H

OH
OH
metabolites

Article
Impact of Maternal Weight Gain on the Newborn Metabolome
Teresa Guixeres-Esteve 1 , Francisco Ponce-Zanón 1,2,3 , José Manuel Morales 2,4 , Empar Lurbe 1,2,3 ,
Julio Alvarez-Pitti 1,2,3, * and Daniel Monleón 2,4,5, *

1 Pediatric Department, Consorcio Hospital General, University of Valencia, 46014 Valencia, Spain;
maguies3@uv.es (T.G.-E.)
2 INCLIVA Biomedical Research Institute, Hospital Clínico, University of Valencia, 46010 Valencia, Spain
3 Biomedical Research Networking Center for Physiopathology of Obesity and Nutrition (CIBEROBN),
Institute of Health Carlos III (ISCIII), 28029 Madrid, Spain
4 Department of Pathology, University of Valencia, 46010 Valencia, Spain
5 CIBER of Frailty and Healthy Aging (CIBERFES), Institute of Health Carlos III (ISCIII), 28029 Madrid, Spain
* Correspondence: alvarez_jul@gva.es (J.A.-P.); daniel.monleon@uv.es (D.M.); Tel.: +34-96-1820772 (J.A.-P.);
+34-96-3864145 (D.M.)

Abstract: Pre-pregnancy obesity and excessive gestational weight gain (GWG) appear to affect birth
weight and the offspring’s risk of obesity and disease later in life. However, the identification of
the mediators of this relationship, could be of clinical interest, taking into account the presence
of other confounding factors, such as genetics and other shared influences. The aim of this study
was to evaluate the metabolomic profiles of infants at birth (cord blood) and 6 and 12 months after
birth to identify offspring metabolites associated with maternal GWG. Nuclear Magnetic Resonance
(NMR) metabolic profiles were measured in 154 plasma samples from newborns (82 cord blood
samples) and in 46 and 26 of these samples at 6 months and 12 months of age, respectively. The
levels of relative abundance of 73 metabolomic parameters were determined in all the samples.
We performed univariate and machine-learning analysis of the association between the metabolic
levels and maternal weight gain adjusted for mother‘s age, Body Mass Index (BMI), diabetes, diet
adherence and infant sex. Overall, our results showed differences, both at the univariate level and in
the machine-learning models, between the offspring, according to the tertiles of maternal weight gain.
Citation: Guixeres-Esteve, T.; Some of these differences were resolved at 6 and 12 months of age, whereas some others remained.
Ponce-Zanón, F.; Morales, J.M.; Lactate and leucine were the metabolites with the strongest and longest association with maternal
Lurbe, E.; Alvarez-Pitti, J.; Monleón, weight gain during pregnancy. Leucine, as well as other significant metabolites, have been associated
D. Impact of Maternal Weight Gain in the past with metabolic wellness in both general and obese populations. Our results suggest that
on the Newborn Metabolome. the metabolic changes associated to excessive GWG are present in children from early life.
Metabolites 2023, 13, 561. https://
doi.org/10.3390/metabo13040561
Keywords: metabolomics; gestational weight gain; offspring; newborn; umbilical cord
Academic Editors: Susanne Aufreiter
and Bo Li

Received: 20 March 2023

1. Introduction
Revised: 6 April 2023
Accepted: 11 April 2023 In 1989, Barker et al. hypothesized that an unfavorable environment in utero can
Published: 15 April 2023 cause programmed adaptations in fetal development that persist into extrauterine life,
creating a phenotype that is more susceptible to cardiovascular disease [1]. An example of
an unfavorable intrauterine milieu is fetal overnutrition, defined as fetal exposure to excess
maternal fuels due to maternal obesity, gestational diabetes (GD), or excessive gestational
Copyright: © 2023 by the authors. weight gain (GWG). Many epidemiological, clinical, and animal-model studies strongly
Licensee MDPI, Basel, Switzerland. suggest that mothers’ prenatal obesity and high-fat dietary intake are associated with
This article is an open access article cardiometabolic morbidity in their progeny, including obesity, hypertension, hyperglycemia
distributed under the terms and and insulin resistance, hyperlipidemia and non-alcoholic fatty-liver disease [2,3].
conditions of the Creative Commons Many studies suggest that cord-plasma-metabolite profiles (resulting from fetal re-
Attribution (CC BY) license (https://
sponse to in utero exposure) can serve as early-life biomarkers of the risk of metabolic
creativecommons.org/licenses/by/
disease in the long term. Hence, interest has grown in the investigation of metabolomic
4.0/).

Metabolites 2023, 13, 561. https://doi.org/10.3390/metabo13040561 https://www.mdpi.com/journal/metabolites

Metabolites 2023, 13, 561 2 of 15

profiles in the cord blood of mother-child pairs with inconsistent results produced by the
studies conducted thus far.
Perng et al. reported that the metabolites in energy-production and DNA/RNA
turnover pathways and the branched-chain amino acids (BCAAs) in cord blood were
associated with larger neonatal size (a known risk factor for poor cardiovascular health
later in life), and that BCAAs and androgen hormone patterns were higher in obese
compared with lean school-age children (6–10 years). Furthermore, higher factor scores for
these patterns were correlated with continuous measures of overall and central adiposity
as wellas other cardiometabolic biomarkers, such as higher HOMA-IR [4,5]. Kadakia et al.
found that cord-blood BCAAs and ketone-body metabolites were positively correlated with
newborn adiposity [6]. Cao et al. linked cord-plasma metabolites with the longitudinal
BMI trajectories of 946 children from birth up to the age of 18 years. These trajectories
were categorized into three patterns: early-onset overweight and obesity (early-OWO),
late-onset OWO (late-OWO), and normal weight trajectory (NW). These results suggested
that the cord-blood metabolome was most useful factor in identifying children at risk of
early-OWO, with twenty-two triacylglycerols and diacylglycerols negatively associated
with early-OWO, and five cholesterol esters positively associated [7].
Similarly, Perng et al. did not find any significant associations between cord-blood-
metabolite patterns and maternal characteristics, except for cesarean delivery, some metabo-
lites of energy production, and cell-proliferation pathways, which were associated with
larger size at birth. On the other hand, they observed that the BCAA scores were higher in
the cord plasma of overweight women before pregnancy [5]. Lowe et al. found an associa-
tion between maternal BMI and cord-plasma levels of BCAAs, their metabolic bioproducts,
and phenylalanine [8]. Francis et al. noted differences in fifty-two metabolites between
infants who were exposed to fetal overnutrition and those who were not exposed, with a
small amount of variation between maternal obesity only, GD only, and both [9].
However, while obesity and GD are widely studied as conditions in fetal overnutrition,
few metabolomic studies focus on GWG. To gain a better understanding of the latter’s
influence, we performed a longitudinal study on obese and lean pregnant women classified
by tertiles of GWG. The aim of our study was to identify differences between offspring
metabolite profiles, from the newborn (cord blood) to the 12-months stage, with respect to
maternal GWG.

2. Materials and Methods

2.1. Study Population: Clinical and Biochemical Measurements
This study hypothesized that maternal-dependent factors, especially GWG, lead to
metabolomic changes in newborns and during the first year of life.
For this purpose, a prospective observational cohort study was designed, beginning
at birth and following up to 12 months of life. Eighty-three mother-newborn pairs were
divided into three groups, according to tertiles of weight gain, to analyze the impact of the
mother’s gestational weight gain. To participate in the study, the following inclusion and
exclusion criteria were set:: newborns born at full term (over 37 in weeks gestational age at
birth), ascertained according to the Ballard method [10], by normal delivery or by cesarean
section, at the Hospital General Universitario of Valencia, Spain. Preterm newborns or
newborns with any perinatal pathology were excluded. Mothers included were normal
weight, obese or obese with gestational diabetes except mothers with gestational diabetes
treated with insulin or any other illness.
All participating mothers received written information about the study and signed
their informed consent to participate. The study was approved by the hospital’s review
board and was carried out in accordance with the Declaration of Helsinki. Both the samples
(plasma from umbilical cord of the newborns and plasma from peripheral veins from the
mothers), and the collected data were stored according to the directives dictated by the law
of Biomedical Investigation of 2007 (Law 14/2007) and all applicable rules.
Metabolites 2023, 13, 561 3 of 15

(A) From mothers, age, height, and weight measurements prior to gestation as recorded
in their pregnancy booklet by the midwife, and weight at the end of gestation were
collected. The GWG was calculated and its conformity with the 2009 recommenda-
tions of the Institute of Medicine (IOM) was verified [11]. Regarding the evolution of
the pregnancy, the presence or absence of gestational diabetes, the degree of maternal
adherence to the Mediterranean diet (AMD) using the validated questionnaire devel-
oped by Trichopoulos et al. [12] and smoking habits were recorded. Finally, the type
of delivery was also recorded (vaginal or cesarean section).
(B) From all newborns, the following information was recorded:
- Somatometry (weight, length, head circumference and ponderal index and per-
centiles according to Intergrowth-21st), performed by trained nurses in the mater-
nity ward. Weight was measured with ADE scale model M112600 (ADE GmbH
& Co, Hamburg, Germany). Length was measured in the supine position using a
neonatometer. Head circumference was measured with a tape measure at the max-
imum circumference. The ponderal index, also known as the corpulence index or
Rohrer’s index, was calculated with the following formula PI = weight/length3
× 100. Newborns were classified as small for gestational age (SGA), appropriate
for gestational age (AGA) or large for gestational age (LGA) [13].
- Blood pressure (BP) was obtained by taking 3 measurements, using oscillometric
method, with System 7100 Non-invasive Blood Pressure AMI (Advanced Medical
Instruments Inc., Broken Arrow, OK, USA) and heart rate (HR).
- Type of feeding (breastfeeding or infant formula feeding); as well as the need for
supplementation with artificial formula during the first days of life in those who
were breastfed.
- Umbilical cord blood samples were obtained from the clamped umbilical cord
immediately after delivery for metabolomic studies.
(C) Blood sample were collected at 6 months of age to conduct metabolomic study.
(D) In infants, at 12 months of life, weight, length, head circumference and BMI were
recorded, and their percentiles were calculated according to the WHO 2006/2007
curves [14]. The type of feeding at 12 months was recorded. Finally, blood samples
were collected to perform metabolite and biochemical studies.

2.2. NMR Metabolomics

Umbilical-venous-cord blood was collected in EDTA tubes, centrifuged to yield plasma,
stored at −80 ◦ C and thawed before use. For NMR analysis, 500 µl of plasma was mixed
with 50 µl of D2O (as a field lock). A total of 500 µl of the mixture of each sample was then
individually transferred into a 5-mm high-quality NMR tube. A single-pulse presaturation
NMR spectrum was acquired from all samples, with 256 transients collected into 65 k data
points for all experiments. Nuclear magnetic resonance is a reproducible and accessible
technique that has been applied successfully to the metabolic profiling of a variety of
samples [15–17]. Spectra were processed using MestReNova 8.1 (Mestrelab Research S.L.,
A Coruña, Spain). They were then chemical-shift-referenced on the alanine CH3 doublet
signal at 1.475 ppm, normalized to the total aliphatic area, lipid excluded, and transferred
to MATLAB (MathWorks, Natick, MA, USA, 2012). We analyzed the chemical-shift region,
including resonances of 0.50–4.70 ppm (the aliphatic region) and 5.20–10.00 ppm (the
aromatic region). Resonances were annotated by research data and Chenomx resonances
database (Chenomx NMR 7.6). The NMR peaks were quantified using semi-automated
in-house MATLAB peak-fitting routines. The final metabolite relative spectral abundance
was calculated. Machine learning and regression models were calculated in R 4.1 with
the package ‘mdatools’. Finally, each metabolic feature was normalized to the standard
deviation in all the samples from the analysis group (birth, 6 months, or 12 months) to
obtain z-scores.
Partial least-squares discriminant analysis (PLS-DA) was applied to maximize the
separation between samples and to identify discriminant patterns [18]. The PLSDA models
Metabolites 2023, 13, 561 4 of 15

were designed for child sex and weight at birth and for maternal obesity, adherence to
the Mediterranean diet, and diabetes by calculating a linear-regression model with these
variables for each metabolic feature and using the residues for the PLS-DA analysis. A
permutation test was performed to check the potential overfitting of the PLS-DA models.
The chemometric models were cross-validated with 10-fold Venetian blind cross-validation.
In each replicate, 10% of the data were left out of the training calculations and used as a
test dataset. Variable importance in projections (VIP) coefficients were calculated for all
the models to select spectral features with strongest contributions to the models. Spectral
features with high VIP coefficients contributed more to class separation during analysis,
while those with very small VIP coefficients provided little contribution to classification. A
Metabolite Set Enrichment Analysis (MSEA) of metabolites with VIPs scores higher than
1 and p-values below 0.05 was performed with MetaboAnalyst and the Small Molecule
Pathway Database (SMPDB). Metabolite set enrichment analysis is conceptually similar to
Gene Set Enrichment Analysis. A collection of predefined metabolite sets is used by MSEA
algorithms to rank the lists of metabolites obtained in the analysis. This prior knowledge
about metabolite makes it possible to identify significant and coordinated changes in
metabolic networks and obtain biological insights.

2.3. Statistical Analysis

Demographics and Clinical Data Comparison
The IBM SPSS Statistics v.26 statistical program was used for data analysis. First, the
database was segmented according to the 3 study groups, and the Kolmogorov Smirnov
normality test was performed to verify that the population sample conformed to a normal
distribution for the quantitative variables in the 3 groups. When a normal distribution of
variables was not observed in any of the groups, the means of the variables in the 3 groups
were compared with the nonparametric Kruskal Wallis test. In cases in which a variable
had a normal distribution in the 3 groups, the means of the variable in the 3 groups were
compared with ANOVA. The data on the quantitative variables were summarized in simple
statistics (mean ± standard deviation). Differences between qualitative variables were
analyzed using the Chi-square test and the data were expressed as percentages.

3. Results
The samples were divided according to the tertiles of the mothers’ absolute GWG. The
maternal characteristics were comparable among the three groups except for BMI prior to
gestation which was higher in the group in the first tertile of GWG (p = 0.004) and GWG
adequacy according to the Institute of Medicine’s recommendations [11], which was higher
in the first group, as expected. Table 1 shows the clinical differences between the mothers.

Table 1. Characteristics of mothers, according to absolute maternal GWG tertiles. Data are presented
as either mean ± SD or no. (%). } Analysis of variance (ANOVA). ¥ The Kruskal Wallis test. • Chi-
squared test. Data with significant p-values are labeled as follows: ** for p < 0.01 and *** for p < 0.001.

1st Tertile GWG 2nd Tertile GWG 3rd Tertile GWG

Mothers’ Variables p Value
(N = 28) (N = 27) (N = 28)
Age (years) ¥ 31.5 (± 8.48) 34.11 (± 5.17) 33.18 (± 5.41) 0.429
Yes 75% Yes 48.1% Yes 57.1%
Maternal obesity 0.116
No 25% No 51.9% No 42.9%
BMI prior to
33.61 (±7.31) 28.34 (± 7.07) 28.07 (± 6.03) 0.004 **
pregnancy (Kg/m2 ) ¥
GWG (Kg) } 2.33 (± 4.31) 10.11 (± 1.12) 16.78 (± 3.91) <0.001 ***
GWG adequacy
Yes 100% Yes 66.7% Yes 21.4%
according to IOM <0.001 ***
No 0% No 33.3% No 78.6%
recommendations •
AMD score ¥ 6.96 (± 2) 7.76 (± 1.7) 7.46 (± 1.6) 0.396
Metabolites 2023, 13, 561 5 of 15

Table 1. Cont.

1st Tertile GWG 2nd Tertile GWG 3rd Tertile GWG

Mothers’ Variables p Value
(N = 28) (N = 27) (N = 28)
High 12.5% High 23.5% High 8.3%
Degree of AMD • Medium 58.3% Medium 70.6% Medium 75% 0.266
Low 29.2% Low 5.9% Low 16.7%
Diabetes during Yes 35.7% Yes 11.1% Yes 17.9%
0.072
pregnancy No 64.3% No 88.9% No 82.1%
Yes 14.3% Yes 18.5% Yes 21.4%
Smoking habits • 0.78
No 85.7% No 81.5% No 78.6%
Vaginal 50% Vaginal 44.4% Vaginal 60.7%
Type of delivery • 0.469
Cesarean 50% Cesarean 55.6% Cesarean 39.3%

There were no clinical differences between the offspring at birth according to the
tertiles of the maternal GWG, as shown in Table 2.

Table 2. Characteristics of children at birth, according to absolute maternal GWG tertiles. Data are
presented as either mean ± SD or no. (%). } Analysis of variance (ANOVA). ¥ The Kruskal Wallis
test. • Chi-squared test. Data with significant p-values are labelled as follows.

Newborns’ 1st Tertile GWG 2nd Tertile GWG 3rd Tertile GWG
p Value
Variables (N = 28) (N = 27) (N = 28)
Birth weight (BW) (g)
3363 (±552) 3311 (±557) 3454 (±449) 0.593
}
Weight percentile } 53.45 (±31.14) 52.4 (±31.1) 58 (±30.4) 0.773
SGA 7.1% SGA 11.1% SGA 10.7%
BW Classification • AGA 75% AGA 70.4% AGA 60.7% 0.8
LGA 17.9% LGA 18.5% LGA 28.6%
Length (cm) } 49.37 (±2.08) 48.6(±1.94) 49.67 (±2.13) 0.149
Length percentile } 48.81 (±28.2) 39.78 (±25.71) 53.33 (±31.8) 0.21
Head circumference
34.46 (±1.52) 34.38 (±1.57) 34.97 (±1.41) 0.351
(HC) (cm) ¥
HC percentile¥ 62.8 (±27.01) 64 (±28.79) 71.68 (±27.67) 0.323
ponderal index ¥ 2.77 (±0.267) 2.85 (±0.24) 2.81 (±0.31) 0.406
Systolic blood
78.63 (±87) 81.5 (±12.5) 78.89 (±12.66) 0.605
pressure (mmHg) }
Diastolic blood
50.66 (±10) 49.79 (±9.63) 49.5 (±9.45) 0.899
pressure (mmHg) }
Heart rate (bpm) } 126.81 (±14.69) 129.65 (±12.65) 132.69 (±16.93) 0.366
breast 67.9% breast 88.9% breast 75%
Type of feeding • 0.169
formula 32.1% formula 11.1% formula 25%
Formula Yes 31.6% Yes 29.2% Yes 19%
0.62
Supplementation • No 68.4% No 70.8% No 81%

Table 3 shows the characteristics of the children at 12 months of age according to the
tertiles of the absolute maternal GWG. There were no clinical differences at 12 months of
age except for length (p = 0.039). There were also no statistically significant differences
between the children’s blood tests at 12 months of age.
Metabolites 2023, 13, 561 6 of 15

Table 3. Characteristics of children at 12 months of life according to tertiles of absolute maternal

GWG. Data are presented as either mean ± SD. } Analysis of variance (ANOVA). ¥ The Kruskal
Wallis test. Data with significant p-values are labeled as follows: * for p < 0.05.

1st Tertile GWG 2nd Tertile GWG 3rd Tertile GWG p Value
12 Month
(N = 27) (N = 24) (N = 25)
Somatometry
Weight (g) } 10,509 (±1516) 9736 (±1193) 10,293 (±1242) 0.114
Weight percentile ¥ 71.14 (±28.75) 58.54 (± 29.43) 61.92 (± 27.87) 0.214
Length (cm) } 76.74 (±3.4) 74.83 (±2.64) 76.64 (±2.4) 0.039 *
Length percentile ¥ 60.4 (±32) 48.79 (±28.72) 60.08 (±26.68) 0.128
Head circumference
46.38 (±1.32) 45.81 (±1.15) 46.80 (±1.69) 0.094
(HC) (cm) ¥
HC percentile ¥ 64.64 (±27.89) 57.83 (±24.94) 68.2 (±29.1) 0.216
BMI (Kg/m2 ) } 17.65 (±1.53) 17.29 (±1.76) 17.38 (±1.69) 0.722
67.86
BMI percentile ¥ 6(±28.24) 60.07(±29.59) 0.516
(±29.172.63)
12 Month
(N = 12) (N = 8) (N = 12)
Blood Test
Leptin (pg/ml) ¥ 452 (±613) 691 (±380) 513 (±626) 0.378
Total cholesterol
160.6 (±32) 158 (±38.2) 152.75 (±25.64) 0.827
(mg/dl) }
HDL cholesterol
40.5 (±9.4) 44.75 (±10.91) 43.16 (±10.66) 0.64
(mg/dl) }
LDL cholesterol
95.75(±34.6) 100.2 (±33.8) 96.5 (±20.5) 0.94
(mg/dl) }
Triglycerides (mg/dl)
183 (±90) 173 (±90.7) 119 (±45) 0.115
}
HOMA index ¥ 0.955 (±0.77) 1.99 (±1.58) 1.34 (±1.15) 0.126
Uric acid (mg/dl) } 3.15 (±0.64) 3.22 (±0.49) 3.05 (±0.669) 0.822

We quantified 43 metabolic spectral features in 82 blood-serum samples from the

newborns (0 months), 46 samples at 6 months of age, and 26 samples at 12 months. We
analyzed the data by age, exploring the associations between the metabolic profiles and
maternal weights at the three different timepoints, adjusting for sex, weight at birth, and
lactation of the newborn, as well as for maternal obesity, maternal diabetes, and adherence
to the Mediterranean diet. The adjusted results were calculated on the subset, with all the
covariates measured, which included 56 samples at 0 months, 42 at 6 months and 26 at
12 months.
The adjusted linear regression between the metabolite levels and the maternal GWG
was significant for different metabolites at different ages (Figure 1). Interestingly, the
significant associations at birth were strongly dominated by short-chain fatty acids; this
influence decreased at 6 months of age and disappeared at 12 months of age.
To further explore the metabolic impact of maternal weight gain on offspring metabolism
and identify non-linear relationships that may not have been detected by the linear re-
gression, we split the children into groups according to the maternal-weight-gain tertiles
and used partial least-squares discriminant analysis (PLS-DA) to maximize the separation
between the samples and to identify discriminant patterns. We adjusted the analysis for the
same confounders as the linear-regression analysis by calculating a linear-regression model
with confounders for each metabolic feature and using the residues for the PLS-DA analysis.
A permutation test was performed to check for the over fitting of the PLS-DA models.
The multivariate chemometric models were cross-validated with 10-fold Venetian blind
cross-validation. In each run, 10% of the data were left out of the training and used to test
the model. Spectral regions with high variable importance in projections (VIP) coefficients
obtained during PLS-DA were more critical in providing class separation during analysis.
In contrast, those with very small VIP coefficients contributed less to the classification. We
Metabolites 2023, 13, 561 7 of 15

Metabolites 2023, 13, x FOR PEER REVIEW 7 of 15

applied the complete PLS-DA analysis to the data at birth (Figure 2A,B), at 6 months of age
(Figure 3A,B), and at 12 months of age (Figure 4A,B).

Figure 1. Linear association between maternal weight gain and metabolite levels. Linear regression
beta coefficients
coefficients and
and 95%95%confidence
confidenceintervals
intervals(CI)
(CI)for
forthe
theassociation
associationatatbirth (orange),
birth 6 months
(orange), 6 months of
age (green),
of age andand
(green), 12 months
12 monthsof ageof (blue) between
age (blue) maternal
between weight
maternal gain and
weight gainindividual child serum
and individual child
metabolites adjusted
serum metabolites for child
adjusted forsex,
child child
sex, weight at birth,
child weight maternal
at birth, obesity,
maternal maternal
obesity, diabetes,
maternal and
diabetes,
maternal
and maternal adherence to the Mediterranean diet. Each square represents the beta coefficient for aa
adherence to the Mediterranean diet. Each square represents the beta coefficient for
single metabolite, and the horizontal line indicates the 95% CI. Positive beta coefficients indicate a
single metabolite, and the horizontal line indicates the 95% CI. Positive beta coefficients indicate a
positive association with the outcome, while negative beta coefficients indicate a negative associa-
positive association with the outcome, while negative beta coefficients indicate a negative association.
tion. Metabolites with a statistically significant association (adjusted p value < 0.05) are marked as
Metabolites
“a” (birth), with a statistically
“b” (6 months ofsignificant
age), and/or association
“c” (12 (adjusted
months of p value
age).<Label
0.05) are marked
keys: CCC,ascho-
“a”
(birth), “b” (6 months of age), and/or “c” (12 months of age). Label keys: CCC,
line-containing compounds; FA1, CH3 groups in fatty acids; FA2, beta CH2 groups in all, saturated choline-containing
compounds;
and FA1, CH3
unsaturated, fatty groups in fatty
acids; FA3, =CH acids;
2-CHFA2,
2-CHbeta
2-CH=CH2 groups
in fatty in all,
acids; saturated
FA4, =CH-CH and unsaturated,
2-CH= in fatty
acids; FA5, all
fatty acids; -CH=CH-
FA3, =CH2 -CH in fatty
2 -CHacids;
2 -CH=ApoHDL, apolipoproteins
in fatty acids; FA4, =CH-CH in HDL
2 -CH= lipoparticles.
in fatty acids; FA5, all
-CH=CH- in fatty acids; ApoHDL, apolipoproteins in HDL lipoparticles.
To further explore the metabolic impact of maternal weight gain on offspring me-
The score
tabolism plot of the
and identify PLS-DArelationships
non-linear analysis of the metabolome
that at birth
may not have (Figure
been 2A) by
detected shows
the
some moderate discrimination and a continuous trend from tertile 1 to tertile 3 of the
linear regression, we split the children into groups according to the maternal-weight-gain
maternal
tertiles weight
and usedgain.
partial least-squares discriminant analysis (PLS-DA) to maximize the
separation between the samples and to identify discriminant patterns. We adjusted the
analysis for the same confounders as the linear-regression analysis by calculating a line-
ar-regression model with confounders for each metabolic feature and using the residues
for the PLS-DA analysis. A permutation test was performed to check for the over fitting
of the PLS-DA models. The multivariate chemometric models were cross-validated with
10-fold Venetian blind cross-validation. In each run, 10% of the data were left out of the
training and used to test the model. Spectral regions with high variable importance in
projections (VIP) coefficients obtained during PLS-DA were more critical in providing
class separation during analysis. In contrast, those with very small VIP coefficients con-
tributed less to the classification. We applied the complete PLS-DA analysis to the data at
 birth (Figures 2A,B), at 6 months of age (Figures 3A,B), and at 12 months of age (Figures
4A,B).
The score plot of the PLS-DA analysis of the metabolome at birth (Figure 2A) shows
Metabolites 2023, 13, 561 some moderate discrimination and a continuous trend from tertile 1 to tertile 3 of 8 ofthe
15

maternal weight gain.

Figure
Figure 2. 2. PLSDA
PLSDA analysis
analysis and
and z-scores
z-scores ofof newborn
newborn metabolome
metabolome at at birth
birth by
by maternal-weight
maternal-weight gain gain
tertile. (A)
tertile. (A)Score
Scoreplot
plotwith
with95%
95% confidence
confidence ellipse
ellipse of the
of the PLS-DA
PLS-DA analysis
analysis of theofmetabolome,
the metabolome, ad-
adjusted
justed
for forsex,
child child sex,weight
child child weight
at birth,atmaternal
birth, maternal
obesity,obesity,
maternal maternal
diabetes, diabetes, and maternal
and maternal adherence ad-
herence
to to the Mediterranean
the Mediterranean diet fordiet
the for the discrimination
discrimination between between
tertilestertiles
(tertile(tertile
1(T1) 1(T1)
gray, gray,
tertiletertile
2 (T2)2
(T2) black, tertile 3 (T3) red). Cross-validation parameters: RMSECV 0.304,
black, tertile 3 (T3) red). Cross-validation parameters: RMSECV 0.304, R2CV: 0.582; ROC curve R2CV: 0.582; ROC curve
AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same PLS-DA metabolome
AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same PLS-DA metabolome
model. (C) Z-scores and 95% confidence intervals of individual metabolites in offspring grouped
model. (C) Z-scores and 95% confidence intervals of individual metabolites in offspring grouped
by tertile. Metabolites with statistically significant pairwise differences between tertiles (adjusted
by tertile.
p-value Metabolites
< 0.05) withasstatistically
are labelled “a” (tertile significant pairwise
1 vs. tertile2), differences
“b” (tertile between
1 vs. tertile tertiles
3), and/or “c”(adjusted
(tertile 2
p-value
vs. tertile< 0.05) are labelled
3). Label keys areas
the“a” (tertile
same 1 vs. in
as those tertile2),
Figure“b”1. (tertile 1 vs. tertile 3), and/or “c” (tertile
2 vs. tertile 3). Label keys are the same as those in Figure 1.
These trends disappeared at 6 months (Figure 3A) and 12 months (Figure 4A) of age,
These trends
suggesting disappeared
the influence at 6factors.
of other monthsHowever,
(Figure 3A)
theand 12 months at
metabolomes (Figure 4A) of
6 months of age,
age
suggesting the influence of other factors. However, the metabolomes at 6 months
still showed some differences between the middle tertile and the extreme tertiles. These of age
still showeddisappeared
differences some differences
again between the middle
at 12 months tertile
of age in favorand
of the extreme
a specific tertiles. profile
metabolic These
differences disappeared again at 12 months of age in favor of a specific metabolic profile
for tertile 3, indicating larger maternal weight gain. The VIPscores (Figures 2B, 3B and
for tertile 3, indicating larger maternal weight gain. The VIPscores (Figures 2B, 3B and 4B)
4B) also showed that the contributions to the models of the different metabolites were
also showed that the contributions to the models of the different metabolites were different
different at birth and at 6 and 12 months of age. Despite these variations, some metabo-
at birth and at 6 and 12 months of age. Despite these variations, some metabolites were
lites were present in all the sets of VIP scores higher than 1. Glycoprotein fragments, fatty
present in all the sets of VIP scores higher than 1. Glycoprotein fragments, fatty acids, and
acids,and choline-containing compounds were among the most significant contributors
choline-containing compounds were among the most significant contributors in all the
in all the models. Additionally, branched-chain amino acids such as isoleucine and leu-
models. Additionally, branched-chain amino acids such as isoleucine and leucine, butyrate
cine, butyrate derivatives and lactate were also present in most of the sets.
derivatives and lactate were also present in most of the sets.
 Metabolites2023,
Metabolites 2023,13,
13,561
x FOR PEER REVIEW 99 of
of1515

Figure3.3.PLSDA
Figure PLSDAanalysis
analysisandandz-scores
z-scoresof of newborn
newborn metabolome
metabolome at 6atmonths
6 months of age,
of age, by mater-
by maternal-
nal-weight-gain
weight-gain tertile.
tertile. (A) (A)
ScoreScore
plotplot
withwith
95% 95% confidence
confidence ellipse
ellipse of ofthethePLS-DA
PLS-DAanalysis
analysisofofthethe
metabolome, adjusted for child sex, child weight at birth, maternal obesity, maternal
metabolome, adjusted for child sex, child weight at birth, maternal obesity, maternal diabetes, and diabetes, and
maternaladherence
maternal adherencetotothe theMediterranean
Mediterraneandiet dietfor
forthe
thediscrimination
discriminationbetween
betweentertiles
tertiles(tertile
(tertile11(T1)
(T1)
gray, tertile 2 (T2) black, tertile 3 (T3) red). Cross-validation parameters: RMSECV 0.304, R2CV:
gray, tertile 2 (T2) black, tertile 3 (T3) red). Cross-validation parameters: RMSECV 0.304, R2CV: 0.582;
0.582; ROC Curve AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same
ROC Curve AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same PLS-DA
PLS-DA model of the metabolome. (C) The Z-scores and 95% confidence intervals of individual
model of the metabolome.
metabolites (C) The Z-scores
in offspring grouped and
by tertile. 95% confidence
Metabolites intervals of significant
with statistically individual pairwise
metabolitesdif-
inferences
offspring grouped
between by tertile.
tertiles Metabolites
(adjusted p-valuewith statistically
< 0.05) significant
are labeled pairwise
as “a” (tertile differences
1 vs. tertile 2),between
“b” (ter-
tertiles (adjusted
tile 1 vs. p-value
tertile 3), and/or<“c”0.05) are labeled
(tertile as “a”
2 vs. tertile 3).(tertile 1 vs. are
Label keys tertile
the 2), “b”as(tertile
same those 1invs. tertile
Figure 1. 3),
and/or “c” (tertile 2 vs. tertile 3). Label keys are the same as those in Figure 1.

In addition to the VIP contributions, we calculated the z-scores 95% confidence inter-
vals for each group and built logistic-regression models for pairwise group comparisons
between the tertile groups which were also adjusted by the same confounders as the
linear-regression models for a simpler visualization of the differences between the groups
(Figures 2C, 3C and 4C). The analysis by tertile revealed that many of the strongest contrib-
utors to the PLS-DA models at the three time-points did not exhibit statistically significant
pairwise associations between any of the three tertile groups. According to the mean
differences analysis, the influence of maternal weight gain on the offspring metabolome
was greatest at 6 months of age, with sixteen significant differences between tertile 1 and
2 and two differences between tertile 1 and 3, compared with the differences at birth, at
which point a total of five statistically significant differences occurred, and at month 12
when only two statistically significant differences occurred, although the sample size was
rather low at this time point, which may have precluded the achievement of statistical
significance. At all the ages, most of the statistically significant associations corresponded
to comparisons between tertile 1 and tertile 2. Interestingly, a few of these statistically
significant associations expanded from birth to 6 months of age and included fatty acids,
branched-chain amino acids and choline-containing compounds.
 Metabolites2023,
Metabolites 2023,13,
13,561
x FOR PEER REVIEW 10 ofof1515
10

Figure PLSDA
Figure4. 4. PLSDA analysis and and
analysis z-scores of child
z-scores of metabolome at 12 months
child metabolome at 12 of age, byof
months maternal-weight-
age, by mater-
gain tertile. (A) Score
nal-weight-gain plot(A)
tertile. with 95%plot
Score confidence
with 95% ellipse of the PLS-DA
confidence ellipse ofanalysis of theanalysis
the PLS-DA metabolome,of the
metabolome,
adjusted adjusted
for child for child
sex, child sex, child
weight weight
at birth, at birth,
maternal maternal
obesity, obesity,
maternal maternal
diabetes, diabetes,
and maternal and
maternal to
adherence adherence to the Mediterranean
the Mediterranean diet for the discrimination
diet for the discrimination between
between tertiles tertiles
(tertile 1 (T1)(tertile 1 (T1)
gray, tertile
2 gray, tertiletertile
(T2) black, 2 (T2) black,
3 (T3) tertile
red). 3 (T3) red). parameters:
Cross-validation Cross-validation
RMSECV parameters: RMSECV
0.304, R2CV: 0.582; 0.304,
ROC CurveR2CV:
0.582; ROC Curve AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same
AUC: 0.96. (B) Metabolites with PLS-DA VIP score higher than 1 for the same PLS-DA model of the
PLS-DA model of the metabolome. (C) The Z-scores and 95% confidence intervals of individual
metabolome. (C) The Z-scores and 95% confidence intervals of individual metabolites in offspring
metabolites in offspring grouped by tertiles. Metabolites with statistically significant pairwise dif-
grouped
ferencesby tertiles.tertiles
between Metabolites withp-value
(adjusted statistically
< 0.05)significant
are labeledpairwise
as “b” differences
(tertile 1 vs.between
tertile 3),tertiles
and/or
(adjusted p-value < 0.05) are labeled as “b” (tertile 1 vs. tertile
“c” (tertile 2 vs. tertile 3). Label keys are the same as those in Figure 1. 3), and/or “c” (tertile 2 vs. tertile 3).
Label keys are the same as those in Figure 1.
In addition to the VIP contributions, we calculated the z-scores 95% confidence in-
Ourfor
tervals metabolite set enrichment
each group analysis (Figure 5)models
and built logistic-regression suggests that
for studying
pairwise hypoxic-like
group compari-
metabolism, ketone bodies production, and ammonia-related metabolic pathways may
sons between the tertile groups which were also adjusted by the same confounders as the
help to understand this connection.
linear-regression models for a simpler visualization of the differences between the
groups (Figures 2C, 3C and 4C). The analysis by tertile revealed that many of the
strongest contributors to the PLS-DA models at the three time-points did not exhibit sta-
tistically significant pairwise associations between any of the three tertile groups. Ac-
cording to the mean differences analysis, the influence of maternal weight gain on the
offspring metabolome was greatest at 6 months of age, with sixteen significant differ-
ences between tertile 1 and 2 and two differences between tertile 1 and 3, compared with
the differences at birth, at which point a total of five statistically significant differences
occurred, and at month 12 when only two statistically significant differences occurred,
although the sample size was rather low at this time point, which may have precluded
the achievement of statistical significance. At all the ages, most of the statistically signif-
icant associations corresponded to comparisons between tertile 1 and tertile 2. Interest-
ingly, a few of these statistically significant associations expanded from birth to 6 months
of age and included fatty acids, branched-chain amino acids and choline-containing
compounds.
Metabolites 2023, 13, x FOR PEER REVIEW 11 of 15

Our metabolite set enrichment analysis (Figure 5) suggests that studying hypox-
Metabolites 2023, 13, 561 ic-like metabolism, ketone bodies production, and ammonia-related metabolic pathways
11 of 15
may help to understand this connection.

Figure 5. Metabolite set enrichment analysis of the association between maternal weight gain and
Figure 5. Metabolite set enrichment analysis of the association between maternal weight gain and
offspring metabolome. The metabolite set enrichment analysis of metabolites with PLS-DA scores
offspring metabolome. The metabolite set enrichment analysis of metabolites with PLS-DA scores
higher
higherthan
than11and adjustedppvalues
andadjusted values<<0.05
0.05at
atbirth,
birth,66months
monthsofofage,
age,or
or12
12months
monthsof ofage.
age.Metabolic
Metabolic
pathways
pathways whose names are indicated are significant (p-values lower than 0.05 after theadjustment
whose names are indicated are significant (p-values lower than 0.05 after the adjustment
using
using the
theHolm-Bonferroni
Holm-Bonferroni method
method andand False
False Discovery
Discovery Rate)
Rate) and
and have
have aa pathway
pathway impact
impact value,
value,
calculatedthrough
calculated throughpathway-topology
pathway-topologyanalysis,
analysis, over
over 0. The
0. The pathways
pathways are represented
are represented as circles.
as circles. The
The circle
circle colourcolour indicates
indicates the significance
the significance level,highest
level, from from highest
(red) to(red) to (white)
lowest lowest in(white) in the en-
the enrichment
richment The
analysis. analysis.
circleThe
sizecircle size is proportional
is proportional to the value
to the impact impactofvalue
each of each pathway
pathway from the from the to-
topology
pology
analysis. analysis.

4.
4. Discussion
Discussion
Maternal
Maternal GWG
GWG during
during pregnancy
pregnancy is is an
animportant
important determinant
determinant of of fetal
fetal growth
growth and and
development
developmentand andcancanhave
havea asignificant
significantimpact
impact ononthethe
metabolic
metabolic health of offspring
health of offspringlater in
later
life. OurOur
in life. study aimed
study to explore
aimed the metabolomic
to explore the metabolomic profilesprofiles
of children at birth,at
of children at birth,
6 months,
at 6
and at 12 and
months, months
at 12ofmonths
age, and
ofto analyze
age, and tothem withthem
analyze respect
withto the maternal
respect to theGWG.
maternalAlthough
GWG.
previous studies explored the metabolomic profiles of offspring and
Although previous studies explored the metabolomic profiles of offspring and their as- their association
with maternal
sociation with metabolic states, thisstates,
maternal metabolic is the this
firstisstudy in which
the first studythe profilesthe
in which areprofiles
analyzed are
with respect to maternal GWG using both regression models and multivariate PLS-DA
models adjusted for confounders. Adjusting for confounders in both the mothers and the
children allowed for a more accurate detection of associations and reduced false positive
discoveries. Although many metabolic associations were detected using our combined
Metabolites 2023, 13, 561 12 of 15

analysis, most of them were present either in the tertiles analysis or in the linear-regression
analysis, suggesting a complex, nonlinear association that was probably modulated by
many external factors. In fact, at birth only, the three tertiles followed a progressive trend
from tertile 1 to tertile 2 and, later, tertile 3 whereas at 6 and 12 months of age, there seems
to have been discrimination between the high and the low tertiles with respect to the
others. The interpretation of these results is far from simple, but the score plots suggest
that some metabolic shifts took place during the first 12 months of life, which may have
been modulated by the maternal GWG.
The infant metabolome is influenced by a range of factors, including genetics, diet, and
environmental exposures, and undergoes significant changes during the first year of life as
the infant grows and develops [19]. Maternal GWG has been associated with alterations
in the infant amino acid metabolome [20] and lipid metabolome [21]. The metabolites
examined in this study cover a wide and rather comprehensive spectrum of compounds.
By studying 43 metabolites, including amino acids, sugars, ketone bodies, lipid moieties,
short-chain fatty acids, and general cellular and circulating compounds, at birth and at two
more early ages, we aimed to provide a broader view of the impact of maternal weight
gain during pregnancy on the infant metabolome. Our study revealed that the influence
of maternal weight gain during pregnancy on the early metabolic profiles of offspring is
not linear and is likely to be multifactorial. Although some differences between the tertiles
of maternal weight gain appeared during birth, their influence peaked at 6 months of age,
and most were resolved at 12 months of age. The impact in the long term and on other
biological processes of these changes is unknown and deserves further investigation.
Among the metabolites affected by maternal weight gain in early life, butyrate deriva-
tives and, specifically, 3-hydroxybutyrate, appear to be predominant. During the first
few days of life, new-borns experience a physiological state known as neonatal ketosis,
characterized by high levels of circulating ketone bodies, including 3-hydroxybutyrate.
This is a normal response to the sudden cessation of glucose supply from the mother, which
after a few days stabilizes to low levels. However, infants who are born prematurely may
have abnormal levels of 3-hydroxybutyrate during their first year of life [19]. The organic
compound 3-hydroxybutyrate has been shown to inhibit the activity of histone deacety-
lases, which are enzymes that remove acetyl groups from histone proteins and therefore
regulate epigenetic modifications [22]. It also has important cell-signaling and regulatory
functions, including pathways involved in reducing oxidative stress and inflammation,
and it is potentially linked to the differences between the glycoprotein levels observed in
our study [23]. Glycoproteins, which appear to be affected by maternal weight gain at birth
and at 6 and 12 months, reflect leukocyte activation and are associated with inflammation.
A previous study found that higher maternal weight gain during pregnancy was associated
with an increase in leucocyte activation in the umbilical cord blood of newborns [24], which
may be related to our findings on glycoproteins and 3-hydroxybutyrate. More recently,
Jacopo et al. found lower levels of beta-hydroxybutyrate in both SGA and low-birth-weight
placenta metabolomes, which may reflect poorer tolerance to hypoxia [25,26]. Finally,
butyrate derivatives, such as aminobutyrates and oxobutyrates, are bacterial co-metabolites
that are initially produced by the gut microbiota and further processed by human cells [27].
These facts combined with our results suggest a relevant role of this metabolite in the
potential impact of maternal weight gain on infant health.
Our analysis revealed the association between other, less frequently studied, metabo-
lites in serum and maternal weight gain. Lactate and acetate at birth appear as to be linearly
correlated with maternal weight gain. In a previous study, higher maternal weight gain was
associated with higher lactate levels in the cord blood of new-borns [28]. Phenylalanine,
which showed statistically significant differences between tertiles at birth in our study, is
an essential amino acid that is necessary for proper growth and development in infants.
There is some evidence to suggest that maternal phenylalanine intake during pregnancy
may be associated with increased birth weight and adiposity in offspring [29], but the
underlying causes are unclear. Leucine is a branched-chain amino acid (BCAA) that is
Metabolites 2023, 13, 561 13 of 15

typically associated with metabolic diseases e.g., type 2 diabetes or obesity in adults [30].
However, its role in early life is controversial. In our study, the babies from mothers with
lower maternal weight gain exhibit lower levels of leucine. Despite high levels of BCAAs
in breast milk, the concentration of leucine and isoleucine in the blood of newborns is
relatively low. Leucine levels increase rapidly during the first few months of life, reaching
adult levels by around six months of age but their association with maternal weight gain
and with infant health is poorly understood.
The reported levels of choline in newborns are relatively high compared to those in
adults because choline is essential for brain development, which is rapid during the first
few months of life [31]. By the time a baby is one year old its choline requirements are
similar to those of an adult. Our results show that these findings may extend to other
choline-containing related compounds, such as phosphocholine and phosphatidylcholine.
Phosphocholine is also a metabolite that partially arises from the processing in the gut
of dietary carnitine into trimethylamines and choline-containing compounds [32]. Our
results suggest that maternal weight gain during pregnancy affects the bacterial ecosystem
of the baby. Microbial co-metabolism can affect nutrient availability and absorption, as
well as immune-system development, and may be pivotal in many of the metabolic effects
we observed in this study. While the initial colonization of the gut microbiota is strongly
influenced by the mother’s microbiota during delivery and breastfeeding, the microbiota
evolves and becomes more diverse based on a variety of factors throughout life.
On one hand, the main strength of our study lies in the novelty of its analysis of
offspring metabolomic profiles according to maternal GWG and their adjustment according
to other common confounders of both the mother (obesity, diabetes during gestation and
adherence to the Mediterranean diet) and the child (sex, weight at birth and type of feeding).
Moreover, this analysis was not limited to the moment of birth, but instead continued up to
the age of one year, identifying what may be markers of cardiovascular risk later in life.
On the other hand, our study has several limitations. First, the high cost of metabolomic
studies and the difficulty in obtaining subjects has caused the initial sample size to be mod-
est, which could have played a role in the failure to find clinical differences and in the
missing of some metabolomic differences between the three groups. Second, the progres-
sive loss of subjects during follow-up, which was particularly affected by the COVID
pandemic, lmeant that the sample sets at different ages (at 6 months and, especially at 12
months of age) had a smaller number of subjects. Finally, we also lacked information about
other potential co-variables such the infant microbiome, the exact duration of exclusive
breastfeeding, the total formula intake, and the order of introduction and quantity of other
complementary foods.
Overall, the mechanisms underlying the relationship between maternal weight gain
and metabolic profiles in infants are not well understood, but it has been hypothesized that
higher maternal weight gain may lead to alterations in placental function and metabolism,
which could in turn affect fetal and infant metabolism. We identified several metabolites
associated with maternal weight gain either linearly or non-linearly by tertiles which
could help to identify metabolic pathways and clusters involved in long-term implications.
Our metabolite set enrichment analysis (Figure 5) suggests that studying hypoxic-like
metabolism, ketone bodies production, and ammonia-related metabolic pathways may help
to understand this connection. The presence of high amino derivatives of short-chain fatty
acids, methylamines, and choline-containing compounds in all our significant metabolic
sets suggests the relevant role of nitrogen metabolism in this network of interactions.
Further research is needed to elucidate the mechanisms through which maternal GWG
modulates these changes in children’s metabolome, as well as the long-term effects in later
life. It is very difficult to translate these metabolomic findings into clinical routine with our
current knowledge, but further investigation may help to identify at-risk infants for whom
to target more personalized dietary and growth monitoring interventions.
Metabolites 2023, 13, 561 14 of 15

Author Contributions: Conceptualization, T.G.-E., D.M., J.A.-P. and E.L.; methodology, D.M., J.A.-P.
and E.L.; validation T.G.-E., F.P.-Z., J.M.M. and D.M. formal analysis, J.M.M., T.G.-E. and D.M.;
investigation, T.G.-E., F.P.-Z., J.M.M., J.A.-P. and D.M.; resources, T.G.-E., F.P.-Z. and J.A.-P.; data
curation, T.G.-E., F.P.-Z. and J.A.-P.; writing—original draft preparation, T.G.-E., J.A.-P. and D.M.;
writing—review and editing, T.G.-E., J.A.-P., E.L. and D.M.; supervision, T.G.-E., J.A.-P., E.L. and
D.M.; project administration, J.A.-P. and E.L.; funding acquisition, E.L. All authors have read and
agreed to the published version of the manuscript.
Funding: The work was funded by grants PID2019-108973RB-C22 and PCIN2017-117 from the
Ministerio de Ciencia e Innovación of Spain, GV/2020/048 and IDIFEDER/2021/072 from the
Generalitat Valenciana of Spain, AP-2021-016-BREIC from the VLC-BIOCLINIC initiative of the
University of Valencia and the INCLIVA, GUTMOM (INTIMIC-085) from the EU Joint Pro-gramming
Initiative Healthy Diet Healthy Life (HDHL). The Institute of Health Carlos III (Plan Nacional de I +
D + I) cofounding FEDER (Grant numbers PI17/01517 and PI20/00269 to E.L.)
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee of the General University Hospital
Consortium of Valencia (CHGUV) (reference number: 01517/2017).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to ethical reasons.
Acknowledgments: We are grateful to Christine Deutsch for her manuscript editing. We also
acknowledge support from, the INCLIVA Biomedical Research Institute. Finally, we thank the
patients involved in the study for their generous contribution.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Barker, D.J.; Osmond, C.; Golding, G.; Kuh, D. Wadsworth ME Growth in utero, blood presure in childhood and adult life, and
mortality from cardiovascular disease. BMJ 1989, 294, 564–567. [CrossRef] [PubMed]
2. Kislal, S.; Shook, L.L.; Edlow, A.G. Perinatal exposure to maternal obesity: Lasting cardiometabolic impact on offspring. Prenat.
Diagn. 2020, 40, 1109–1125. [CrossRef]
3. Perng, W.; Oken, E.; Dabelea, D. Developmental overnutrition and obesity and type 2 diabetes in offspring. Diabetologia 2019, 62,
1779–1788. [CrossRef] [PubMed]
4. Perng, W.; Gillman, M.W.; Fleisch, A.F.; Michalek, R.D.; Watkins, S.M.; Isganaitis, E.; Patti, M.; Oken, E. Metabolomic profiles and
childhood obesity. Obesity (Silver Spring Md.) 2014, 22, 2570–2578. [CrossRef]
5. Perng, W.; Rifas-Shiman, S.L.; McCulloch, S.; Chatzi, L.; Mantzoros, C.; Hivert, M.; Oken, E. Associations of cord blood metabolites
with perinatal characteristics, newborn anthropometry, and cord blood hormones in project viva. Metab. Clin. Exp. 2017, 76,
11–22. [CrossRef]
6. Kadakia, R.; Talbot, O.; Kuang, A.; Bain, J.R.; Muehlbauer, M.J.; Stevens, R.D.; Ilkayeva, O.R.; Lowe, L.P.; Metzger, B.E.; Newgard,
C.B.; et al. Cord Blood Metabolomics: Association With Newborn Anthropometrics and C-Peptide Across Ancestries. J. Clin.
Endocrinol. Metab. 2019, 104, 4459–4472. [CrossRef]
7. Cao, T.; Zhao, J.; Hong, X.; Wang, G.; Hu, F.B.; Wang, X.; Liang, L. Cord Blood Metabolome and BMI Trajectory from Birth
to Adolescence: A Prospective Birth Cohort Study on Early Life Biomarkers of Persistent Obesity. Metabolites 2021, 11, 739.
[CrossRef] [PubMed]
8. Lowe, J.; William, L.; Bain, J.R.; Nodzenski, M.; Reisetter, A.C.; Muehlbauer, M.J.; Stevens, R.D.; Ilkayeva, O.R.; Lowe, L.P.;
Metzger, B.E.; et al. Erratum. Maternal BMI and Glycemia Impact the Fetal Metabolome. Diabetes Care 2017;40:902–910. Diabetes
Care 2018, 41, 640. [CrossRef]
9. Francis, E.C.; Kechris, K.; Cohen, C.C.; Michelotti, G.; Dabelea, D.; Perng, W. Metabolomic Profiles in Childhood and Adolescence
Are Associated with Fetal Overnutrition. Metabolites 2022, 12, 265. [CrossRef]
10. Ballard, J.L.; Novak, K.K.; Driver, M. A simplified score for assessment of fetal maturation of newly born infants. J. Pediatr. 1979,
95, 769–774. [CrossRef]
11. Rasmussen, K.M.; Yaktine, A.L. Weight Gain During Pregnancy: Reexamining the Guidelines; National Academy of Sciences:
Washington, DC, USA, 2009.
12. Trichopoulou, A.; Costacou, T.; Bamia, C.; Trichopoulos, D. Adherence to a Mediterranean diet and survival in a Greek population.
N. Engl. J. Med. 2003, 348, 2599–2608.
13. Battaglia, F.C.; Lubchenco, L.O. A practical classification of newborn infants by weight and gestational age. J. Pediatr. 1967, 71,
159–163. [CrossRef] [PubMed]
Metabolites 2023, 13, 561 15 of 15

14. De Onis, M. WHO Child Growth Standards—Length/Height-for-Age, Weight-for-Age, Weight-for-Length, Weight-for-Height and Body
Mass Index-for Age; World Health Organization: Geneva, Switzerland, 2006; Available online: https://www.who.int/publications/
i/item/924154693X (accessed on 15 April 2022).
15. Pichler, G.; Amigo, N.; Tellez-Plaza, M.; Pardo-Cea, M.A.; Dominguez-Lucas, A.; Marrachelli, V.G.; Monleon, D.; Martin-Escudero,
J.C.; Ascaso, J.F.; Chaves, F.J.; et al. LDL particle size and composition and incident cardiovascular disease in a South-European
population: The Hortega-Liposcale Follow-up Study. Int. J. Cardiol. 2018, 264, 172–178. [CrossRef] [PubMed]
16. Galbis-Estrada, C.; Pinazo-Durán, M.D.; Martínez-Castillo, S.; Morales, J.M.; Monleón, D.; Zanon-Moreno, V. A metabolomic
approach to dry eye disorders. The role of oral supplements with antioxidants and omega 3 fatty acids. Mol. Vis. 2015, 21,
555–567. [PubMed]
17. García-Villaescusa, A.; Morales-Tatay, J.M.; Monleón-Salvadó, D.; González-Darder, J.M.; Bellot-Arcis, C.; Montiel-Company, J.M.;
Almerich-Silla, J.M. Using NMR in saliva to identify possible biomarkers of glioblastoma and chronic periodontitis. PLoS ONE
2018, 13, e0188710. [CrossRef]
18. Trygg, J.; Wold, S. Orthogonal projections to latent structures (O-PLS). J. Chemom. 2002, 16, 119–128. [CrossRef]
19. Bhatia, J.; Gates, A. The Newborn Infant. In Clinical Biochemistry: Metabolic and Clinical Aspects; Elsevier: Amsterdam, The
Netherlands, 2015; pp. 701–718.
20. He, X.; Ji, Y.; Li, H. Maternal gestational weight gain and offspring’s amino acid metabolome in the first year of life: A prospective
birth cohort study. JAMA Pediatr. 2020, 6, 570–578.
21. Lu, Q.; Cheng, Y.; Chen, L.; Chen, X.; Wang, X.; Wang, S.; Yan, W. Associations of maternal gestational weight gain with newborn
lipid levels and the risk of macrosomia: A prospective cohort study. BMC Pregnancy Childbirth 2021, 1, 1–9. [CrossRef]
22. Newman, J.C.; Verdin, E. β-hydroxybutyrate: Much more than a metabolite. Diabetes Res. Clin. Pract. 2014, 106, 173–181.
[CrossRef]
23. Newman, J.C.; Verdin, E. Ketone bodies as signaling metabolites. Trends Endocrinol. Metab. 2014, 25, 42–52. [CrossRef]
24. Harreiter, J.; Simmons, D.; Desoye, G.; Corcoy, R. Adverse pregancy outcomes and metabolic syndrome in women with gestational
diabetes treated with metformin or insulin: A randomised trial. Diabetologia 2019, 4, 601–612. [CrossRef]
25. Troisi, J.; Symes, S.J.K.; Lombardi, M.; Cavallo, P.; Colucci, A.; Scala, G.; Adair, D.C.; Guida, M.; Richards, S.M. Placental
Metabolomics of Fetal Growth Restriction. Metabolites 2023, 13, 235. [CrossRef]
26. Rising, C.L.; D’Alecy, L.G. Hypoxia-induced increases in hypoxic tolerance augmented by beta- hydroxybutyrate in mice. Stroke
1989, 20, 1219–1225. [CrossRef]
27. Tremaroli, V.; Bäckhed, F. Functional interactions between the gut microbiota and host metabolism. Nature 2012, 489, 242–249.
[CrossRef] [PubMed]
28. Masuyama, H.; Hiramatsu, Y. Relationships of maternal weight gain during pregnancy with pacental and fetal weight, placental
and fetal ratio and fetal lactate levels in normal and abnormal pregnancy. J. Obstet. Gynaecol. Res. 2010, 3, 457–465. [CrossRef]
29. Goldin, K.R. Maternal and infant serum phenylalanine levels and pregnancy outcome in infants of nonphenylketonuric mothers.
J. Natl. Med. Assoc. 1993, 85, 617–620.
30. Zhang, Z.; Monleon, D.; Verhamme, P.; Staessen, J.A. Branched-Chain Amino Acids as Critical Switches in Health and Disease.
Hypertension 2018, 72, 1012–1022. [CrossRef]
31. Zeisel, S.H.; Da Costa, K. Choline: An essential nutrient for public health. Nutr. Rev. 2009, 67, 615–623. [CrossRef]
32. Tang, W.H.W.; Wang, Z.; Levison, B.S.; Koeth, R.A.; Britt, E.B.; Fu, X.; Wu, Y.; Hazen, S.L. Intestinal Microbial Metabolism of
Phosphatidylcholine and Cardiovascular Risk. N. Engl. J. Med. 2013, 368, 1575–1584. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.