value over cross- models, GBTM classifications were used in all subsequent analyses. Details
sectional measures for future body composition and obesity [16]. on trajectory modeling can be found in the Supplementary Material.
Furthermore, obesity in the first years of life has been associated
to genetic and early-life environmental factors such as pre- Stool sample collection and microbiota analysis
pregnancy maternal body mass index, gestational weight gain, Fecal samples were collected at a home visit (3–4 months; mean [SD], 3.7
feeding practices (i.e., breastfeeding and formula use), and [1.1] months) and a clinic visit (12 months; mean [SD], 12.5 [1.7] months);
socioeconomic adversity [17, 18]. Early life also represents a DNA was extracted using the commercial kits (Qiagen Mo Bio PowerSoil)
critical window for microbiota maturation, when exogenous optimized for the Thermofisher KingFisher® robot.
factors such as mode of delivery, diet, antibiotics exposure, and Our approach to defining the gut microbiota of infants of the CHILD
cohort has been previously described [24, 25]. Briefly, the V4 hypervariable
built environment heavily influence bacterial membership and
region of the 16S rRNA gene of fecal DNA was amplified by PCR using
composition within the gut [19]. This results in a dramatic universal bacterial primers (V4-515f: V4-806r). Paired-end sequences were
expansion of diversity as well as fluctuations in bacterial pre-processed using Dada2 in Qiime2 v.2018.6 (www.qiime2.org). Taxo-
colonization that stabilizes after 1–3 years of age [19, 20]. The nomic identity was assigned to the resulting Amplicon Sequence Variants
early-life microbiota has been associated with a number of by alignment to the Greengenes reference (v13.8) database at 99%
disorders that appear later in childhood, however only a few sequence similarity. Sequences were further filtered to remove those that
studies have focused on longitudinal changes within the infant were present at less than 0.005% of the total sequences. Demultiplexed
bacterial community. Since transitions within the infant diet also sequencing data used in this study are deposited into the Sequence Read
occur during this time (e.g., solid food introduction and Archive of NCBI and can be accessed via accession numbers PRJNA657821.
reduction of breastmilk/formula), understanding how shifts Samples were rarefied to 8000 sequencing reads per sample prior to
computing all diversity metrics. Gut microbiota α-diversity was measured
within the early-life microbiota relate to rapid growth or obesity by Shannon index. Changes in microbiota diversity over time were
may be particularly useful for identifying potential preventive calculated in paired samples as the difference between 3 months and 1
interventions. year of age. Analyses were completed in R software, version 4.0.2.
This study will investigate whether BMIz trajectories of growth
up to age 5 years are associated with longitudinal shifts within the
Statistical analyses
bacterial community between 3 months and 1 year of life. We Differences in continuous measures between weight trajectories are
used a data-driven approach to develop longitudinal preschool presented as median [Interquartile range] and evaluated by Wilcoxon rank
BMIz trajectories in children enrolled in the CHILD Cohort Study. In test or Kruskal–Wallis test were appropriate. Differences in categorical
a subsample of the cohort, we quantified changes in the infant gut variables are presented as frequencies (%) and evaluated by Pearson’s Chi-
microbiota diversity and composition and determined whether square test, post hoc tests for multiple comparisons of groups were
these changes were associated with rapid growth culminating in performed by Dunn test. p values were adjusted for multiple comparisons
overweight/obesity by age 5 years. using the Benjamini–Hochberg procedure.
After identification of BMIz trajectories, adjusted odds ratios (OR) and
95% confidence intervals (CI) from weighted multinomial regression were
used to examine factors associated with each trajectory. Weights were
METHODS based on individual posterior probabilities. All models were adjusted for
Study design prenatal smoke exposure, self-reported ethnicity (Caucasian vs. non-
The CHILD Cohort Study is a population-based prospective birth cohort Caucasian), study site, and postpartum maternal BMI (measured when the
study in Canada. It recruited 3621 pregnant mothers across four sites in child was 1 year of age). Next, the relationship between significant risk
Canada (Vancouver, Edmonton, Manitoba, and Toronto) between 2008 and factors from adjusted models in relation to BMIz trajectories and the gut
2012, from which 3455 delivered healthy, full-term infants, and were microbiota was analyzed by Kruskal–Wallis test for categorical factors and
eligible to commence the study. The CHILD Cohort Study has been Spearman correlation for continuous factors.
described in detail elsewhere [21]. This study was approved centrally by To identify potential bacterial genera driving significant differences
the Hamilton Integrated Research Ethics Board (HiREB #07-2929) and all between trajectories, DESeq2 on raw count data with relative log
local ethics boards, and informed consent was obtained from a parent or expression normalization was applied, models were adjusted by self-
legal guardian. reported ethnicity and study site. Genera that statistically discriminated
between children with normative and high stable or rapid growth
trajectories were selected. Ordinal logistic regression adjusted for
Anthropometric measurements
ethnicity and study site was applied to analyze diversity trends of the
Birthweight of all children was collected from birth charts. All subsequent
weight and height measurements were performed by trained research selected genera across BMIz trajectories. Changes in genera with
assistants using standardized procedures, at a home visit when children significant trends were further contrasted to the rapid growth trajectory.
were 3 months of age, and at clinic visits when children were 1, 3 and 5
years old. Shoes and outerwear were removed for weight (Scaletronix
scale) and height (standard stadiometer) measurements. RESULTS
BMI at each time point was calculated and age and sex BMI z-scores Growth trajectories
(BMIz) were derived according to the World Health Organization (WHO) We modelled BMIz trajectories for 3059 participants within the
child growth standards for children younger than 5 years [22], and for 5–19 CHILD Study (53.1% boys) who had at least three BMIz
years for those children that were slightly older than 5 years old at the date measurements available between birth and age 5 years. BIC and
of measurement [23].
AIC statistics improved with increased number of classes in both
LCMM and GBTM. The four-trajectory model from GBTM was
Growth trajectories favored based on approximation to the log of the Bayes factor,
Growth trajectories were identified by group-based trajectory models and median membership probability >0.70 for all classes in both
(GBTM) and latent class mixed models (LCMM). Exact age at the time of boys and girls (Supplementary Table 1). Trajectories from both
measurement (and gestational age at birth) and BMIz at birth, 3 months, 1, sexes had similar trends over time and were pooled together into
3 and 5 years were included in the analysis. Modeling was restricted to one dataset for further analyses (Supplementary Fig. 1). The four
subjects with measurements available for at least three of the five time
trajectories were designated: low stable, normative, high stable
points, and was performed separately for boys and girls. Model
performance was evaluated from two to eight trajectories. Optimal
and rapid growth (Fig. 1).
number of classes was chosen based on Bayes information criteria (BIC), The normative trajectory contained 60% of participants (n =
Akaike information criteria (AIC), Log Bayes Factor (2loge(B10)), median 1849), mean BMIz in this group started at −0.5 BMIz and trended
posterior probability of assignment of at least 0.70, and on trajectories toward zero by age 5 years. A total of 289 (9.4%) participants
being distinct and interpretable. After comparison of trajectories between were placed in the low-stable weight trajectory, which followed a

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 M.E. Reyna et al.
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Fig. 1 BMIz trajectories in the CHILD Cohort Study. a Predicted BMI z-score trajectories obtained from group-based trajectory modeling, and
b proportion of overweight, obese children at 5 years per World Health Organization classification of BMI z-scores.

steady trend of −1 z-scores up to 5 years of age. The high stable diversification may limit overall diversity at 1 year. Moreover, after
trajectory had 782 (25.6%) participants and a mean BMIz of 0.5 quantifying changes in bacterial diversity over time, we observed
up to 3 months, which increased to 1 BMIz at age 1 year. By age 3 that infants in the rapid growth trajectory gained less diversity (as
years, 12% of subjects in this trajectory were classified as measured by Shannon Index) between 3 months and 1 year of age
overweight, and 0.7% as obese. Finally, the rapid growth compared to the normative BMIz trajectory (Fig. 3b, c). These
trajectory contained 139 (4.5%) participants and was character- differences in diversity were not significant at either 3 months
ized by a rapid increase in BMIz from birth, with a mean over 2.5 (p = 0.10) or 1 year (p = 0.65) time points alone, but instead
BMIz by age 3 years. Over 70% children in the rapid growth represent a cumulative change in diversification within the gut
trajectory were overweight or obese by age 3 years, and 89% by and emphasize the value of longitudinal sampling.
age 5 years (Fig. 1). We performed a differential abundance analysis to understand
We analyzed familial and environmental risk factors of early-life whether the reduced diversification observed in the rapid growth
overweight and obesity to identify whether they were significantly trajectory coincided with changes in specific bacterial taxa. A
associated to a certain BMIz trajectory. Adjusted models showed number of genera were significantly altered at the 1 year visit, but
that children not breastfed at 3 or 6 months of age had 70% none at the 3 months visit, including Akkermansia, unclassified
higher odds of belonging to the rapid growth trajectory, Enterobacteriaceae members, Clostridium, Sutterella, and Rumino-
compared to the normative trajectory (3 months BF aOR 1.70, coccus (Fig. 4a).
95% 1.09, 2.64; 6 months BF aOR 1.67, 95% CI 1.13, 2.47) while To understand whether any of the observed taxa may be
increased duration of breastfeeding (in months) was associated associated with the other weight trajectories in these children,
with decreased risk of rapid growth (Fig. 2). We observed similar we performed ordinal logistic regression, including all BMI-z
associations in the high stable trajectory, although only nominally trajectory groups as outcome and the change in abundance of
significant for both 3 months (aOR 1.27, 95% CI 0.99, 1.64) and each of the genera above. Only the relative abundance of
6 months breastfeeding (aOR 1.20, 95%CI 0.97, 1.48). Increase in Akkermansia had a significantly decreasing trend at 1 year (p =
maternal BMI was significantly associated with 31% higher risk of 0.01) and across time (p = 0.009) between the four BMIz
belonging to the high stable trajectory (interquartile aOR 1.31, trajectories (Fig. 4b, c). This was accompanied by a reduction in
95% CI 1.19, 1.45), and 63% for the rapid growth trajectory (aOR overall colonization accumulation between 3 months and 1 year
1.63, 95%1.39, 1.91). Prenatal smoke exposure, delivery mode, in the rapid growth trajectory, suggesting that Akkermansia
reported antibiotic use in the first year of life, and race were not abundance may have a direct impact on overall BMIz gaining
significantly associated to BMIz trajectories (Fig. 2 and Supple- trajectories beginning in infancy.
mentary Table 2).
Gut microbiota diversity and risk factors for rapid growth
Growth trajectories and gut microbiota diversity Maternal BMI and breastfeeding during early life were both
In order to determine whether components of the infant associated with BMIz trajectories, and recent studies have
microbiota differed among BMIz trajectories, we utilized results implicated these in influencing the early-life microbiota composi-
from 988 participants with stool collected at 3 months (n = 826) tion as well [24, 29, 30]. We therefore investigated whether
and/or 1 year (n = 842). A total of 680 participants contained maternal BMI, breastfeeding duration and presence of breastfeed-
paired data from both timepoints. The microbiota subgroup ing were associated with microbiota diversification or changes in
was similar to those in the overall cohort, including the taxonomic relative abundance related to BMIz trajectories.
proportion of subjects in each BMIz trajectory (Table 1 and Breastfeeding at 6 months of age was significantly associated
Supplementary Table 3). with increased diversification of the infant gut over the first year of
Previous studies have demonstrated the importance of bacterial life (Fig. 5a, b). By comparison, postpartum maternal BMI was not
diversity for maintaining healthy weight in both humans and associated with diversification (data not shown). Influences of
mouse models, and much of this diversification of the microbiota breastfeeding were also seen on relative abundance of genera
occurs during the first year of life as infants are exposed to new associated with rapid growth at 1 year of age (Fig. 5c). These
colonizing microbes [7, 26, 27]. A published analysis from the include Ruminococcus and Clostridium, which were negatively
CHILD study linked increased diversity at 3 months with greater correlated with breastfeeding duration, and Sutterella, which
risk of weight gain [28]. Supporting this, we found a significantly increased with breastfeeding duration. We did not identify any
negative correlation between diversity at 3 months and diversity significant correlations with maternal BMI and any of the genera
at 1 year (Spearman, ρ = −0.2, p < 0.0001) (Fig. 3a). Thus, early associated with rapid growth.

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 M.E. Reyna et al.
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Fig. 2 Adjusted odds ratio and 95% CI for the associated risk of individual factors on BMIz trajectories from weighted multinomial
regression (normative trajectory used as reference group). All models adjusted for maternal BMI, prenatal smoke exposure, study site, and
race. ^Odds ratio per interquartile increase of exposure variable (in maternal postpartum BMI and breastfeeding duration models).

Table 1. Characteristics of all participants (N = 3059) in the CHILD Cohort Study and by BMIz trajectory groups.
Overall Cohort Low-stable Normative High stable Rapid growth
(N = 3059) (N = 289, 9.4%) (N = 1849, 60.4%) (N = 782, 25.6%) (N = 139, 4.5%)
Sex (Male) 1624 (53.1) 167 (57.8) 973 (52.6) 448 (57.3) 36 (25.9)
Race (Caucasian) 1957 (64.7) 158 (55.2) 1204 (65.8) 510 (65.9) 85 (63.0)
Delivery mode (Vaginal) 2253 (74.6) 224 (77.5) 1378 (76.0) 550 (70.3) 101 (72.7)
Gestational age 9.10 (0.31) 9.08 (0.32) 9.08 (0.32) 9.15 (0.28) 9.10 (0.30)
(Months)
Prenatal smoke 547 (18.2) 41 (14.9) 322 (17.7) 151 (19.7) 33 (24.6)
exposure (Yes)
Maternal BMI (kg/m2) 25.38 (5.86) 23.29 (4.60) 24.94 (5.48) 26.54 (6.33) 29.07 (7.53)
Antibiotic use—birth 573 (19.4) 51 (18.1) 338 (19.0) 154 (20.1) 30 (22.4)
to age 1
Breastfeeding duration 10.70 (6.82) 12.19 (6.36) 11.04 (6.90) 9.71 (6.55) 8.73 (6.98)
(Months)
Breastfeeding at 3 months
Exclusive 1775 (60.2) 190 (67.6) 1113 (62.8) 410 (53.7) 62 (47.7)
Partial 768 (26.1) 70 (24.9) 437 (24.7) 224 (29.3) 37 (28.5)
None 404 (13.7) 21 (7.5) 222 (12.5) 130 (17.0) 31 (23.8)
Breastfeeding at 6 months
Exclusive 530 (18.4) 56 (20.2) 351 (20.3) 110 (14.6) 13 (10.2)
Partial 1694 (58.7) 184 (66.4) 1009 (58.4) 437 (58.1) 64 (50.4)
None 661 (22.9) 37 (13.4) 369 (21.3) 205 (27.3) 50 (39.4)
Annual family income
<$50K 358 (13.3) 29 (12.0) 213 (13.0) 91 (13.1) 25 (20.8)
$50K–<$100K 915 (34.0) 89 (36.9) 552 (33.7) 230 (33.2) 44 (36.7)
$100K–<$150K 756 (28.1) 59 (24.5) 460 (28.1) 207 (29.9) 30 (25.0)
≥$150K 661 (24.6) 64 (26.6) 411 (25.1) 165 (23.8) 21 (17.5)
BMI-z classification at 3 years
Underweight 22 (0.8) 15 (5.6) 6 (0.4) 1 (0.1) 0 (0.0)
Normal range 2562 (92.1) 255 (94.4) 1651 (98.6) 621 (87.2) 35 (28.0)
Overweight 166 (6.0) 0 (0.0) 16 (1.0) 85 (11.9) 65 (52.0)
Obese 32 (1.2) 0 (0.0) 2 (0.1) 5 (0.7) 25 (20.0)
BMI-z classification at 5 years
Underweight 24 (0.9) 12 (4.5) 12 (0.7) 0 (0.0) 0 (0.0)
Normal range 2159 (79.1) 253 (95.5) 1516 (91.7) 376 (54.7) 14 (11.6)
Overweight 379 (13.9) 0 (0.0) 112 (6.8) 232 (33.7) 35 (28.9)
Obese 166 (6.1) 0 (0.0) 14 (0.8) 80 (11.6) 72 (59.5)

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 M.E. Reyna et al.
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DISCUSSION The associations between obesity and rapid weight gain in
In this study, we harnessed the power of the longitudinal CHILD infancy and its tracking to subsequent obesity in later life are well
Cohort Study to provide new insights into the relationship established [2, 36, 37], however studies aiming to generate
between the gut microbiota composition in infancy and growth mechanistic insights into childhood obesity have focused on snap
trajectories beginning at birth and up to 5 years of life. Using a shots of children who are already experiencing clinical signs of
cohort of full-term, healthy children, we identified four BMIz metabolic disorders. Here we coupled growth trajectories that
trajectories that coincided with different rates of growth based on exist between birth and 5 years with changes in microbiota
BMI z-scores: low stable, normative growth, high stable, and rapid composition between ages 3 months and 1 year, when the
growth from birth. Importantly, while our observed trajectories microbiota is undergoing its most dynamic changes in diversity
were not fully in line with clinical BMIz outcomes at 3 and 5 years and colonization in response to environmental exposures and
of age (per WHO standards), they were similar to those observed in dietary changes. We found that infants in the rapid growth
other studies, such as a consortium study of eight European birth trajectory experienced significantly less diversification of their gut
cohorts [31], as well as a study of 1957 children from the Quebec microbiota during the first year of life and displayed an altered
Longitudinal Study of Child Development [32]. Furthermore, these microbiota composition by 1 year when compared to normative
trajectories were associated with familial and environmental infants. Many of these genera have been identified in other
factors previously reported in the literature [33–35]. Thus, the human and mouse studies of obesity, however, whether some of
utilization of these growth trajectories that exist prior to obvious these are contributors or bystanders of weight gain has been
differences in BMIz allow for early identification of at-risk children difficult to parse out [38, 39]. We therefore investigated whether
during infancy in order to address modifiable factors involved in the colonization of any of these genera trended over all growth
development of childhood obesity. trajectories. In particular, the expansion of Akkermansia, the genus

a b c
spearman (rho=-0.2, ****) 4 *
4
ns
ns
3

Shannon Index Change

3
2
Shannon Index

BMIz Trajectory
Shannon Index

Low Stable
2 Normative
2 High Stable
0 Rapid Growth

1 1

−2

0 0

3 month 1 year 5 10 15
Visit Age (months)

Fig. 3 Alpha diversity of the gut microbiome over time in CHILD Cohort Study infants. a Paired Shannon index measures at 3 months and 1
year and corresponding Spearman correlation. Darker color indicates lower diversity at 3 months. b Shannon index over time by BMI-z
trajectory groups. c Boxplot of changes in Shannon index between paired 3 months and 1 year samples. p values calculated from Wilcoxon
rank-sum tests. ****p value <0.0001, *p value <0.05, ns = p value >0.05.

a Significant Genera at 1 year

b c trend p-value =0.01
−2
10
Change in Akkermansia relative abundance

Prevotella

Akkermansia −2.5 0.1

10
Akkermansia RelA (log10)

BMIz Trajectory
Sutterella Low Stable
−3 Normative
Streptococcus 10
High Stable
0.0 Rapid Growth
Ruminococcus
(Lachnospiraceae) −3.5
10
Enterobacteriaceae
(unclass.)
Clostridium −4 −0.1
10
(Erysipelotrichaceae)
5 10 15
−6 −4 −2 0 2 Age (months)
Log2 Fold Change
Rapid Growth vs. Normative

Fig. 4 Differential abundance analysis on BMIz trajectories in the CHILD Cohort Study. a Log2 fold change of differential abundance at 1
year visit between normative and rapid growth trajectory. Only significant genera are shown. b Akkermansia relative abundance over time in
each BMI-z trajectory. c Crossbar plots of mean and standard deviation reflecting change in relative abundance for Akkermansia between
3 months and 1 year in each BMI-z trajectory. p value corresponds to coefficient across BMIz trajectories from ordinal logistic regression
adjusted for ethnicity and study site.

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 M.E. Reyna et al.
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predominantly comprised of Akkermansia muciniphila, was 3 months was higher in non-breastfed children aligns with that of
blunted throughout the first year of life in infants experiencing a separate subset of 1087 children from the CHILD cohort
rapid BMIz gain and displayed a significantly inverse trend in published previously [28]. Similar to our findings, Forbes et al.
expansion with overall BMIz gain. Our findings that a significant additionally showed that infants who were overweight or at risk of
overlap exists between Akkermansia abundance and patterns of being overweight at 1 year of age (as defined per WHO weight for
growth supports a mechanistic link between this commensal and length z-scores) had significantly higher richness of microbiota at
longitudinal weight gains that exist prior to obesity and may be 3 months of age [28], although the association was not significant
sensitive to modification during infancy. Notably, Akkermansia is in our analysis. While they postulated that increased energy
currently being investigated as a live biotherapeutic product, extraction from a more diverse gut early in life increases the risk of
which may complement current family-based life style interven- being overweight at 1 year, their analysis did not include the
tions that have shown only modest effect on weight loss [40]. longitudinal impact of early diversification on the microbiota.
In accordance to previous studies, we found that breastfeeding Indeed, our analyses indicate that early, high diversity may
status and duration were significantly associated with different impede newly colonizing bacteria within the first year and limit
BMIz trajectories, particularly in the rapid growth trajectory [33]. total diversification. While we did not find direct associations
Previous studies observed a dominant influence of breastfeeding between Akkermansia and breastfeeding duration, our analyses
on the infant microbiome when compared to maternal BMI, and did not incorporate the heterogeneity of breastmilk metabolites,
our findings support this [29]. While we did not identify correlation which have been demonstrated to influence Akkermansia in
between maternal BMI and the infant microbiota composition, we animal models [14]. Furthermore, Akkermansia expansion in our
did observe significant associations between occurrence and study was weakly correlated with diversification (spearman: ρ =
duration of breastfeeding and both diversification and the relative 0.27, p = 4.6e−11), suggesting that they are intertwined.
abundance of genera that were increased within the rapid growth Following the definitions used in Forbes et al. in our study
trajectory. Moreover, our finding that microbiota diversity at sample, 62% (n = 412) and 22% (n = 147) of children classified as

Fig. 5 Gut microbiome diversity, composition and risk factors for rapid growth. a Shannon index over time by breastfeeding status at
6 months. b Boxplot of changes in Shannon index between paired 3 months and 1 year samples by breastfeeding status at 6 months.
c Spearman-rank correlation of maternal BMI or breastfeeding duration and relative abundance of genera associated with rapid growth.

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 M.E. Reyna et al.
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overweight or at risk of overweight at 1 year were placed into the 7. Petersen C, Bell R, Klag KA, Lee S-H, Soto R, Ghazaryan A, et al. T cell-mediated
high stable and normative trajectories, respectively. The remaining regulation of the microbiota protects against obesity. Science. 2019;365:
16% (n = 106) of children were classified in the rapid growth eaat9351.
trajectory in which we observed most relevant changes in 8. Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. The gut microbiota
as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA.
microbiota. The fundamental differences in our findings may
2004;101:15718–23.
suggest that although overweight measures restricted to the 9. Kim KN, Yao Y, Ju SY. Short chain fatty acids and fecal microbiota abundance in
infancy period may point to specific perturbations of the humans with obesity: a systematic review and meta-analysis. Nutrients.
microbiota in early life, the identification of longitudinal pheno- 2019;11:2512.
types of obesity described in this study provide additional 10. Sender R, Fuchs S, Milo R. Revised estimates for the number of human and
information about the role of the infant microbiota in preschool bacteria cells in the body. PLoS Biol. 2016;14:e1002533.
obesity that could otherwise be missed. 11. Macchione IG, Lopetuso LR, Ianiro G, Napoli M, Gibiino G, Rizzatti G, et al.
The CHILD cohort is unique in that it comprises a general Akkermansia muciniphila: key player in metabolic and gastrointestinal disorders.
population of multi-ethnic children, but considering it is an Eur Rev Med Pharmacol Sci. 2019;23:8075–83.
12. Schneeberger M, Everard A, Gómez-Valadés AG, Matamoros S, Ramírez S, Delz-
observational study, it is important to address the limitations of
enne NM, et al. Akkermansia muciniphila inversely correlates with the onset of
our study. Despite the clear associations between early-life BMI inflammation, altered adipose tissue metabolism and metabolic disorders during
and chronic co-morbidities presented in other studies [5], there is obesity in mice. Sci Rep. 2015;5:16643.
ongoing debate as to the utility of BMI as an indicator of adiposity 13. Depommier C, Everard A, Druart C, Plovier H, Van Hul M, Vieira-Silva S, et al.
in infancy. Future studies should analyze gut microbiota regula- Supplementation with Akkermansia muciniphila in overweight and obese human
tion in early life with respect to body composition measures. volunteers: a proof-of-concept exploratory study. Nat Med. 2019;25:1096–103.
Secondly, to minimize the inherent limitations of mixture 14. Ribo S, Sánchez-Infantes D, Martinez-Guino L, García-Mantrana I, Ramon-Krauel
modeling for identifying trajectories of growth, we relied on M, Tondo M, et al. Increasing breast milk betaine modulates Akkermansia
statistical criteria for class enumeration, performed stratified abundance in mammalian neonates and improves long-term metabolic health.
Sci Transl Med. 2021;13:eabb0322.
modeling by sex, and incorporated posterior probabilities into
15. White RA, Bjørnholt JV, Baird DD, Midtvedt T, Harris JR, Pagano M, et al. Novel
regression modeling [41]. Nevertheless, the associations reported developmental analyses identify longitudinal patterns of early gut microbiota
here need to be validated by experimental models and replicated that affect infant growth. PLoS Comput Biol. 2013;9:e1003042.
in independent human populations. 16. Aris IM, Chen L-W, Tint MT, Pang WW, Soh SE, Saw S-M, et al. Body mass index
The burden in quality of life, mental and physical health as early trajectories in the first two years and subsequent childhood cardio-metabolic
as preschool age due to childhood obesity co-morbidities are well outcomes: a prospective multi-ethnic Asian cohort study. Sci Rep. 2017;7:8424.
documented, but the underlying mechanisms and the role of the 17. Hemmingsson E. Early childhood obesity risk factors: socioeconomic adversity,
microbiota maturation in childhood obesity regulation remain family dysfunction, offspring distress, and junk food self-medication. Curr Obes
unclear. This study provides evidence of the underlying biological Rep. 2018;7:204–9.
18. Mameli C, Mazzantini S, Zuccotti GV. Nutrition in the first 1000 days: the origin of
alterations in children with different BMIz trajectories over time.
childhood obesity. Int J Environ Res Public Health. 2016;13:838.
The most striking associations were found in those children with a 19. Tamburini S, Shen N, Wu HC, Clemente JC. The microbiome in early life: impli-
steep increase in BMI during the first year of life that continues up cations for health outcomes. Nat Med. 2016;22:713–22.
to 5 years of age. Our findings support a novel avenue of research 20. Petersen C, Turvey SE. Can we prevent allergic disease? Understanding the links
on the human microbiota, targeted on tangible interventions to between the early life microbiome and allergic diseases of childhood. Curr Opin
reduce childhood overweight and obesity. Pediatr. 2020;32:790–7.
21. Subbarao P, Anand SS, Becker AB, Befus AD, Brauer M, Brook JR, et al. The
Canadian Healthy Infant Longitudinal Development (CHILD) study: examining
DATA AVAILABILITY developmental origins of allergy and asthma. Thorax. 2015;70:998–1000.
A list of variables available in the CHILD Cohort Study is available at https:// 22. De Onis M, editor. WHO child growth standards: length/height-for-age, weight-
childstudy.ca/for-researchers/study-data/. Researchers interested in collaborating on for-age, weight-for-length, weight-for-height and body mass index-for-age;
a project and accessing CHILD Cohort Study data should contact the Study’s National methods and development. Geneva: WHO Press; 2006. p. 312.
Coordinating Centre (NCC) to discuss their needs before initiating a formal request. 23. de Onis M. Development of a WHO growth reference for school-aged children
To contact the NCC, please email [email protected]. More information about data and adolescents. Bull World Health Organ. 2007;85:660–7.
access for the CHILD Cohort Study can be found at https://childstudy.ca/for- 24. Fehr K, Moossavi S, Sbihi H, Boutin RCT, Bode L, Robertson B, et al. Breastmilk
researchers/data-access/. feeding practices are associated with the co-occurrence of bacteria in mothers’
milk and the infant gut: the CHILD cohort study. Cell Host Microbe.
2020;28:285–297.e4.
25. Patrick DM, Sbihi H, Dai DLY, Al Mamun A, Rasali D, Rose C, et al. Decreasing
REFERENCES antibiotic use, the gut microbiota, and asthma incidence in children: evidence
1. Tackling obesity in Canada: Childhood obesity and excess weight rates in Canada from population-based and prospective cohort studies. Lancet Respir Med.
—Canada.ca [Internet]. 2021. https://www.canada.ca/en/public-health/services/ 2020;8:1094–105.
publications/healthy-living/obesity-excess-weight-rates-canadian-children. 26. Le Chatelier E, Nielsen T, Qin J, Prifti E, Hildebrand F, Falony G, et al. Richness
html#shr-pg0. of human gut microbiome correlates with metabolic markers. Nature.
2. Smego A, Woo JG, Klein J, Suh C, Bansal D, Bliss S, et al. High body mass index in 2013;500:541–6.
infancy may predict severe obesity in early childhood. J Pediatr. 2017;183:87–93.e1. 27. Lim ES, Zhou Y, Zhao G, Bauer IK, Droit L, Ndao IM, et al. Early life dynamics of the
3. Garden FL, Marks GB, Simpson JM, Webb KL. Body mass index (BMI) trajectories human gut virome and bacterial microbiome in infants. Nat Med. 2015;21:1228–34.
from birth to 11.5 years: relation to early life food intake. Nutrients. 2012;4:1382–98. 28. Forbes JD, Azad MB, Vehling L, Tun HM, Konya TB, Guttman DS, et al. Association
4. van Rossem L, Wijga AH, Brunekreef B, de Jongste JC, Kerkhof M, Postma DS, et al. of exposure to formula in the hospital and subsequent infant feeding practices
Overweight in Infancy: Which Pre- and Perinatal Factors Determine Overweight with gut microbiota and risk of overweight in the first year of life. JAMA Pediatr.
Persistence or Reduction A Birth Cohort Followed for 11 Years. Ann Nutr Metab. 2018;172:e181161.
2014;65:211–99. 29. Stewart CJ, Ajami NJ, O’Brien JL, Hutchinson DS, Smith DP, Wong MC, et al.
5. Sharma V, Coleman S, Nixon J, Sharples L, Hamilton‐Shield J, Rutter H, et al. A Temporal development of the gut microbiome in early childhood from the
systematic review and meta-analysis estimating the population prevalence of TEDDY study. Nature. 2018;562:583–8.
comorbidities in children and adolescents aged 5 to 18 years. Obes Rev. 30. Gohir W, Ratcliffe EM, Sloboda DM. Of the bugs that shape us: maternal obesity,
2019;20:1341–9. the gut microbiome, and long-term disease risk. Pediatr Res. 2015;77:196–204.
6. Ziyab AH, Karmaus W, Kurukulaaratchy RJ, Zhang H, Arshad SH. Developmental 31. Rzehak P, Wijga AH, Keil T, Eller E, Bindslev-Jensen C, Smit HA, et al. Body mass
trajectories of Body Mass Index from infancy to 18 years of age: prenatal index trajectory classes and incident asthma in childhood: results from 8 Eur-
determinants and health consequences. J Epidemiol Community Health. opean Birth Cohorts—a Global Allergy and Asthma European Network initiative. J
2014;68:934–41. Allergy Clin Immunol. 2013;131:1528–1536.e13.

International Journal of Obesity (2022) 46:1351 – 1358

Content courtesy of Springer Nature, terms of use apply. Rights reserved

 M.E. Reyna et al.
1358
32. Pryor LE. Developmental trajectories of body mass index in early childhood and final version of the manuscript. PS and SET conceptualized and designed the study,
their risk factors: an 8-year longitudinal study. Arch Pediatr Adolesc Med. critically reviewed the manuscript for important intellectual content and approved its
2011;165:906. final version. RCTB and BBF processed and analyzed 16S data and approved the final
33. Azad MB, Vehling L, Chan D, Klopp A, Nickel NC, McGavock JM, et al. Infant version of the manuscript. RD and DLL substantially contributed to data acquisition,
feeding and weight gain: separating breast milk from breastfeeding and formula defined variables used in the study and approved its final version. ABB, MBA, KM,
from food. Pediatrics. 2018;142:e20181092. TJM, PJM, ES, and ALK contributed to the design of the study and critically revised
34. Stuart B, Panico L. Early-childhood BMI trajectories: evidence from a prospective, and approved the final version of the manuscript. WL critically reviewed the
nationally representative British cohort study. Nutr Diabetes. 2016;6:e198. methodology used in the manuscript, assisted in data analysis and approved the final
35. Mattsson M, Maher GM, Boland F, Fitzgerald AP, Murray DM, Biesma R. Group‐ version of the manuscript.
based trajectory modelling for BMI trajectories in childhood: a systematic review.
Obes Rev. 2019;20:998–1015.
36. Butler ÉM, Pillai A, Morton SMB, Seers BM, Walker CG, Ly K, et al. A prediction COMPETING INTERESTS
model for childhood obesity in New Zealand. Sci Rep. 2021;11:6380. The authors declare no competing interests.
37. Das T, Ostbye T, Chan YH, Tan NC, Chew E. Maternal and infant predictors of
growth trajectories in singapore children in the first 18 months of life. BMJ
Pediatrics Open. 2021;5:A110.
ADDITIONAL INFORMATION
38. Mbakwa CA, Hermes GDA, Penders J, Savelkoul PHM, Thijs C, Dagnelie PC, et al.
Gut microbiota and body weight in school-aged children: the KOALA Birth Cohort Supplementary information The online version contains supplementary material
Study. Obesity. 2018;26:1767–76. available at https://doi.org/10.1038/s41366-022-01117-z.
39. Tun MH, Tun HM, Mahoney JJ, Konya TB, Guttam DS, Becker AB, et al. Postnatal
exposure to household disinfectants, infant gut microbiota and subsequent risk Correspondence and requests for materials should be addressed to Padmaja
of overweight in children. CMAJ. 2018;190:11. Subbarao.
40. Kumar S, Kelly AS. Review of childhood obesity: from epidemiology, etiology,
and comorbidities to clinical assessment and treatment. Mayo Clin Proc. Reprints and permission information is available at http://www.nature.com/
2017;92:251–65. reprints
41. van der Nest G, Lima Passos V, Candel MJJM, van Breukelen GJP. An overview of
mixture modelling for latent evolutions in longitudinal data: modelling approa- Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
ches, fit statistics and software. Adv Life Course Res. 2020;43:100323. in published maps and institutional affiliations.

ACKNOWLEDGEMENTS
We thank the founding director of the CHILD Cohort Study, Dr. Malcolm R. Sears for Open Access This article is licensed under a Creative Commons
his contributions and continuous guidance during the development of this Attribution 4.0 International License, which permits use, sharing,
manuscript. We thank the CHILD Cohort Study participant families for their adaptation, distribution and reproduction in any medium or format, as long as you give
dedication and commitment to advancing health research. CHILD was initially appropriate credit to the original author(s) and the source, provide a link to the Creative
funded by CIHR and AllerGen NCE Inc. Visit CHILD at childcohort.ca. This research was Commons license, and indicate if changes were made. The images or other third party
undertaken, in part, thanks to funding from Genome Canada and the Canada material in this article are included in the article’s Creative Commons license, unless
Research Chairs Program. indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons.
AUTHOR CONTRIBUTIONS org/licenses/by/4.0/.
MER and CP carried out the analysis and interpretation of data, contributed to the
design of the study, and drafted the initial manuscript. DLYD carried out the analysis
and interpretation of data, defined variables used in the study, and approved the © The Author(s) 2022

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