Biology of Reproduction, 2018, 98(6), 795–809

doi:10.1093/biolre/ioy010
Research Article
Advance Access Publication Date: 18 January 2018

Research Article

Diet-induced obesity alters the maternal

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metabolome and early placenta transcriptome
and decreases placenta vascularity in
the mouse†
Tami J. Stuart1 , Kathleen O’Neill2,‡ , David Condon3 , Issac Sasson2 ,
Payel Sen4 , Yunwei Xia5 and Rebecca A. Simmons1,∗
1
Department of Pediatrics, Division of Neonatology, Children’s Hospital of Philadelphia, University of Pennsylvania,
Philadelphia, Pennsylvania, USA; 2 Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia,
Pennsylvania, USA; 3 Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania, Philadelphia,
Pennsylvania, USA; 4 Epigenetics Center, Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, Pennsylvania, USA and 5 College of Arts and Sciences, Cornell University, Ithaca, New York, USA
∗
Correspondence: Center for Research on Reproduction and Women’s Health, Biomedical Research Building II/III 1308,
Perelman School of Medicine, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA. Children’s
Hospital of Philadelphia, Philadelphia, PA, USA. Email: rsimmons@pennmedicine.upenn.edu
‡
Kathleen O’Neill is co-first author.
†
Grant Support: Funding for this research was provided by K12 HD000849-28 (KEO RSDP), T32 HD040135 (KEO),
T32HD060556 (TS), R01DK062965 (RAS).
Conference Presentation: Presented in part at the 2016 American Society for Reproductive Medicine, Salt Lake City, Utah,
the 2017 Pediatric Academic Societies Meeting, San Francisco, California, and the 2017 Society for Reproductive
Investigation Meeting, Orlando, Florida.
Edited by Dr. Sarah Kimmins, PhD, McGill University

Received 28 August 2017; Revised 19 December 2017; Accepted 17 January 2018

Abstract
Maternal obesity is associated with an increased risk of obesity and metabolic disease in offspring.
Increasing evidence suggests that the placenta plays an active role in fetal programming. In this
study, we used a mouse model of diet-induced obesity to demonstrate that the abnormal metabolic
milieu of maternal obesity sets the stage very early in pregnancy by altering the transcriptome of
placenta progenitor cells in the preimplantation (trophectoderm [TE]) and early postimplantation
(ectoplacental cone [EPC]) placenta precursors, which is associated with later changes in placenta
development and function. Sphingolipid metabolism was markedly altered in the plasma of obese
dams very early in pregnancy as was expression of genes related to sphingolipid processing in the
early placenta. Upregulation of these pathways inhibits angiogenesis and causes endothelial dys-
function. The expression of many other genes related to angiogenesis and vascular development
were disrupted in the TE and EPC. Other key changes in the maternal metabolome in obese dams
that are likely to influence placenta and fetal development include a marked decrease in myo and
chiro-inositol. These early metabolic and gene expression changes may contribute to phenotypic
changes in the placenta, as we found that exposure to a high-fat diet decreased placenta microves-
sel density at both mid and late gestation. This is the first study to demonstrate that maternal
obesity alters the transcriptome at the earliest stages of murine placenta development.

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796
Summary Sentence
Obesity in a mouse model leads to alterations in the maternal metabolome and early placenta
transcriptome as well as changes in vascularity later in gestation which may provide a mechanism
for decreased fetal growth.
Key words: angiogenesis, developmental origins of health and disease, gene expression, metabolism, placenta,
trophectoderm.
Introduction
The rate of obesity has more than doubled in the past 35 years, with
one-third of adults in the Unites States now classified as obese [1].
Not surprisingly, this overall increase in the prevalence of obesity
has been accompanied by an equivalent rise in the number of obese
pregnant women. While obese women are more likely to give birth
to large for gestational age babies [2], they may also give birth to
infants that are small for gestational age (SGA) [3], and recent stud-
ies show that SGA infants born to obese women face higher rates
of neonatal morbidity than appropriately grown infants [4]. More-
over, higher maternal weight before pregnancy is associated with the
development of obesity, insulin resistance, and dyslipidemia in the
offspring [5].
The mechanisms underlying the development of these diseases
later in life remain to be fully elucidated. However, it is becoming
more evident that the placenta is playing an active role in the pro-
gramming of offspring of obese mothers. Survival and growth of the
fetus are critically dependent on the placenta, which acts as the in-
terface between the maternal and fetal circulation to maintain fetal
homeostasis and supply nutrients for optimal fetal growth. Impor-
tantly, nutrient transport and hormonal secretion can be altered by
the placenta in response to alterations in the maternal environment
such as obesity, hypoxia, and inflammation [6].
The window of time during which a fetus is exposed to an obese
milieu is wide, from conception through birth. Therefore, attempts to
elucidate the impact of maternal obesity at different developmental
stages are paramount to understanding this complex process. While
the mouse model of diet-induced obesity using a high-fat diet does
not directly mimic a Western diet, it is the most common animal
model used to mimic morbid obesity in humans and results in very
similar features of human obesity, i.e. insulin and leptin resistance
and hyperlipidemia [7–10]. Using this mouse model of diet-induced
obesity, we previously showed that marked maternal obesity is asso-
ciated with fetal growth restriction and abnormal placentation and
ultimately leads to the development of obesity and impaired glucose
tolerance in the offspring, similar to what has been observed in hu-
man pregnancies complicated by morbid obesity [11]. Further, in
embryo transfer studies, we found that exposure to maternal obesity
prior to implantation was sufficient to result in placental dysfunction
and fetal growth restriction, suggesting that the early embryonic pe-
riod represents a critical window of susceptibility [11]. At embryonic
day 6.5 (e6.5), implantation is fully established and the ectoplacental
cone (EPC) is formed, but its cells are still pluripotent. Therefore,
the window between implantation at e4.5 and formation of the EPC
at e6.5 provides an ideal time to investigate early alterations of the
placenta that are direct consequences of maternal exposures.
The mechanisms underlying abnormal placenta function and de-
velopment in maternal obesity have been extensively studied [6, 11,
12], yet the precise mechanisms are not fully understood. Maternal
metabolic function is altered in the obese state [13], which is likely to
have a major impact on the developing conceptus. Complex diseases,
such as obesity, require the use of integrated approaches to fully un-
T. J. Stuart, 2018, Vol. 98, No. 6
derstand the way in which the condition alters normal metabolic and
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physiologic processes resulting in adverse outcomes [14]. Integration
of “-omic” data may identify potential causal pathways underlying
abnormal placenta function and later development of obesity and
metabolic disorders in the offspring. Therefore, we hypothesized that
obesity prior to pregnancy and during very early pregnancy alters the
maternal metabolome which in turn adversely impacts placenta de-
velopment as evidenced by changes in the transcriptome, and leads
to downstream effects on placenta development.
Materials and Methods
Animals and Diet
All experimental procedures involving the use of live animals were
approved by the Institutional Animal Care and Use Committee re-
view boards of the Children’s Hospital of Philadelphia and the Uni-
versity of Pennsylvania. Female C57BL/6J mice (JAX stock #000664)
at 4 weeks were housed (five animals per cage) and given free ac-
cess to feed and water. Animals were randomly selected to be fed
one of two diets: either standard control diet (Lab Diet 5015) or
a high-fat diet (HFD; Harlan TD.06414). The control diet is 4.7
kcal/gm, with calories provided by 19.8% protein, 54.1% carbo-
hydrates, and 26.1% fat (∼37% saturated, 40% monounsaturated,
and 23% polyunsaturated). The HFD is 5.1 kcal/gm, with calo-
ries provided by 18.4% protein, 21.3% carbohydrates, and 60.3%
fat (37% saturated, 47% monounsaturated, and 16% polyunsat-
urated). Female animals were permitted to feed ad libitum for at
least 12 weeks prior to mating and were continued on control or
HFD throughout gestation. Females were mated with control male
C57BL/6J mice, and mating was confirmed via presence of a vaginal
plug (e0.5).
Transcriptome analysis of trophectoderm (e4.5)
Four days after mating, blastocysts were harvested. The uterine horn
of the pregnant mouse was removed from the peritoneal cavity
and placed in cold calcium and magnesium-free phosphate buffered
saline (PBS). A 30 G needle was attached to a 1 mL syringe filled
with PBS, and the needle was inserted into the end of the uter-
ine horn. Blastocysts were flushed from the uterine horn into a 60
mm dish filled with cold PBS. Under a dissecting microscope, mi-
croneedles were used to dissect the trophectoderm (TE) mechani-
cally from the abembryonic pole. Trophectoderm from five blasto-
cysts were pooled and preserved in RNAlater Stabilization Solution
(Ambion) to produce a single sample. Trophectoderm cDNA from
four replicates of either control or HFD-derived blastocysts was uti-
lized for gene expression profiling. Total RNA was isolated from TE
(PicoPure RNA Isolation Kit, Arcturus KIT0202). Complementary
DNA was generated and linearly amplified (Ovation PICO WTA
System, Nugen). A subsequent round of linear amplification was
performed (TransPlex WTA Kit, Rubicon) to generate an adequate
sample for microarray hybridization. Samples were hybridized to
Obesity alters early placenta gene expression, 2018, Vol. 98, No. 6 797

the GeneChip Mouse Exon 1.0ST array (Affymetrix 901168) and
scanned (GeneChip Scanner 3000 7G System, Affymetrix). Probe
intensity data were normalized and summarized to transcript cluster
ID level using RMA as implemented in the Partek Genomics Suite.
Absence of embryonic tissue in samples was confirmed by lack of
expression of octamer-binding transcription factor 4 (Oct4) using
quantitative PCR. Pairwise comparisons were made between groups
and false discovery rates (FDR) were calculated along with adjusted
P values by the Benjamini and Hochberg step-up method. Ingenuity
Pathway Analysis (IPA) software was used to evaluate functional
clipping (∼0.3 mL/mouse). The blood was collected in EDTA tubes,
and samples were centrifuged for 10 min at 1500× g. Plasma from
10 animals on each diet (n = 20) was isolated and stored at –80◦ C
until pregnancy was confirmed by delivery of pups. Samples were
sent to Metabolon Inc. (Durham, NC, USA) for analysis and were
extracted and prepared using Metabolon’s standard solvent extrac-
tion method. The extracted samples were split into equal parts for
analysis on complementary GC/MS (gas chromatography mass spec-
trometry) and LC/MS (liquid chromatography mass spectrometry)
platforms. Following normalization to volume extracted (plasma),

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pathways. log transformation and imputation of missing values, if any, with
the minimum observed value for each compound, Welch two-sample
t-test and ANOVA contrasts were used to identify biochemicals that
Transcriptome analysis of EPC (e6.5)
differed significantly between experimental groups. Analysis by two-
Postimplantation embryos were isolated at embryonic day 6.5 (e6.5).
way ANOVA identified biochemicals exhibiting significant interac-
The uterine horn of the pregnant mouse was removed from the peri-
tion and main effects for experimental parameters of diet. An esti-
toneal cavity and placed in cold calcium and magnesium-free PBS.
mate of the FDR (q-value) was also calculated to take into account
The decidua was freed from the muscular uterine lining, and the
for multiple comparisons.
embryo was shelled out from the decidua using forceps under a dis-
secting microscope. Reichert’s membrane was removed [15], and
embryos were divided at the embryonic/extraembryonic junction
Immunohistochemistry
(circumferential groove) [16]. The embryonic portion, or epiblast,
Pregnant female mice were sacrificed at e12.5 or e18.5 using CO2
was placed in 50 μl extraction buffer (Arcturus PicoPure RNA Iso-
and cervical dislocation. The gravid uterus was dissected free and
lation Kit), incubated at 42◦ F for 30 min, and then snap frozen and
placed in cold PBS on ice. Individual implantation sites were sep-
stored at –140◦ F. Similarly, the extraembryonic portion which in-
arated, and placentas were dissected from the fetus, divided in
cludes the EPC and extra-embryonic ectoderm [17] was separately
half, placed in 10% formalin, and sent to the Children’s Hospi-
processed in the same manner. Total RNA was isolated from tissues
tal of Philadelphia Pathology core for staining with plasmalemma
using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher), in-
vesicle-associated protein (PLVAP) antibody. PLVAP antibody is a
cluding an on-column DNase digestion (Qiagen). RNA quality was
mouse-specific antibody that stains endothelial cells in embryonic
assessed using the Agilent Bioanalyzer RNA 6000 Pico Kit (Agilent)
and adult tissues and can therefore be used to quantify vascular-
ensuring all RNA Integrity Numbers were >8. Real-time quanti-
ity via the endothelial cells lining the vascular beds. Placentas were
tative reverse-transcription polymerase chain reaction (qPCR) was
embedded in paraffin, sectioned, and stained with PLVAP antibody
used on the RNA extracted from the corresponding epiblast to de-
(Biorad MCA2539T, RRID AB 1102821, Supplemental Table 1).
termine the sex of the embryo using TaqMan probes for inactive X
Staining was performed on a Bond Max automated staining system
specific transcripts (Xist) as a positive control (Mm01232884 m1)
(Leica Biosystems). The Bond Intense R staining kit (Leica Biosys-
and eukaryotic translation initiation factor 2, subunit 3, structural
tems DS9263) was used. Standard protocol was followed with the
gene Y-linked (Eif2s3y) (Mm01210630 m1) as a marker of male sex
exception of the primary antibody incubation, which was extended
(Applied Biosystems). Ribosomal and mitochondrial RNA were re-
to 1 h at room temperature. Avidin Biotin Blocking (Vector Labs
moved from total RNA with the RiboGone—Mammalian kit (Clon-
SP-2001) and a Peptide Blocking step were added (DAKO X0909).
tech). RNA-Seq libraries were prepared on individual sexed EPC (n
PLVAP antibody was used at a 1:100 dilution, and antigen retrieval
= 5 for female embryos and n = 4 for male embryos from each
was performed with E1 (Leica Biosystems) retrieval solution for 20
diet group) using the SMARTer Stranded RNA-Seq kit (Clontech).
min. Biotinylated Anti-Rat Secondary (Vector BA-4001, Supplemen-
Twenty-five million 75 base pair (bp) paired-end reads were ob-
tal Table 1) was used at a 1:200 dilution. Slides were rinsed, dehy-
tained for each biologic replicate on the NextSeq500 platform (Illu-
drated through a series of ascending concentrations of ethanol and
mina), aligned to the mm10 genome using STAR [18]. Genes were
xylene, then coverslipped. Slides were scanned on a Scanscope CS-O
counted using featureCounts [19] with Gencode M9, and differ-
slide scanner at 20× magnification and viewed with Aperio Im-
ential expression was done using EdgeR and corrected for multi-
ageScope software (Leica Biosystems). The Microvessel Analysis V1
ple testing using the Bonferroni correction [20]. qPCR was used to
algorithm (Leica Biosystems) was used to quantify vascularity. This
replicate RNA-Seq findings on a separate cohort of EPCs derived
algorithm detects and quantifies microvessels on slides stained with
from control and obese mice (n = 10 EPCs in each group, total 20
endothelial markers, such as PLVAP antibody. Microvessel density
mice). Select differentially expressed genes involved in early vascular
was calculated by the algorithm as the number of vessels per unit
development (core 1 synthase, glycoprotein-N-acetylgalactosamine
of analysis area in square microns. The analysis area included the
3-beta-galactosyltransferase, 1 (C1galt1), endothelial PAS domain
entire placenta. Placentas were sexed using genomic DNA isolated
protein 1 (Epas), platelet derived growth factor, alpha (Pdgfa), and
from the tails of corresponding fetuses via the QIAamp DNA Mini
transforming growth factor, beta receptor II (Tgfbr2)) identified in
kit (Qiagen), and qPCR was performed with Taqman probes for
RNA-Seq studies were examined with qPCR using TaqMan probes
Xist (Mm01232884 m1) and sex determining region of Chr Y (Sry)
(Mm00473987, Mm01236112, Mm01205760, Mm03024091).
(Mm00441712 s1). Results from the quantification of vascularity
were analyzed using two-way ANOVA (PRISM software version
Metabolomic analysis in maternal plasma 7.0a; GraphPad), and significance was defined as P value ≤ 0.05. At
Six days after detection of a vaginal plug (e6.5) female mice were e12.5, one slide each from seven control males, five control females,
briefly anesthetized with isoflurane, and blood was collected via tail seven HFD males, and seven HFD females were analyzed. At e18.5,
798
Table 1. Weights of control and HFD dams, placentas, and fetuses. Data are presented as mean ± SEM.
Control Diet
Dam weight after 12 weeks on diet 22.41 g ± 0.349 (n = 40)
E18.5 fetal weight 1.163 g ± 0.028
E18.5 female fetus weight 1.178 g ± 0.033 (n = 22)
E18.5 male fetus weight 1.182 g ± 0.044 (n = 16)
E18.5 placenta weight 0.089 g ± 0.002
E18.5 female placenta weight 0.087 g ± 0.003 (n = 22)
E18.5 male placenta weight 0.095 g ± 0.004 (n = 16)
one slide each from five control males, six control females, three
HFD males, and five HFD females were analyzed.
Uterine and umbilical artery doppler imaging
All doppler imaging was performed by the Small Animal Imag-
ing Facility at the University of Pennsylvania using the Vevo
2100 Imaging System (FUJIFILM Visual Sonics) as described by
Hernandez-Andrade et al. [21]. Mice were anesthetized with 2%
isoflurane, and abdominal hair was removed using a chemical hair
remover (Nair). Mice were positioned on a heated pad and body tem-
perature and respiratory rate were continuously monitored. Umbili-
cal artery flow was measured at the level of umbilical cord insertion
at the abdomen of the fetus. Three measurements of the peak systolic
velocity (PSV) and end diastolic velocity (EDV) were taken per cord,
and the mean of each was used to calculate the resistive index (RI)
using the standard calculation of (PSV-EDV)/PSV. All images were
obtained at <60o . At e12.5–13.5, umbilical cord measurements were
taken on 12 HFD fetuses and 16 control fetuses (from three litters
per diet), and bilateral uterine artery measurements were taken on
two control dams and two HFD dams. At e18.5, umbilical cord
measurements were taken on six HFD fetuses and four control fe-
tuses (from three litters per diet), and uterine artery measurements
were taken on two control dams and three HFD dams. Results were
compared using an unpaired t-test (PRISM software version 7.0a;
GraphPad).
Results
Maternal, fetal, and placenta characteristics
At the time of mating, mice fed an HFD were significantly heav-
ier, had elevated 2 h glucose tolerance tests (GTT), and increased
fasting leptin, as we have previously described [11]. Overall, there
were no differences in resorption rates at e6.5 (2.5% control vs
4.5% HFD), litter size at delivery (6.3 control pups vs 7.6 HFD
pups), or the male offspring to female offspring ratio between the
two groups. Similar to previous studies [11, 22], male and fe-
male HFD e18.5 fetuses were significantly growth restricted; how-
ever, placentas did not differ in weight (Table 1). We did not as-
sess fertility or fetal malformations in this study; however, previ-
ous studies demonstrate that female mice fed a 60% HFD have
decreased fertility and higher rates of fetal developmental defects
[8, 22, 23].
Maternal obesity alters transcriptomic pathways
related to inflammation and vascular development in
the TE
To elucidate the underlying mechanisms linking maternal obesity
to altered offspring development and identify pathways that were
T. J. Stuart, 2018, Vol. 98, No. 6
High-Fat Diet P-value
29.58 g ± 0.696 (n = 38) <0.0001
1.045 g ± 0.017 0.0003
1.040 g ± 0.019 (n = 22) 0.0007
1.051 g ± 0.028 (n = 21) 0.0127
0.083 g ± 0.002 0.067
0.082 g ± 0.004 (n = 22) 0.2947
0.088 g ± 0.002 (n = 21) 0.0998
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disrupted at the earliest stage of placenta development, gene ex-
pression profiling was performed on TE of e4.5 blastocysts from
control and HFD dams. Using an FDR of <0.05, 5285 genes were
differentially expressed between HFD and controls (1 gene down-
regulated and 5284 genes upregulated) (Supplemental Table 2). IPA
revealed disruption of over 400 canonical and biologic pathways
including endothelin-1 (ET-1) signaling, leptin signaling in obesity,
pigment epithelium-derived factor (PEDF) signaling, corticotropin-
releasing hormone (CRH) signaling, eNOS signaling, protein kinase
A signaling, p38 mitogen-activated protein kinase (p38 MAPK) sig-
naling, fatty acid α-oxidation, and IL-12 signaling and production
in macrophages (Supplemental Table 3).
The ET-1 signaling pathway was upregulated in HFD TE. A to-
tal of 77 of 181 genes in this pathway were differentially expressed
(P = 2.76E-05). Genes such as endothelin 1 (Edn1), fibroblast
growth factor receptor 3 (Fgfr3), and mitogen-activated protein ki-
nase 3 (Mapk3) were all significantly upregulated compared to con-
trols. ET-1 is a potent vasoconstrictor, and elevated levels in the
placenta are thought to play a role in the development of preeclamp-
sia that is associated with maternal obesity [24].
A total of 42 of 109 genes in the p38 MAPK signaling pathway
were differentially expressed leading to predicted activation by an
HFD (P = 1.33E-02). This pathway is triggered by stress stimuli
such as death ligands, inflammatory cytokines, and oxidative stress
and leads to transcription of genes responsible for apoptosis and
production of proinflammatory cytokines, such as IL-6 [25].
Importantly, we found that the pathway for leptin signaling in
obesity was upregulated in TE of mice fed an HFD (34/84 genes al-
tered, P = 1.08E-02). Genes such as thymoma viral proto-oncogene
1 (Akt1), growth factor receptor bound protein 2 (Grb2), and
phosphoinositide-3-kinase regulatory subunit 5 (Pik3r5) were all
significantly upregulated.
Maternal obesity alters upstream regulators and
regulatory networks
IPA can identify the cascade of upstream transcriptional regu-
lators that may explain changes in gene expression and illumi-
nate the biological activities occurring in the tissue or cell of in-
terest. We identified differential expression of multiple upstream
transcriptional regulators including catenin (cadherin associated pro-
tein), beta 1 (CTNNB1) (Wnt signaling), p38 MAPK, eph receptor
B2 (ERK) 1/2, IL4, toll-like receptor 2 (TLR 2), and CREB binding
protein (CREBBP), which were all activated. Important upstream
regulators that were inhibited included RE1-silencing transcription
factor (REST), protein kinase, AMP-activated, alpha 2 catalytic sub-
unit (PRKAA2), and additional sex combs like 1 (ASXL1). REST re-
presses the expression of the cholinergic gene in non-neuronal cells.
Interestingly, the placenta contains large amounts of acetylcholine
and choline acetyltransferase, which are thought to regulate blood
Obesity alters early placenta gene expression, 2018, Vol. 98, No. 6
flow in the placenta [26, 27]. However, its biological functions in
the TE are still unclear. PRKAA2 mediates differentiation of tro-
phoblast stem cells [28]. Reduction in activity of PRKAA1/2 inhibits
the production of hormones that are necessary for differentiation
of trophoblast stem cells, thereby inhibiting further differentiation
[29]. ASXL1, a polycomb protein, is obligate for normal embryonic
development.
Individual genes of interest that were significantly altered in
TE of HFD dams include LYR motif containing 4 (Lyrm4) (−27
fold change, q < 0.15), OPA1, mitochondrial dynamin like GTPase
(Opa1) (–2.072 fold change, q < 0.15), phosphatidylinositol-4,5-
bisphosphate 3-kinase catalytic subunit beta (Pik3cb) (–2.75 fold
change, q < 0.15), and fibroblast growth factor receptor 2 (Fgfr2)
(–3.428 fold change, q < 0.15). Lyrm4 encodes for an iron–sulfur
cluster protein important in oxidative phosphorylation [30]. Opa1
encodes for a mitochondria-shaping protein that controls efficiency
of steroidogenesis in trophoblasts [31], and Pik3cb is involved in
trophoblast differentiation [32]. The fibroblast growth factor (FGF)
signaling pathway has been shown to be critical for trophoblast pro-
liferation and differentiation [33]. Fgfr2 mutant mice display multi-
ple placental defects, including a failure of the labyrinthine layer to
develop, and die shortly after implantation [33], therefore downreg-
ulation of Fgfr2 in TE of HFD dams is of particular interest.
Maternal obesity alters multiple pathways in EPC
To determine the progression of changes that were identified early
in development in HFD TE, we generated strand-specific total RNA-
Seq libraries after ribosomal RNA depletion of EPC from mouse
embryos at e6.5. 32,193 unique transcripts were identified in the
EPC. Using an FDR < 0.05, 350 genes were differentially expressed
between control and HFD EPCs (89 genes downregulated and 261
genes upregulated), and using an FDR < 0.1, 595 genes were
differentially expressed (188 downregulated and 407 upregulated)
(Supplemental Figure 1), (Supplemental Table 4).
We again utilized IPA to highlight key alterations between all
(males and females combined) HFD mice and all control mice. Key
top canonical pathways disrupted included the signaling pathways
of sphingosine-1-phosphate, sphingomyelin metabolism, p21 acti-
vated kinases (PAK), ceramide, integrin-linked kinases (ILK), and
peroxisome proliferator-activated receptor (PPAR) (Supplemental
Table 3).
Sphingosine-1-phosphate (S1P) is a bioactive lipid derived from
the metabolism of sphingomyelin [34], and it plays a crucial role
in vascular development, immune cell trafficking, and neurogenesis.
The sphingosine-1-phosphate signaling pathway was significantly
altered in EPC of obese dams (P = 1.9E-03). A total of 10 of 125
genes comprising this pathway were differentially expressed. Many
of the genes in this pathway, including Pik3cb, N-acylsphingosine
amidohydrolase 1 (Asah1), sphingomyelin phosphodiesterase 1, acid
lysosomal (Smpd1), sphingomyelin phosphodiesterase 2, neutral
(Smpd2), and growth factor receptor bound protein 2 (Grb2) have
been correlated with human obesity and insulin resistance [35–38].
Of note, S1P is elevated in the plasma of obese mice and humans,
and plasma S1P concentrations positively correlate with measures of
metabolic disease in humans [39].
PAK signaling was activated by maternal obesity (P = 3.63E-04).
PAKs are protein kinases involved in a wide array of biologic func-
tions including gene transcription, apoptosis, and cellular morphol-
ogy [40]. A total of 10 of 101 genes in this pathway were differen-
tially expressed, including Rac/Cdc42 guanine nucleotide exchange
799
factor (GEF) 6 (Arhgef6), fibroblast growth factor receptor 1 (Fgfr1),
integrin alpha 2 (Itga2), and platelet derived growth factor receptor,
beta polypeptide (Pdgfrb).
ILK signaling was downregulated with 12 of 196 genes differ-
entially expressed including myosin, heavy polypeptide 3, skeletal
muscle, embryonic (Myh3) and myelocytomatosis oncogene (Myc)
(P = 6.46E-03). ILK is a ubiquitously expressed protein that acts in
the critical phases of cell polarization, migration, proliferation, and
survival [41]. It is highly expressed in villous cytotrophoblast cells
in first trimester human chorionic floating villi, and its activity is
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increased during syncytialization [42].
The PPAR signaling pathway was inhibited in HFD EPCs with
a total of nine genes differentially expressed (P = 9.74E-04). These
genes include interleukin 1 receptor-like 2 (Il1rl2), Pdgfrb, tumor
necrosis factor receptor superfamily, member 1b (Tnfrsf1b), and nu-
clear receptor co-repressor 2 (Ncor2). PPARs are known to modulate
inflammatory responses and energy homeostasis and are critically
important to early placenta development [43, 44].
Additionally, upstream analysis using IPA revealed activation of
transforming growth factor, beta 1 (TGF-b1), tumor necrosis factor
(TNF), interleukin 6 (IL-6), and SMAD family member 4 (SMAD
4). Examples of individual genes that were significantly altered in
EPC of HFD dams include chemokine (C-C motif) receptor-like 2
(Ccrl2), a chemoattractant receptor, which was downregulated 2.46
log2FC in HFD EPCs, and phospholipase A2, group IIF (Pla2g2f),
which is involved in lipoprotein modification [45] and was downreg-
ulated 2.26 log2FC in HFD EPCs. fibroblast growth factor 4 (Fgf4),
which is vital to for the maintenance of undifferentiated trophoblast
stem cells, was upregulated 5.08 log2FC, and bone morphogenetic
protein 7 (Bmp7), which can induce trophoblast differentiation, was
upregulated 1.99 log2FC.
Sex-specific differences in the transcriptome of the EPC
It is well established that the placenta is a sexually dimorphic or-
gan [46]. However, the timing at which this dimorphism first de-
velops is not known nor whether this process occurs prior to the
production of sex hormones and is thus dependent only on sex chro-
mosomes. Therefore, we next stratified our RNA-Seq data by sex.
Using an FDR of <0.05, there were 14 genes that were differentially
expressed between male control and male HFD EPCs, including 3-
hydroxybutyrate dehydrogenase, type 1 (Bdh1) which is important
in fatty acid catabolism (–1.47 log2FC in HFD males) and coagula-
tion factor II (thrombin) receptor (F2r), which is a thrombin receptor
gene (1.42 log2FC in HFD males). By expanding the FDR to < 0.15,
we identified 50 genes that were differentially expressed between
male control and male HFD EPCs, of which 17 genes were down-
regulated and 33 were upregulated (Supplemental Table 5). Of major
significance, there were no genes that were differentially expressed
between female control and female HFD EPCs, suggesting that males
are much more susceptible to the effects of maternal obesity.
Canonical pathways altered by an HFD in male EPCs as com-
pared to control male EPCs included pathways previously identified
such as axonal guidance, vitamin D receptor/retinoid X receptor
(VDR/RXR) activation, PPAR signaling, and PAK signaling, as well
as pathways found to be unique to the males such as sertoli cell-
sertoli cell junction signaling, ketogenesis, and androgen biosynthe-
sis (Supplemental Table 3). Upstream regulators that were activated
included TGF-β1, parathyroid hormone (PTH), and endothelin-1
(EDN1). PPARG and Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)
were repressed by an HFD in EPC of male embryos.
800
Figure 1. Venn diagram of some of the most significant canonical pathways found using IPA that were either unique to TE (e4.5) or EPC (e6.5) or overlapped.
When we compared all female EPCs to all male EPCs, we found
68 genes differentially expressed with an FDR < 0.05. Of these
68 genes, 12 are known to be sex-linked. When we opened the
FDR to 0.15 (326 genes, Supplemental Table 6) and performed IPA,
we found that several canonical pathways were downregulated in
males including glutamate receptor signaling (Supplemental Table 3).
Downregulation of this pathway was largely in part to decreased ex-
pression of glutamate receptor, metabotropic 1 (Grm1), which had a
–6.8 log2FC (FDR 0.05) as compared to females. Inhibited upstream
regulators included Sonic hedgehog (SHH) and brain-derived neu-
rotrophic factor (BDNF). Activated upstream regulators included
Growth factor receptor bound protein 2 (GRB2) and SRY-box 7
(SOX7).
Multiple pathways overlapped between HFD TE and
HFD EPC
There was considerable overlap in canonical pathways altered by
an HFD between TE at e4.5 and EPC at e6.5, including VDR/RXR
activation, ET-1 signaling, axonal guidance signaling, p38 MAPK
signaling, and PPARα/RXRα activation suggesting the importance
of these pathways in the development of abnormal placenta function
in an obese pregnancy (Figure 1). Additionally, there were changes
in expression of genes at e4.5 that persisted at e6.5. For example,
several antiangiogenic genes, including angiopoietin 2 (Angpt2), ar-
restin, beta 1 (Arrb1), collagen, type IV, alpha 1 (Col4a1), collagen,
type IV, alpha 2 (Col4a2), serine (or cysteine) peptidase inhibitor,
clade E, member 1 (Serpine1), and serine (or cysteine) peptidase in-
hibitor, clade F, member 1 (Serpinf1) were significantly increased by
an HFD (P < 0.05 vs. control) in both TE and EPC.
Obesity alters the metabolome in maternal plasma at
e6.5
Given that our transcriptome results implicated early changes in
lipid metabolism, we next performed metabolic profiling in ma-
T. J. Stuart, 2018, Vol. 98, No. 6
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ternal plasma at e6.5 to investigate whether changes in the ma-
ternal metabolic profile correlated with changes in the early pla-
centa transcriptome. The results of global metabolic profiling of
maternal plasma demonstrated that an HFD has a profound effect
on metabolism, with greater than a third of metabolites measured
demonstrating a significant difference between obese and control
dams. A total of 522 compounds of known identity were detected in
plasma samples. Levels of 192 biochemicals were significantly differ-
ent between HFD and control samples (P ≤ 0.05); 89 were present in
higher levels and 103 were present in lower levels in plasma derived
from obese dams compared to controls (Supplemental Table 7).
Principal components analysis revealed distinct separation be-
tween the plasma samples derived from the control and obese ani-
mals (Figure 2). The large degree of separation between these sample
types indicates that global metabolism changed in a significant man-
ner with diet.
Multiple lipid species in maternal plasma are increased
by obesity
Exposure to an HFD resulted in significant changes in the lev-
els of biochemicals involved in fatty acid metabolism. There were
significant increases in the levels of several fatty acid species in plasma
(Table 2) in the HFD dams. Levels of long-chain fatty acids (LCFA)
myristate, myristoleate, palmitate, and stearate were significantly el-
evated in the plasma of HFD dams. Similar to LCFA, several polyun-
saturated fatty acids (PUFA) were also significantly increased by an
HFD. Specifically, in HFD maternal plasma, the levels of ω-6 fatty
acids (dihomo-linolenate, adrenate, docosapenaenoate, and dihomo-
linoleate) were elevated (>2 fold) compared to controls, while the
level of ω-3 fatty acids did not differ between the groups.
When dietary fats are in abundance, animals can metabolize them
in multiple ways. They can store them in adipose tissue, shunt them
to other complex lipids (phospholipids, sphingolipids), or they can
oxidize them for energy production via mitochondrial β-oxidation.
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Figure 2. Principal component analysis of plasma samples from control (pink) and HFD (red) pregnant dams at e6.5. N = 10 per diet intervention.

Table 2. Fatty acids altered in maternal plasma at e6.5 as a result of diet-induced obesity as detected using liquid chromatography/mass
spectroscopy. N = 10 per diet intervention for plasma analyses. Red and green shaded cells indicate P ≤ 0.05 (red indicates that the mean
values are significantly higher; green values significantly lower). Light red and light green shaded cells indicate 0.05 < P < 0.10.

The increases in glycerophospholipids and sphingomyelin species Finally, the levels of metabolites involved in sphingolipid
together with decreases in carnitine and its metabolic precursor de- metabolism were dramatically altered in the plasma of obese dams.
oxycarnitine detected in the plasma of obese dams suggest significant Of the 24 metabolites in this pathway, >50% were significantly
alterations in β-oxidation. higher in HFD dams (Table 3).
802 T. J. Stuart, 2018, Vol. 98, No. 6

Table 3. Sphingolipid metabolites altered in maternal plasma at e6.5 as a result of diet-induced obesity as detected using liquid chromatog-
raphy/mass spectroscopy. N = 10 per diet intervention. Red and green shaded cells indicate P ≤ 0.05 (red indicates that the mean values
are significantly higher; green values significantly lower). Light red and light green shaded cells indicate 0.05 < P < 0.10.

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Table 4. Lysine metabolites altered in maternal plasma at e6.5 as a result of diet-induced obesity as detected using liquid chromatogra-
phy/mass spectroscopy. N = 10 per diet intervention. Red and green shaded cells indicate P ≤ 0.05 (red indicates that the mean values are
significantly higher; green values significantly lower). Light red and light green shaded cells indicate 0.05 < P < 0.10.

Multiple amino acid pathways are influenced by
maternal obesity
Plasma of obese dams was enriched in several lysine catabolites
(e.g. 2-aminoadipate, 2-oxoadipate, glutarylcarnitine), suggesting
that maternal obesity alters lysine availability and/or utilization
(Table 4). Acetylated lysine metabolites were also significantly lower
in plasma of HFD dams.
Glycine and several glycine metabolites were significantly re-
duced in plasma of HFD dams. Interestingly, recent studies have
shown that low glycine levels are associated with insulin re-
sistance and increased visceral adiposity [47, 48]. Because the
utilization of carbohydrates is impaired when insulin resistance
is present, the oxidation of amino acids becomes an alterna-
tive energy source by entering the citric acid cycle at different
points.
Maternal HFD is associated with altered microbial
metabolites in plasma
It is well established that the gut microbiota is altered by obe-
sity [49–51]. In our study, HFD dams exhibited alterations in
microbial metabolites derived from tyrosine and tryptophan (phe-
nol sulfate, p-cresol sulfate, p-cresol-glucuronide, indolepropionate,
and 3-indoxyl sulfate) in their plasma (Figure 3). Further evidence for
Obesity alters early placenta gene expression, 2018, Vol. 98, No. 6
Figure 3. Microbial metabolites significantly altered (P < 0.05) using Welch’s two-sample t-test in maternal plasma at e6.5 as detected using liquid chromatogra-
phy/mass spectroscopy. N = 10 per diet intervention.
an altered microbiome was manifested in the bile acid profiles. Bile
acids play an important role in lipid metabolism and obesity. While
the levels of multiple primary (taurocholate, chenocdeoxycholate)
and secondary bile acids (taurodeoxycholate ursodeoxycholate, tau-
roursodeoxycholate, hyodeoxycholate) were altered by exposure to
HFD in plasma, the changes in secondary bile acids were more pro-
nounced. Secondary bile acids are generated when microbiota in the
gut deconjugate and dehydroxylate bile acids synthesized by the host.
Taken together, these findings strongly suggest that the microbiome
is altered by exposure to an HFD, and importantly, the early embryo
may be exposed to altered microbial metabolites generated by the
maternal microbiome in the setting of maternal obesity.
The results of the above transcriptomic and metabolomic studies
are summarized in Table 5.
Vascular development is impaired in placenta of HFD
dams
Given that maternal exposure to an HFD altered several path-
ways and metabolites vital to vascular development such as
sphingosine-1-phosphate, the ET-1 signaling pathway, and sphin-
golipid metabolism, we sought to explore downstream phenotypic
changes in placenta vasculature. Therefore, we next stained mid and
late gestation placentas from control and HFD dams with PLVAP
antibody to quantify vascularity. At e12.5, placentas of control males
had the highest microvessel density, followed by control females and
HFD females. HFD males had the lowest microvessel density, which
was 31.4% lower than control males (P < 0.05) (Figure 4). The
changes in microvessel density persisted throughout the remainder of
gestation, and e18.5 male placentas from HFD dams had ∼30% less
microvessel density when compared to both control male and con-
trol female placentas (P < 0.05). HFD female placentas had ∼20%
decreased microvessel density when compared to controls (Figure 5).
Given that we found changes in microvessel density of HFD pla-
centas, we sought to determine whether an HFD altered arterial flow
and resistance in the placenta and uterus. Umbilical artery and uter-
ine artery dopplers were performed on HFD and control dams and
fetuses at e12.5 and e18.5. The average RI measured in the umbilical
artery of fetuses at e12.5 was slightly increased in the HFD group as
803
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compared to the controls (0.8731 ± 0.017 vs 0.8552 ± 0.013, P =
0.408). At e18.5, the RI of the umbilical artery in the control group
was 0.9031 ± 0.0107 vs 0.917 ± 0.0113 in the HFD group (P =
0.421). The average RI of the uterine arteries in the control group at
e12.5 was 0.6097 ± 0.0532 vs 0.6713 ± 0.1254 in the HFD group
(P = 0.635), and at e18.5, the control group uterine artery average
RI was 0.5634 ± 0.0424 vs 0.5954 ± 0.0059 in the HFD group (P
= 0.398). Therefore, the RI was slightly, but not significantly, ele-
vated in the uterine arteries of dams fed an HFD by 10.1% at e12.5
and 5.7% at e18.5. Additionally, the RI of the umbilical arteries of
HFD fetuses also trended upward slightly compared to controls by
2.1% at e12.5 and 1.5% at e18.5. However, these results were not
statistically significant.
Discussion
In this study, we demonstrated that maternal obesity alters the ma-
ternal metabolome very early in pregnancy and is associated with
marked changes in expression of genes involved in key pathways such
as sphingolipid metabolism, angiogenesis, and fatty acid metabolism
in cells that will eventually give rise to the placenta. While multiple
studies have demonstrated that obesity in pregnancy alters placenta
structure and function [11] [52–54], our data surprisingly show that
the abnormal metabolic milieu of maternal obesity sets the stage
very early in pregnancy by altering the transcriptome of placenta
progenitor cells in the TE and EPC, which in turn are associated
with profound implications for placenta development and function.
In the mouse, by embryonic day 4 (e4), the totipotent cells of the
morula differentiate into two distinct lineages: the outer layer of
cells, the TE, develops into the extraembryonic tissue, and the inner
cell mass gives rise to the fetal structures [55]. The multipotent layer
of TE cells anchors to the uterus and divides to form the extraem-
bryonic ectoderm and EPC which then forms the spongiotrophoblast
and trophoblast giant cells [56]. Therefore, the window between em-
bryonic days 4 and 6.5 provides an ideal time to investigate early
alterations of the placenta that are direct consequences of maternal
exposures.
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T. J. Stuart, 2018, Vol. 98, No. 6

Table 5. Summary of major metabolomic and transcriptomic results.

804
Obesity alters early placenta gene expression, 2018, Vol. 98, No. 6 805

e12.5 Placenta Microvessel Density Perhaps most striking was our finding of markedly altered sphin-
golipid metabolism in plasma of obese dams very early in pregnancy.
0.0008
* This directly correlated with changes in gene expression regulating
Vessels per unit area (um2) * pvalue <0.05 sphingosine-1-phosphate and ceramide signaling pathways in EPC,
0.0006 which persisted in placenta at e12.5 as shown in our previous studies
[11]. Upregulation of these pathways inhibit angiogenesis and result
0.0004 in endothelial dysfunction by abolishing vascular endothelial growth
factor (VEGF) or insulin-stimulated endothelial nitric oxide synthase
activation and subsequent nitric oxide production from endothelial
0.0002
cells [62]. Indeed, many of these key pathways were disrupted in TE

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and EPC. Further, excess ceramides play a major role in lack of re-
0.0000 modeling of the maternal spiral arteries that occurs in preeclampsia,
which is associated with maternal obesity [63]. Thus, changes in ma-
e

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al

al

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M

m
ternal sphingolipid metabolism could be a mechanism through which
Fe

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obesity results not only in inflammation and insulin resistance in the

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mother but also activates a transcription program in placenta pro-

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genitors resulting in abnormal vascularization in placenta of obese

Diet and Sex
dams.
Figure 4. Microvessel density was decreased in male HFD placenta at e12.5 Multiple phospholipids, including 1-stearoyl-2-arachidonoyl-
as measured via immunohistochemistry staining with PLVAP antibody (N = GPC, were elevated in maternal plasma of obese dams at e6.5. This
5–7 per group, ∗ P < 0.05). key phospholipid is an important regulator of p38 MAPK activity
under conditions of cell stress and lipotoxicity [64], and the p38
MAP kinase pathway was significantly elevated in EPC at e6.5. The
e18.5 Placenta Microvessel Density p38 group of MAP kinases serve as a nexus for signal transduction
* and play a vital role in numerous biological processes including in-
0.0006 * * p value < 0.05 flammation and placental angiogenesis [65], thus providing another
Vessels per unit area (um2)

link between changes in the maternal metabolome and changes in

gene expression in the EPC.
0.0004 HFD dams exhibited differences in several lysine metabolites
compared to controls. Acetylated lysine molecules are commonly
found in histone proteins; the acetylation status of lysine in histones
0.0002 is correlated with active gene expression as acetylation of lysine ef-
fectively removes the unmodified amino acid’s positive charge and
hence reduces the interaction between the histone tail and nucle-
0.0000 osome [66]. Additionally, high-throughput proteomic assays have
demonstrated that lysine acetylation of proteins involved in immune
e

e
al

al

al

al

modulation and metabolism occurs which can alter function [67].

M

m
Fe

Fe
ol

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Therefore, changes in acetylation of lysine could have profound

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effects on development-related gene expression patterns and post-

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translational effects by altering the function of proteins involved in

Diet and Sex
Figure 5. Microvessel density remained decreased in male HFD placentas at
e18.5 as measured via immunohistochemistry staining with PLVAP antibody
(N = 3–6 per group, ∗ P < 0.05).
Not surprisingly, an HFD induced marked elevations in satu-
rated fatty acids, including palmitate, myristoleate, myristate, and
stearate, in plasma of obese dams in early pregnancy. Levels of these
pro-inflammatory fatty acids are elevated in obese humans and are
linked to the development of insulin resistance and other metabolic
disorders that accompany obesity [57]. Additionally, we observed a
disrupted balance of ω-6:3 ratio in obese dams. ω-6 fatty acids are
pro-inflammatory and enhance adipogenesis, whereas ω-3 fatty acids
are associated with improved insulin action and decreased adipos-
ity [58–60]. We have previously shown that this pro-inflammatory
metabolic milieu in the mother results in marked oxidative stress in
the preimplantation embryo [61], and prevention of oxidative stress
precludes the development of obesity in the offspring, thus demon-
strating the central role of oxidative stress and inflammation in the
programming of the offspring [61].
metabolic signaling.
Another key change in the maternal metabolome of obese dams
that is likely to influence placenta and fetal development was a
marked decrease in myo and chiro-inositol. Inositols are a family
of simple carbohydrates, and these two stereoisomers play a key role
in the insulin pathway, acting synergistically as insulin-sensitizing
agents through the enhancement of glucose peripheral tissue up-
take and glycogen synthesis [68]. Of note, myo-inositol/D-chiro-
inositol supplementation in pregnant mice with a metabolic-like syn-
drome phenotype improves blood pressure, glucose tolerance, and
leptin levels and prevents increased maternal weight gain [69]. Myo-
inositol also regulates endothelial function by decreasing oxidative
stress, enhancing endothelial nitric oxide synthase and nitric oxide
activity [70]. Thus, low levels of these important metabolites may
contribute to insulin resistance associated with maternal obesity.
Multiple studies have demonstrated that maternal obesity re-
sults in a pro-inflammatory state in the placenta [71, 72]. These
studies have all been performed in term or near term placenta, yet
our transcriptome data showing activation of multiple inflammatory
pathways and genes including IL-6 signaling, suggest that this pro-
cess occurs very early in gestation. The inflammatory state in TE
806
and EPC of HFD dams is likely due to the marked changes in lipid
species such as palmitate, omega-6 fatty acids, sphingolipids, and
their metabolites, like eicosanoids, that are significantly elevated in
the plasma of obese dams during early pregnancy.
There was significant overlap in the gene expression pathways
that were altered by an HFD in TE and EPC, implying that these
particular pathways may be especially important to early placenta
development (Figure 1). These pathways include ET-1 signaling, p38
MAPK signaling, as well as PPARα/RXRα activation. ET-1 is an ex-
tremely potent vasoconstrictor that acts directly on vascular smooth
muscle cells and has been linked to diseases such as hypertension
and atherosclerosis [73]. It is well known that obesity is associated
with endothelial dysfunction, and ET-1-mediated vasoconstriction
is increased in obese humans [74]. Additionally, elevated circulating
plasma ET-1 levels have been positively correlated with preeclamp-
sia [75] and the production of reactive oxygen species that promote
apoptosis of trophoblasts [76]. This correlates with the finding that
placentas of mice with diet-induced obesity display both oxidative
stress and decreased number of trophoblasts [77]. These changes
may be another mechanism by which an HFD leads to decreased
microvessel density later in gestation.
The PPARα/RXRα activation pathway was predicted to be in-
hibited by an HFD in both TE and EPC, and the more generalized
PPAR signaling pathway was also predicted to be inhibited, though
this was only statistically significant in EPC. This was an unexpected
finding given that the physiologic actions of fatty acids are medi-
ated by PPARs. However, it is possible that an overabundance of
free fatty acids leads to a subsequent downregulation of these path-
ways. PPARs are highly expressed in the placenta and thought to be
involved in trophoblast differentiation and function [78], and defi-
ciencies of both PPARγ and PPARδ result in lethal placenta defects
[44]. The placentas of PPARγ null mice display vascular anomalies
and poor differentiation of the trophoblast [78]. Additionally, in hu-
man studies, gestational diabetes mellitus has been associated with a
downregulation in term placental PPARα, PPARγ , and RXRα [79]
In addition to ET-1 and PPAR signaling, there were many other
genes and pathways altered by an HFD in early placenta that are re-
lated to angiogenesis and vascular development, including the PEDF
signaling pathway and the pathway for leptin signaling in obesity.
The PEDF signaling pathway was activated by an HFD in TE. PEDF
is a widely expressed glycoprotein that acts as a potent antiangio-
genic factor [80]. Huppertz et al. recently identified PEDF secretion
as a mechanism by which human trophoblasts restrain angiogenesis
and vascularization later in pregnancy [81]. PEDF binds to VEGFR2
and blocks the stimulatory effect of VEGF. These changes in key
genes regulating vascular development in the TE and EPC were cor-
related with changes we found in microvessel density in mid and late
gestation placenta, suggesting that the mechanism underlying abnor-
mal placenta vascularization occurs prior to placentation in the very
early embryo.
The canonical pathway for leptin signaling in obesity was also
activated by an HFD in TE. Leptin is an abundant circulating
adipokine, and while it is widely known for its role in energy home-
ostasis and metabolism, it also participates in a myriad of other
processes including immune function and inflammation [82]. Acti-
vation of leptin receptors on endothelial cells leads to endothelial
cell dysfunction via increased oxidative stress responses as well as
increases in TNF-α and IL-6 [82], both of which were also predicted
to be activated by an HFD in EPC. In the more mature placenta,
leptin is produced by syncytiotrophoblast and controls growth of
the placenta. High levels of leptin in placentas from obese pregnan-
T. J. Stuart, 2018, Vol. 98, No. 6
cies reduce cytotrophoblast proliferation and placental growth factor
(PlGF) production and are thought to contribute to the development
of preeclampsia [75].
In addition to changes in placenta development and function,
obesity may impact fetal development by altering microbial metabo-
lites and molecules to which the fetus is exposed. In our study,
multiple microbial metabolites were altered in plasma of HFD dams.
Several studies have shown that the composition of gut micro-
biota is altered by obesity [49–51]; however, the functional im-
plications of an altered microbiome on fetal growth and develop-
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ment are not fully understood. Gomez de Aguero et al. showed
that maternal microbiota-derived compounds are transferred from
the mother to the offspring and shape the immune system of the
offspring [83]. Therefore, gaining better understanding of the com-
pounds present in maternal plasma and, thus potentially transferred
to the offspring, is paramount. The obese maternal plasma pro-
files showed altered levels of microbial-derived metabolites pro-
duced from amino acids (tyrosine and tryptophan), which have
been suggested to interact with host signaling pathways and affect
host immunity [84]. Indole, a major tryptophan-derived microbial
metabolite we found to be present in higher levels in plasma of
HFD dams, has been shown to interact with inflammation-related
processes in the human host [85]. Therefore, in addition to altered
placenta function, our studies suggest that obesity alters the compo-
sition of biochemicals to which the embryo is exposed. This will not
only affect fetal development, but also impact long-term offspring
outcomes.
Male offspring are known to be more susceptible to the effects
of intrauterine exposures such as maternal obesity and diabetes
[86, 87], and it is possible that the placenta plays a key role in
this finding. It is recognized that there are sex differences in gene
expression in normal placenta, which have been attributed to sex
hormones, and it is also known that maternal HFD can affect gene
expression in a sex-specific manner [88]. However, given our pre-
vious findings that a pregestational exposure to obesity also results
in sex-specific differences in long-term outcomes between males and
females [11], it is possible that sex-specific developmental program-
ming is occurring even prior to the expression of sex hormones.
When we compared all male EPCs to all female EPCs, we found
alterations in several interesting pathways, many of which are related
to placenta and vascular development. Glutamate receptor signaling
was downregulated in males. Though glutamate signaling was pre-
viously thought to be limited to the central nervous system, it is
now known that functional glutamate receptors are found in many
different organs, including skin and placenta [89]. Grm1 is upreg-
ulated in some forms of cancer, and it has been found to enhance
angiogenic signaling [90]. SHH and BDNF, both upstream regula-
tors, were inhibited in males as compared to females, and these two
regulators also play important roles in placenta formation and an-
giogenesis. Shh helps to control placenta labyrinth development, and
Shh null mice have pronounced defects in yolk sac vasculature [91].
Additionally, studies have shown Shh to be indirectly angiogenic by
upregulating Vegf, angiopoietin 1 (Angpt1), and Angpt2 [92]. Of
note, Vegfc was downregulated in males, which is consistent with
deactivation of the SHH pathway.
BDNF is neurotrophin thought to play key roles in placenta
angiogenesis, cytotrophoblast proliferation, and protection of en-
dothelial progenitor cells via increased expression of superoxide
dismutase, and alterations have been implicated in a wide variety
of diseases [93]. Lower levels of BDNF have been found in both pla-
centa and cord blood of women with preeclampsia [94], and BDNF
Obesity alters early placenta gene expression, 2018, Vol. 98, No. 6
was recently shown to be sexually dimorphic in term human pla-
centas [95]. Baseline differences between male and female EPC may
be one explanation for the increased susceptibility of males to the
effects of maternal obesity.
As mentioned above, we found that exposure to an HFD de-
creased microvessel density at both mid and late gestation. Notably,
this decrease was most pronounced in the placentas of male fetuses.
Additionally, we found increased uterine artery resistance in HFD
dams and a very slight trend toward increased umbilical artery re-
sistance in fetuses of dams fed an HFD. However, these results were
not statistically significant. Given that the histological changes we
noted in microvessel density were more significant in males, and we
were not able to determine the sex of the fetuses undergoing doppler
imaging, we speculate that this may be why the increase in RI in the
uterine and umbilical arteries was not statistically significant.
In conclusion, our data demonstrate that diet-induced obe-
sity in the female mouse profoundly affects the maternal plasma
metabolome and alters gene expression pathways in the TE and EPC
that are vital to placenta development and necessary to support the
developing embryo. This is the first study to demonstrate that mater-
nal obesity alters the transcriptome at the earliest stages of murine
placenta development. IPA highlighted significant clustering of genes
involved in sphingolipid metabolism, vascular development, and in-
flammation, and we speculate that these early changes are directly
associated with the decreased placenta microvessel density we ob-
served later in gestation. These alterations may represent some of
the many complex ways in which maternal obesity exerts its effects
on the offspring via the placenta.
Supplementary data
Supplemental Table 1. Antibodies used for immunohistochemistry
staining.
Supplemental Table 2. Gene expression profiling of trophectoderm
from e4.5 blastocysts from control and HFD dams. Using an FDR of
≤0.05, 5285 genes were differentially expressed between HFD and
controls (1 gene downregulated and 5284 genes upregulated).
Supplemental Table 3. Canonical pathways with P ≤ 0.05 for all
transcriptomic analyses.
Supplemental Figure 1. Volcano plot of all genes sequenced, compar-
ing all control EPC samples to all HFD EPC samples at e6.5. Noted
in red are genes with an FDR ≤0.15.
Supplemental Table 4. RNA-Seq of e6.5 EPC, comparing all control
EPCs to all HFD EPCs. Using an FDR of ≤0.1, 595 genes were
differentially expressed (188 downregulated and 407 upregulated).
Supplemental Table 5. RNA-seq of e6.5 EPCs, comparing male con-
trol EPCs to male HFD EPCs. Using an FDR of ≤0.15, there were
50 genes that were differentially expressed (17 downregulated and
33 upregulated).
Supplemental Table 6. RNA-seq of e6.5 EPCs, comparing all female
EPCs to allmale EPCs. Using an FDR of ≤0.15, there were 326 genes
differentially expressed (277 downregulated and 49 upregulated).
Supplemental Table 7. Global metabolic profiling of e6.5 mater-
nal plasma. Levels of 192 biochemicals were significantly different
between HFD and control samples (P ≤ 0.05); 89 were present in
higher levels, and 103 were present in lower levels.
Acknowledgments
The authors would like to acknowledge John Tobias, Teri Ord, Metabolon Inc,
the Children’s Hospital of Philadelphia Pathology core, University of Penn-
807
sylvania Small Animal Imaging Facility, and the University of Pennsylvania
Epigenetics Program for their help with this research.
Conflict of Interest. The authors have declared that no conflict of interest exists.
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