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Sexually Dimorphic Effect of In Vitro Fertilization
(IVF) on Adult Mouse Fat and Liver Metabolomes
Sky K. Feuer, Annemarie Donjacour, Rhodel K. Simbulan, Wingka Lin,
Xiaowei Liu, Emin Maltepe, and Paolo F. Rinaudo
Departments of Obstetrics, Gynecology, and Reproductive Sciences (S.K.F., A.D., R.K.S., W.L., X.L.,
P.F.R.) and Pediatrics (E.M.), University of California San Francisco, San Francisco, California 94143
The preimplantation embryo is particularly vulnerable to environmental perturbation, such that
nutritional and in vitro stresses restricted exclusively to this stage may alter growth and affect
long-term metabolic health. This is particularly relevant to the over 5 million children conceived by
in vitro fertilization (IVF). We previously reported that even optimized IVF conditions reprogram
mouse postnatal growth, fat deposition, and glucose homeostasis in a sexually dimorphic fashion.
To more clearly interrogate the metabolic changes associated with IVF in adulthood, we used
nontargeted mass spectrometry to globally profile adult IVF- and in vivo-conceived liver and go-
nadal adipose tissues. There was a sex- and tissue-specific effect of IVF on adult metabolite sig-
natures indicative of metabolic reprogramming and oxidative stress and reflective of the observed
phenotypes. Additionally, we observed a striking effect of IVF on adult sexual dimorphism. Male-
female differences in metabolite concentration were exaggerated in hepatic IVF tissue and sig-
nificantly reduced in IVF adipose tissue, with the majority of changes affecting amino acid and lipid
metabolites. We also observed female-specific changes in markers of oxidative stress and adipo-
genesis, including reduced glutathione, cysteine glutathione disulfide, ophthalmate, urate, and
corticosterone. In summary, embryo manipulation and early developmental experiences can
affect adult patterns of sexual dimorphism and metabolic physiology. (Endocrinology 155:
4554–4567, 2014)
The Developmental Origins of Health and Disease
(DOHaD) hypothesis holds that embryonic and fetal ad-
aptation to suboptimal uterine environments can predis-
pose a series of metabolic diseases in adulthood, including
cardiovascular disease, diabetes, hypertension, and stroke
(1). Preimplantation development has been recognized as
a window of notable environmental sensitivity, and many
animal studies have reported that nutritional, oxidative,
and in vitro stresses restricted exclusively to this period are
sufficient to alter developmental growth and metabolic
trajectories, leading to pathologies such as hypertension,
dyslipidemia, and

-cell dysfunction in adulthood (2–4).
This is of particular relevance to the over 5 million
children conceived using assisted reproductive technolo-
gies such as in vitro fertilization (IVF). Because the eldest
IVF individuals are only in their mid-30s, the relationship
between preimplantation embryo manipulation and
adult-onset metabolic pathologies is elusive, although
modest changes in growth kinetics, fasting glucose, blood
pressure, vascular function, and fat deposition have been
reported in IVF adolescents (5–8). To address this con-
troversy, several mouse models of IVF have been devel-
oped and used to demonstrate that even clinically opti-
mized IVF conditions are sufficient to reprogram adult
metabolism (9–11). Our group has shown that female
animals in particular exhibit latent overgrowth, increased
fat accumulation and fasting glucose levels, and impaired
insulin secretion in response to stimulatory levels of glu-
cose. However, these mice are physiologically indistin-
guishable from controls until approximately 17 weeks of
age (Supplemental Figure 1). In contrast, male animals
display no overt phenotype (9).
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received June 9, 2014. Accepted August 19, 2014.
First Published Online September 11, 2014
Abbreviations: CySS, cysteine-glutathione disulfide; DOHaD, Developmental Origins of
Health and Disease; GPE, glycerophosphoethanolamine; GSH, glutathione; hCG, human
chorionic gonadotropin; IVF, in vitro fertilization; MSEA, metabolite set enrichment
analysis.
REPRODUCTION-DEVELOPMENT
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Sex-based differences are present throughout most
mammalian physiologies, behaviors, diseases, and pheno-
types, arising from a variety of immunological, hormonal,
genetic, and epigenetic mechanisms (12). Sexual dimor-
phism is particularly common to several metabolic hall-
marks in adulthood, including glucose homeostasis, insu-
lin sensitivity,

-cell function, and adipose tissue depots,
and therefore can influence disease susceptibility and pro-
gression (13). Because up to one-third of transcripts are
differentially expressed between males and females by the
blastocyst stage (14), it is not surprising that developmen-
tal programming frequently exhibits sex bias, although
this phenomenon is poorly understood (15).
Recent advances in metabolomics technology have per-
mitted comprehensive and systematic analyses of the bio-
chemical fingerprints within cells and tissues, thus pro-
viding an immediate compendium of cellular metabolic
processes (16). Our group previously performed serum
profiling in adult IVF- and control-conceived female mice
and identified several biomarkers of both insulin resis-
tance and impaired glucose handling similarly noted in
metabolomics-based investigations into diabetes and obe-
sity (9). These results have led us to further interrogate the
metabolic changes associated with IVF in adulthood, as
well as dissociate its sex-specific physiological phenotypes
in liver and gonadal fat. These tissues were selected for
their role in metabolism and insulin sensitivity and be-
cause our data have highlighted adipose tissue as a locus
of developmental reprogramming and sex-specific pheno-
typic variation.
Materials and Methods
Animals
All animals were maintained according to institutional reg-
ulations, under a constant 12-hour light, 12-hour dark cycle with
ad libitum access to water and standard chow (23% protein,
22% fat, and 55% carbohydrate, number 5058; PicoLab). Be-
ginning at 24 weeks, all animals were placed on a high-fat diet
(20% protein, 60% fat, and 20% carbohydrate, number D12492;
Research Diets, Inc) (17) until time of death at 30 weeks.
IVF, embryo culture, and transfer
IVF, embryo culture, and embryo transfer experiments were
performed as previously described (9). Briefly, C57BL/6J females
aged 6– 8 weeks were injected with 5-IU pregnant mare’s serum
gonadotropin followed 46– 48 hours later by 5-IU human cho-
rionic gonadotropin (hCG) to induce superovulation. Thirteen
to fifteen hours after hCG administration, cumulous-oocyte-
complexes were isolated from ampullae and incubated 4– 6
hours in human tubal fluid medium (MR-070-D; Millipore) with
capacitated (1 h) cauda epididymal sperm from C57BL/6J males.
Fertilized zygotes were washed and cultured to the blastocyst
stage in potassium simplex optimization medium (KSOM, con-
taining amino acids and 0.2mM pyruvate, 10mM lactate,
0.2mM glucose, and 1mM glutamine) (MR-106-D; Millipore)
(18), at 37°C under Ovoil (10029; Vitrolife) with 5% CO
2
and
5% O
2
in a modular humidified chamber. To generate postim-
plantation cohorts, pseudopregancy was induced by mating nat-
urally cycling CF-1 females to vasectomized CD-1 males, con-
firmed by the presence of a copulation plug the next morning
(considered d 0.5). Late-cavitating blastocysts were transferred
to the uterine horns of recipients on day 2.5 of pseudopregnancy.
For control experiments, C57BL/6J female mice were superovu-
lated as described above and mated to C57BL/6J males over-
night. Embryonic day 3.5 blastocysts (96 h after hCG administra-
tion) were flushed from the oviducts and transferred immediately to
the uterine horns of CF-1 recipients, thus controlling for litter size
and the embryo transfer procedure. Only animals derived from
litters of 5–7 pups were used in this study. Recipient animals had
similar weight at the time of transfer and gained the same amount
of weight during pregnancy.
Metabolomic profiling
Nonfasted animals were killed by CO
2
exposure followed by
cervical dislocation in the morning, and tissues were harvested
from animals generated by 5 separate IVF and 5 control exper-
iments. At least 3 independent cohorts contributed to each anal-
ysis of tissue and sex. Estrous cycle was monitored using vaginal
smear. Immediately after collection, whole liver (24 samples; n ⫽
6 for each sex and conception condition) and gonadal fat (29
samples; n ⫽7 IVF and 7 control females, n ⫽10 IVF and 5
control males) samples were snap frozen for unbiased metabo-
lomic profiling by Metabolon, Inc, as described in detail else-
where (19, 20). Briefly, samples underwent a series of organic
and aqueous extractions optimized for small molecule recovery
and were then split into equal parts for gas chromatography-
mass spectrometry and liquid chromatography-tandem mass
spectrometry analyses. For the latter platform, samples were
again divided for profiling in both positive (acidic) and negative
(basic) ionization modes.
Bioinformatics and statistics
Mass spectrometry profiles were processed using software
developed by Metabolon, Inc (21). Peaks were called against a
library of 2500 named biochemicals comprised of amino acids,
lipids, carbohydrates, nucleotides, peptides, vitamins, cofactors,
and xenobiotics. For statistical interpretation of detected me-
tabolites, ANOVA contrasts were performed to identify bio-
chemicals that differed significantly between 1) the in vivo and
IVF conception conditions and 2) males vs females, with two-
way ANOVA analyses to describe biochemicals exhibiting a sig-
nificant interaction between sex and conception parameters. For
all comparisons, P⬍.05 was considered significant. Unsuper-
vised Pearson correlations were used to evaluate the relationship
between metabolite concentrations and both percent adiposity
and fasting glucose levels at time of death. For moderately or
strong coefficient values (defined as r⬎0.6), additional cor-
relation analyses were conducted with segregation by sex, con-
ception condition, or both.
Heat maps were generated using GENE-E software developed
by the Broad Institute (available at http://www.broadinstitute.
org/cancer/software/GENE-E/). Heat maps depict the fold-change
difference in metabolite concentration between mean IVF and
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control values, or the z-score (calculated as z ⫽(x ⫺
)/
; where
x⫽the individual scaled metabolite value for an animal,
⫽the
mean value of the metabolite for the defined population, and
⫽
the SD of that population) comparing either metabolite concen-
trations between male and female animals, or individual control
and IVF values to their respective population means.
The web-based metabolomic data processing tool Metabo-
Analyst was used for tissue metabolite data analysis (22, 23). De-
tailed methodology may be found at http://www.metaboanalyst.ca.
Metabolite set enrichment analysis (MSEA) was conducted on
metabolite data mapped according to Human Metabolome Da-
tabase (HMDB) or Kyoto Encyclopedia of Genes and Genomes
(KEGG) identifiers, using the metabolite pathway associated me-
tabolite set library (currently 88 entries).
Results
We conducted global metabolomics profiling of tissues
harvested from mice produced in a previous study of IVF
to model the DOHaD hypothesis (9). Briefly, mice were
generated by IVF under conditions considered optimal for
mouse embryo culture and reflective of current IVF clin-
ical practices (KSOM with amino acids and 5% O
2
ten-
sion) (24). As a control, in vivo-produced blastocysts were
isolated 96 hours after fertilization for transfer to recipi-
ents (flushed blastocyst group), thus accounting for su-
perovulation, litter size, and the embryo transfer proce-
dure (10). To probe the consequences of nutritional stress,
all animals were placed on a high-fat diet beginning at 24
weeks of age until time of death at 30 weeks (6 wk total),
at which point liver and gonadal adipose tissues were har-
vested for experiments. Body weight was similar between
IVF and control mice up through 16 weeks, at which point
IVF females showed a statistical increase in body weight
(before administration of the high-fat diet) that lasted
through 28 weeks. At time of death, there were no signif-
icant changes in body weight or weight-standardized or-
gan sizes between the 2 groups (Supplemental Figure 1).
Dual-energy x-ray absorptiometry at 8, 16, 21, and 28
weeks revealed that IVF females had initially lower per-
cent adiposity but then statistically surpassed control lev-
els of body fat by 21 weeks of age. These weight and fat
percent findings might be partially explained by the fact
that IVF females consumed more food than controls at 7
and 20 weeks but not at 28 weeks.
Metabolic sexual dimorphism in adult fat tissue of
control mice
Unbiased metabolomic investigation of IVF and con-
trol fat samples (n ⫽10 male and 7 female IVF, n ⫽5 male
and 7 female control) identified a total of 231 endogenous
biochemicals comprising all major metabolic groups (Fig-
ure 1A). A complete list of relative metabolite concentra-
tions may be found in Supplemental Table 1. Pearson cor-
relations between metabolite concentrations and percent
adiposity or fasting glucose levels at time of death revealed
no significant relationships. Due to the sex-biased effect of
IVF on adult metabolism (9, 10), we first compared pro-
files between control males and females and observed sig-
nificant sexual dimorphism for 57 metabolites (24.7%,
P⬍.05) (Figure 1B). The dataset was particularly en-
riched for small molecules involved in lipid and amino acid
metabolism. Males exhibited broad increases in metabo-
lite concentration relative to females (Figure 1C). We per-
formed MSEA to determine whether any biologically
meaningful pathways were overrepresented by the altered
metabolites (25), which showed that metabolites involved
in glycerolipid metabolism, the urea cycle, and sphingo-
lipid metabolism exhibited the most significant sexual di-
morphism in control samples (Figure 1D).
Sex-specific effect of IVF on the adult fat
metabolome
In males, 16 metabolites were significantly altered be-
tween IVF and control fat samples (P⬍.05, 2 molecules
increased and 14 decreased), and 9 approached signifi-
cance (.05 ⬍P⬍.1, 0 increased and 9 decreased) (Figure
2, A and B). This included a dramatic reduction in levels
of the glycolytic metabolites glucose and lactate, the pen-
tose phosphate pathway metabolites ribulose and arabi-
tol, as well as the nucleotide precursors inosine 5⬘-mono-
phosphate, GMP, and uridine monophosphate, suggesting a
decreased shunting of glycolytic intermediates through the
pentose phosphate pathway toward nucleotide synthesis.
MSEA highlighted an involvement of the altered metab-
olites with these pathways, although the associations were
not significant after post hoc correction (Figure 2C).
Comparatively, female IVF fat tissue differed from fe-
male controls by 19 metabolites (P⬍.05, 15 increased and
4 decreased), and 21 showed a trend toward significance
(.05 ⬍P⬍.1, 13 increased and 8 decreased) (Figure 2, D
and E). Concentrations of several amino acids were in-
creased in IVF mice, including urea cycle intermediates.
Multiple long-chain (18C) fatty acids were increased,
whereas levels of the glycerophosphoethanolamines (GPEs)
1-arachidonoyl-GPE and 2-docosahexaenoyl-GPE were de-
creased. Further, decreased maltotetraose could reflect
changes in glycogenolysis. There was also evidence of ox-
idative stress in female IVF fat, evidenced by depleted lev-
els of glutathione (GSH) (P⫽.088) (Supplemental Table
1) and a corresponding increase in its oxidized form cys-
teine-glutathione disulfide (CySS) (P⫽.042). MSEA
showed significant alterations to many amino acid and
protein synthesis pathways, as well as a trend toward
4556 Feuer et al IVF Impacts Adult Metabolic Sexual Dimorphism Endocrinology, November 2014, 155(11):4554 – 4567
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Figure 1. Metabolic sexual dimorphism in adult adipose tissue. A, Nontargeted mass spectrometry profiling of 29-week IVF and control fat tissue
(n ⫽5 male and 7 female control animals; 10 IVF males and 7 IVF females; 29 animals total) identified 231 named metabolites comprising all
major metabolic groups. B, The concentrations of 57 metabolites (24.7%) were significantly different between males and females in control
samples (P⬍.05), consisting predominantly of lipid and amino acid derivatives. C, The level of each biochemical in each sample is represented as
the number of SDs above or below the mean level of that biochemical (z-score). Apart from succinylcarnitine and 3-dehydrocarnitine, sexually
dimorphic metabolites displayed uniformly increased concentrations in males. D, Summary plot for MSEA, where pathways are ranked by
Bonferroni-corrected Pvalue with hatched lines depicting Pvalue cutoffs. CMP, cytidine monophosphate; DiHOME, hydroxyoctadec-9(Z)-enolate;
GPI, glycerophosphoinositol; HODE, hydroxyoctadecadienoic acid.
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changes in the malate-aspartate shuttle, urea cycle, am-
monia recycling, and fructose/mannose degradation (Fig-
ure 2F).
The effect of IVF on adult fat metabolite composition
was strikingly sex specific: glycerol was the only metab-
olite significantly different in IVF vs control samples for
Figure 2. Effect of IVF on the adult fat metabolome. A, Categorical distribution of the 16 metabolites significantly altered in IVF males from
controls. B, Heat map depicting fold-change in metabolite concentration between IVF and control metabolite values in male fat samples, including
z-distribution of individual control (blue) and IVF (red) values relative to their respective population means. C, MSEA summary of Bonferroni-
corrected pathways associated with the metabolite changes. D–F, Same as A–C but for the 19 metabolites altered in female IVF fat samples. G and
H, Venn diagrams showing overlap in altered metabolites (G and purple symbols) and MSEA-identified pathways (H and green symbols) between
male and female IVF cohorts. CDP, cytidine diphospho; CMP, cytidine monophosphate; Glu-Gln, glutamylglutamine; IMP, inosine monophosphate;
SSG, glutathione disulfide; UMP, uridine monophosphate.
4558 Feuer et al IVF Impacts Adult Metabolic Sexual Dimorphism Endocrinology, November 2014, 155(11):4554 – 4567
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both males and females. However, the changes occurred in
different directions (1.8-fold decrease from control males,
1.8-fold increase in females) (Figure 2G). Additionally, be-
tween the 13 MSEA-identified pathways enriched in males
and 20 in females, only 5 pathways were altered in both sexes
(Figure 2, H and green symbols in C and F).
Reduced sexual dimorphism in IVF adult fat tissue
We next investigated the effect of IVF on metabolic
sexual dimorphism and observed a striking depletion in
the number of metabolites that differed in concentration
between IVF male and IVF female fat samples. Compared
with the 24.7% (57 of 231 metabolites) dimorphism in
controls, only 33 (14.3%) showed significant male-female
differences (P⬍.05), predominantly for metabolites in-
volved in lipid metabolism (Figure 3, A and B). Only 14
metabolites retained significant sexual dimorphism be-
tween the control and IVF cohorts (purple symbols), in-
dicating that male-female differential concentration was
lost for 43 metabolites and gained for 19 (Figure 3C). Of
these, sexual dimorphism in amino acid and lipid mole-
cules were the most affected by IVF (Figure 3D). MSEA
additionally showed that only steroidogenesis was differ-
ent between sexes (Figure 3E). Overall, there was a dra-
matic reduction in IVF metabolic sexual dimorphism in
gonadal fat tissue (Figure 3, F and green symbols).
Sex-specific effect of IVF on the adult liver
metabolome
We additionally profiled liver samples (n ⫽6 per sex
and per conception condition) and detected a total of 373
endogenous biochemicals (Figure 4A), of which 53
(14.2%) exhibited significant male-female differences in
concentration. As with the control fat tissue, dimorphic
metabolites were predominantly comprised of lipid and
amino acid derivatives (Figure 4B), but increases or de-
creases in metabolite levels were metabolite-specific (Fig-
ure 4C). MSEA classified sphingolipid metabolism, and
glycine, serine, and threonine metabolism as the pathways
most affected by sex differences (Figure 4D). Pearson cor-
relations did not revealed any significant relationships be-
tween metabolite concentrations and percent adiposity or
fasting glucose levels at time of death. Only levels of pyr-
idoxal (one of the 3 forms of vitamin B6) displayed both
a significant sex bias and a high correlation with fasting
glucose levels and its hepatic levels (r ⫽0.67, P⫽.008).
Investigation of IVF liver tissue revealed significant
changes in levels of 32 metabolites between IVF and con-
trol male livers (P⬍.05, 25 molecules increased and 7
decreased), with an additional 30 approaching signifi-
cance (.05 ⬍P⬍.1, 20 increased and 10 decreased) (Fig-
ure 5A). A complete list of relative metabolite concentra-
tions may be found in Supplemental Table 2. The most
striking change was a broad incorporation of dipeptides
into IVF livers, particularly for leucine, alanine, glycine,
and isoleucine-based dipeptides (Figure 5B). There was
also a strong depletion of the bile acid metabolites cholate,

-muricholate, and
␣
-muricholate. Other notable de-
creases in male IVF livers relative to controls included the
ketone body 3-hydroxybutyrate and the active form of
folic acid, 5-methyltetrahydrofolate. Comparative increases
consisted of 3-dehydrocarnitine, the nicotinamide adenine
dinucleotide precursor nicotinamide riboside, as well as the
purine metabolites 5⬘AMP, 5⬘GMP, and guanosine. None of
the changes were associated with any significant MSEA path-
ways (Figure 5C).
In contrast, female IVF livers differed from female con-
trols by 31 metabolites (P⬍.05, 13 increased and 18
decreased), and 32 showed a trend toward significance
(.05 ⬍P⬍.1, 12 increased and 20 decreased). These were
enriched for fatty acid metabolites (Figure 5, D and E)
including long-chain fatty acids and acylglycines; this in
conjunction with a strong depletion of glutamine in all IVF
samples may reflect changes in mitochondrial fatty acid
catabolism. Increased glycogen intermediates maltote-
traose, maltopentaose, and maltohexaose suggests an in-
crease in liver glycogen breakdown in female IVF mice,
which supports the changes in concentrations of lactate
and ribose-5-phosphate that indicate an alternative fate
for glucose. None of the altered metabolites were signif-
icantly associated with any metabolic pathways after post
hoc correction, although MSEA did identify changes in
gluconeogenesis, the pentose phosphate pathway, and long-
chain fatty acid

-oxidation, among others (Figure 5F).
As was observed in the fat, the IVF-associated changes
were relatively sex specific. Five metabolites were signif-
icantly different in IVF vs control samples for both males
and females, including N-acetylalanine, the dipeptides
glycylleucine, glycylphenylalanine and glycyltyrosine and
nicotinamide riboside (Figure 5, G and purple symbols in
B and D). MSEA identified 3 altered pathways in both
sexes, although the changes were not significant (Figure 5,
H and green symbols in C and F).
Exaggerated sexual dimorphism in the IVF adult
liver
In contrast to the IVF fat tissue, sexual dimorphism in
IVF livers was increased compared with controls. A total
of 75 metabolites (20.1% vs 14.2% in controls) displayed
significant male-female differences in concentration, sim-
ilarly enriched for lipid and amino acid metabolites (Fig-
ure 6, A and B). Relative to control samples, 26 metabo-
lites lost significant dimorphic concentrations (P⬎.05),
sex differences were maintained in 27 molecules, and 43
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new metabolites exhibited significantly different male-fe-
male concentrations (Figure 6, C and purple symbols). Of
these, sexual dimorphism was particularly increased for
amino acid metabolites and altered in lipids, with dimor-
phic concentrations shifting away from sterol and fatty
acid metabolism and increasing for glycerophosphocho-
lines and other lysolipids (Figure 6, B and D). MSEA pro-
cessing revealed more significant dimorphism for methi-
onine, 1-carbon folate, and pyrimidine metabolism, as
well as novel dimorphic pathways compared with control
samples, including betaine metabolism, ammonia recy-
cling, glutamate metabolism, and other nonsignificant
glucose handling pathways (Figure 6, E and F and green
symbols).
Taken together, these results demonstrate a sex- and
tissue-specific effect of IVF on adult metabolism.
Figure 3. Reduced sexual dimorphism in IVF fat tissue. A, A total of 33 metabolites (14.3%) were sexually dimorphic (P⬍.05 between males and
females) in fat samples from IVF animals (n ⫽10 males and 7 females). B, Z-score normalized expression of the IVF-associated sexually dimorphic
metabolites, with purple symbols indicating metabolites retaining male vs female differences in both IVF and control samples. C, MSEA
categorization showing a significantly decreased number of dimorphic pathways, with green symbols marking pathways with retained dimorphism
from controls. D and F, Overlap of sexually dimorphic metabolites (D and purple symbols) and MSEA-identified pathways (F and green symbols)
comparing control and IVF cohorts. E, Categorical distribution of the metabolites displaying altered sexual dimorphism. The white bars indicate
metabolites with lost male vs female significance in IVF, and black bars show the percentage of metabolites acquiring significant male vs female
concentrations in IVF compared with controls. For example, of the 41 lipid-categorized metabolites displaying male-female differences in at least 1
conception condition (dark blue section), 21 are no longer dimorphic in IVF tissues (black bar), 12 maintain sexual dimorphism in both control and
IVF groups, and 8 exhibit dimorphism only in IVF samples (white bar). CMP, cytidine monophosphate; E, ethanolamine; GPC,
glycerophosphocholine; GPI, glycerophosphoinositol; IMP, inosine monophosphate.
4560 Feuer et al IVF Impacts Adult Metabolic Sexual Dimorphism Endocrinology, November 2014, 155(11):4554 – 4567
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Discussion
Preimplantation development has been recognized as a
window of notable environmental sensitivity, and several
animal studies have demonstrated that nutritional, oxi-
dative, and in vitro stresses restricted exclusively to this
period are sufficient to predispose growth and metabolic
pathologies (2, 11, 26). Specifically, our group has shown
that both stressful and optimized IVF conditions can re-
program adult mouse growth, fat deposition, and glucose
Figure 4. Metabolic sexual dimorphism in adult liver tissue. A, Nontargeted mass spectrometry profiling of 29-week IVF and control livers (n ⫽6
per sex and conception condition; 24 animals total) identified 373 named biochemicals comprising all major metabolic groups. B, Categorical
distribution of the 53 metabolites with significantly different concentrations between males and females in control samples (P⬍.05). C, Z-
distribution of the sexually dimorphic metabolites in control samples. Of note, concentrations of metabolites were not uniformly changed in one
sex vs the other, as observed in fat tissue. Instead, there are more segmented and pathway-specific changes. D, MSEA summary with pathways
ranked by Bonferroni-corrected Pvalue. GPC, glycerophosphocholine; GPG, glycerophosphoglycerol; GPI, glycerophosphoinositol.
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homeostasis in a sexually dimorphic fashion (9, 10). We
therefore used metabolomics to compare the biochemical
profiles and uncouple the sex specificity of adult mouse
IVF- with in vivo-conceived liver and gonadal adipose
tissues.
One of the novel findings of our study is an expanded
description of naturally occurring metabolite differences
between males and females in liver and adipose tissues.
This is important, because most published metabolomics-
based investigations are either restricted to one sex or not
stratified by sex. Of note, unsupervised hierarchical clus-
tering revealed no correlation between metabolite profile
and estrous cycle at time of death. It is well known that sex
differences between males and females vastly affect mam-
malian phenotypes, behaviors, and disease through a va-
riety of hormonal, immunological, and genetic mecha-
nisms. We observed discordant sterol metabolism, redox
state, mobilization, and oxidation of fatty acids between
Figure 5. Effect of IVF on the adult liver metabolome. A, Categorical distribution of the 32 metabolites significantly altered in IVF males from
controls. B, Heat map depicting fold-change in metabolite concentration between IVF and control metabolite values in male liver samples,
including z-distribution of individual control (blue) and IVF (red) values relative to their respective population means. C, MSEA summary of
Bonferroni-corrected pathways associated with the metabolite changes. D–F, Same as A–C but for the 31 metabolites altered in female IVF liver
samples. G and H, Venn diagrams showing overlap in altered metabolites (G and purple symbols) and MSEA-identified pathways (H and green
symbols) between male and female IVF cohorts.
4562 Feuer et al IVF Impacts Adult Metabolic Sexual Dimorphism Endocrinology, November 2014, 155(11):4554 – 4567
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the 2 sexes. Similar findings have been reported after com-
parison of murine liver and adipose transcriptomes be-
tween males and females (27). In the liver, 14% of detected
metabolites (53 of 373) were differentially concentrated
between the sexes, compared with 24% (57 of 231) in fat,
suggesting that fat tissue is a preferential locus of sexual
Figure 6. Exaggerated sexual dimorphism in IVF liver tissue. A, A total of 75 metabolites (20.1%) exhibited sexually dimorphic concentrations
(P⬍.05 between males and females) in liver samples from IVF animals (n ⫽6 males and 6 females). B, Z-score normalized expression of the IVF-
associated sexually dimorphic metabolites, with purple symbols indicating metabolites retaining male vs female differences in both IVF and control
samples. C, Categorization of enriched pathways in the metabolite set, with green symbols marking pathways with retained dimorphism from
controls. D and F, Overlap of sexually dimorphic metabolites (D and purple symbols) and MSEA-identified pathways (F and green symbols)
comparing control and IVF liver cohorts. E, Categorical distribution of the metabolites displaying altered sexual dimorphism, with white and black
bars indicating metabolites with lost or acquired significant male-female differences, respectively, in IVF vs control livers, as discussed in Figure 3E.
GPC, glycerophosphocholine; GPI, glycerophosphoinositol.
doi: 10.1210/en.2014-1465 endo.endojournals.org 4563
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dimorphism. Indeed, fat mass is largely divergent between
sexes (28) and is a location of sexually dimorphic tran-
scriptional changes in response to nutritional reprogram-
ming (29). Although we are not aware of any analogous
human metabolomics-based studies in these tissues, serum
metabolites are markedly different between the sexes (30),
although it is undocumented which tissue(s) contribute to
these dissimilarities. Interestingly, apart from succinylcarni-
tine and 3-dehydrocarnitine, concentrations of the sexually
dimorphic metabolites in fat tissue were uniformly increased
in males and decreased in females (Figure 1C); by compari-
son, the sex bias in liver tissue was more segmented and
network specific (Figure 4C). The significant male-female
differences observed in control tissues highlights the impor-
tance of controlling for sex in metabolic investigations.
A second important finding is the demonstration of a
tissue- and sex-specific effect of IVF on the adult metabo-
lome. We did not observe uniform or consistent patterns
of change between genders or across tissues suggestive of
an “IVF fingerprint.” One possible explanation is that IVF
male and female blastocysts are differentially affected by
the environment they encounter during early develop-
ment. Subsequently, the additional numerous and com-
plex developmental steps occurring within developing
liver or adipose tissue could be further altered in accor-
dance with new, tissue-specific developmental cues. The
net result would be each tissue adopting a unique and
sex-specific metabolic signature of the developmental
stress encountered, rather than a singly uniform pattern.
Indeed, male-female disparities are apparent even before
gonadal formation and are therefore partially indepen-
dent of sex hormone quality and quantity. For example,
differential expression of several X-linked transcripts, in-
cluding the metabolic genes glucose-6-phosphate dehydro-
genase (G6pd) or phosphoglycerate kinase (Pgk), may be
observed as early as the preimplantation embryo stages
(31). Moreover, up to one-third of transcripts are differ-
entially expressed by sex in the blastocyst, in particular for
glucose and protein metabolic pathways (14). This indi-
cates that the preimplantation embryo is poised already to
differentially respond to environmental changes in a sex-
specific fashion, which may explain the frequent sex bias
observed in various models of DOHaD and metabolic re-
programming (32).
Overall, we observed a striking effect of IVF on adult
metabolic sexual dimorphism, which was increased in IVF
liver and decreased in IVF adipose tissue (Supplemental
Figure 2). As with controls, fat was more susceptible to
change: only 24.6% of metabolites (14 of 57) (Figure 3C)
exhibiting sex bias in control tissues maintained that di-
morphism after IVF, compared with 50.9% (27 of 53)
(Figure 6C) preserved dimorphism between control and
IVF liver samples. Most the changes occurred in lipid and
amino acid metabolites (Figures 3 and 6). Male-female
differences in amino acid concentrations were almost
completely abrogated in IVF adipose tissue (Figure 3B),
whereas IVF livers displayed a shift toward increased di-
morphism in these metabolites, particularly for com-
pounds involved in glutamine, lysine, taurine metabolism,
and the urea cycle (Figure 6B). Sexually dimorphic con-
centrations of glycerolipids and lysolipids were increased
in both tissues, with IVF females displaying significantly
lower levels than males. This is particularly relevant, be-
cause these female IVF animals display similarly strong
decreases in serum concentrations of both glycero- and
lysolipids (9), which has also been observed in metabolo-
mics-based analyses of impaired fasting glucose (33) and
diet-induced obesity (34).
We next investigated whether the metabolites differen-
tially measured between IVF and in vivo tissues could be
used as biomarkers to predict chronic disease susceptibil-
ity, as our physiologic studies indicate that IVF mice are
predisposed to glucose intolerance (9, 10). Indeed, we un-
covered several biochemicals present in IVF tissues that
have been linked with metabolic diseases. Levels of the
purine metabolites AMP, GMP, adenosine, and guanosine
were increased in IVF livers, more so in males. AMP is the
principal activator of AMP kinase, which functions in reg-
ulating energy metabolism and glucose homeostasis in the
liver and other tissues (35). Specifically, activated AMP
kinase conserves cellular resources by promoting ATP-
generating mechanisms and inhibiting anabolic pathways.
The elevation of multiple supporting metabolites suggests
that increased AMP and GMP generation is not derived
exclusively from energy-requiring cellular reactions involv-
ing ATP and GTP, respectively, but additionally through
de novo synthesis and salvage pathways. These pathways
support growth and proliferation through the provision of
nucleotides needed for RNA and DNA synthesis, which
may represent another connection between broad meta-
bolic reprogramming and the disruption of glucose han-
dling in mice conceived by IVF.
Next, aggregate comparison of all IVF with control
liver samples (males and females) showed a striking de-
pletion of several bile acids and salts, with males more
severely affected (Figure 5, B and E, and Supplemental
Table 2). Bile acids have a reciprocal relationship with
both glucose and insulin (36), and it has been demon-
strated that impaired bile acid synthesis and subsequent
reduction in bile acid pool size significantly decreases en-
ergy expenditure and contributes to the pathogenesis of
obesity and diabetes (37). Changes in bile acid metabolism
may therefore be directly connected to perturbed glucose
handling in IVF mice, a hypothesis supported by our
4564 Feuer et al IVF Impacts Adult Metabolic Sexual Dimorphism Endocrinology, November 2014, 155(11):4554 – 4567
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group’s previously reported microarray analysis of IVF
livers and observation of transcriptional changes associ-
ated with bile acid biosynthesis (altered expression of he-
patic Akr1c4,Baat, and Cyp27a1 in IVF mice) and dia-
betes mellitus signaling (including Casp9,Cycs,Fcer1g,
HlaB,HlaC,Ikbkb,Il1rap,Irf1, and Nfkb2) (9).
Additional examples of synergy between IVF transcrip-
tional and biochemical profiles with metabolic disease in-
clude the prominent increase in dipeptide concentration
present in male IVF livers coupled with hepatic misex-
pression of protein ubiquitination pathway genes (Cul1,
Cul2,Dnajb1,Dnajb4,Dnajb9,Dnajb14,Dnajc19,
Dnajc21,HlaB,HlaC,Pan2,Psmc6,Psmd12,Sugt1,
Tap1,Ube2l3,Uchl3,Usp16, and Usp46) (9). Dipeptides
can regulate protein ubiquitination (38), the activity of
which affects hepatic lipid production, insulin resistance,
and secretion (39). Separately, altered expression of genes
involved in mitochondrial function (Casp9,Cox6c,
Cox7a2,Cycs,Gpx4,Ndufa5,Ndufb4,Ndufb6,Ndufs4,
and Uqcrb) (9) in conjunction with decreased levels of
long-chain fatty acids, glutamine, lactate, and increased
ribose-5-phosphate in IVF female livers may reflect changes
in mitochondrial activity and the use of alternative anabolic
branches, such as the shunting of glucose through the pentose
phosphate pathway.
Under the particular conception conditions used in our
study, female animals are predisposed to increased fat ac-
cumulation, and both males and females show fat-exclu-
sive maintenance of epigenetic alterations present in IVF
blastocysts (9). This data suggest that adipose tissue is a
locus of sex- and tissue-specific changes associated with
IVF. Because fat is a primary driver of metabolic dysfunc-
tion (40), it is possible that the acquired sex bias in IVF
tissues contributes to the sex-specific metabolic pheno-
types. Comparison of IVF adipose metabolic profiles
points to altered redox homeostasis, with female-specific
increases in ophthalmate, CySS, urate and corticosterone.
GSH is the primary source of antioxidant reducing power
in animals, and both ophthalmate and CySS are formed
under oxidative stress of GSH. Further, urate and corti-
costerone can induce proinflammatory signaling and in-
crease the production of reactive oxygen species in adi-
pocytes and other tissues (41–43). The relationship
between adipogenesis and redox state is complex, and
emerging evidence suggests that adipogenesis is acceler-
ated by oxidizing conditions. For example, reactive oxygen
species and antioxidant activity show parallel increases with
fat accumulation through adipogenic transcription factor-
dependent mechanisms (44 – 46). It is therefore possible
that the female-specific oxidization in IVF fat tissue is in
part responsible for the increased adiposity in these ani-
mals. Fitting with this hypothesis are the reported tran-
scriptional changes associated with the production of re-
active oxygen species in fat from IVF females (9).
A few factors in this study merit acknowledgment. Our
control group was designed to specifically test the impact
of IVF and embryo culture, while removing variables such
as superovulation and the embryo transfer procedure.
Therefore, comparison of control male and female ani-
mals may not accurately depict the natural sexual dimor-
phism present in adult tissues. Separately, white adipose
tissue depots can vary by adipocyte size, protein compo-
sition, gene expression, and response to gonadal hor-
mones (47, 48), such that our evaluation of gonadal fat
represents a focused analysis of sexual dimorphism and
conception impact on metabolism, and cannot necessarily
be extrapolated to other adipose depots. The analysis
could additionally be limited by outlying data points. For
example, one of the male liver samples contributed ex-
treme values; we consequently displayed the fold-change
and z-score data in heat map form to evaluate variability.
Metabolomics technology has not yet achieved total me-
tabolite coverage, thus creating an intrinsic and unavoid-
able bias toward known compounds. This study should
therefore be regarded as hypothesis generating, rather
than providing a cause-effect relationship. Because a num-
ber of mechanisms may contribute to the observed
changes in metabolite pool size, future studies must focus
on transporter activity and enzyme kinetics to better describe
the causes of the metabolite flux, as well as which aspects of
IVF metabolic signatures are relevant to the underlying eti-
ology of the outward phenotypes.
In summary, in accordance with the DOHaD hypoth-
esis, our data support that preimplantation embryo de-
velopment is a particularly sensitive environmental period
and that in vitro culture can induce permanent changes to
adult metabolism and energy use. Comparison of the IVF
metabolic and transcriptional signatures indicate several
areas of overlap, thus establishing a relationship between
molecular alterations and physiological phenotype. It re-
mains unclear why females specifically are susceptible to
a more severe metabolic phenotype and increased evidence
of oxidative stress, but this may be related to the particular
IVF conditions and/or the significant sexual dimorphism
already present in early embryos. Future studies should
expand the metabolic analysis in additional tissues and
further investigate sexual dimorphic epigenetic differences.
This study underscores the importance of continued and
sex-specific follow-up of IVF-conceived offspring beyond
early postnatal life.
Acknowledgments
We thank Kirk Pappan at Metabolon, Inc for his help processing
the metabolomics data. In memoriam David Barker.
doi: 10.1210/en.2014-1465 endo.endojournals.org 4565
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Address all correspondence and requests for reprints to: Paolo
F. Rinaudo, MD, PhD, Health Sciences West 1464E 513
Parnassus Avenue, San Francisco, CA 94143. E-mail:
rinaudop@obgyn.ucsf.edu.
This work was supported by the National Institute of Child
Health and Human Development Grant RO1:HD 062803-01A1
and by an American Diabetes Association grant (P.F.R.). S.K.F. was
supported by the National Institute of Health Training Fellowship
5T32DK007418-32. R.K.S. was supported by the California Insti-
tute for Regenerative Medicine Grant TB1-01194.
Disclosure Summary: The authors have nothing to disclose.
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