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Journal of D Álvarez, K Ceballo et al. Prenatal metformin improves 239:3 325–338

Endocrinology ovarian function

RESEARCH

Prenatal metformin treatment improves ovarian

function in offspring of obese rats

Daniela Álvarez1,*, Karina Ceballo1,*, Sofía Olguín1, Jonathan Martinez-Pinto2, Manuel Maliqueo3,

Daniela Fernandois1, Ramón Sotomayor-Zárate2 and Gonzalo Cruz1
1Laboratorio de Alteraciones Reproductivas y Metabólicas, Centro de Neurobiología y Plasticidad Cerebral (CNPC), Instituto de Fisiología, Facultad de
Ciencias, Universidad de Valparaíso, Valparaíso, Chile
2Laboratorio de Neuroquímica y Neurofarmacología, Centro de Neurobiología y Plasticidad Cerebral (CNPC), Instituto de Fisiología, Facultad de Ciencias,

Universidad de Valparaíso, Valparaíso, Chile

3Department of Medicine West Division, Endocrinology and Metabolism Laboratory, School of Medicine, University of Chile, Santiago, Chile

Correspondence should be addressed to G Cruz: gonzalo.cruz@uv.cl

*(D Álvarez and K Ceballo contributed equally to this work)

Abstract
Maternal obesity causes a wide range of impairment in offspring, such as metabolic and Key Words
reproductive dysfunctions. We previously demonstrated that female offspring of obese ff maternal obesity
rats have increased serum estradiol levels during early postnatal life, probably because ff metformin
of decreased hepatic cytochrome P450 3A2 levels, which could lead to early onset of ff estradiol
puberty and polycystic ovary condition in adulthood. Using metformin during pregnancy ff sympathetic activity
and nursing to improve the metabolic status of obese mothers could prevent the ff ovarian function
sequence of events that lead to an increase in postnatal serum estradiol levels in female
offspring and, hence, reproductive dysfunction. We found that metformin prevented
an increase in serum estradiol levels at postnatal day 14 in female offspring of obese
mothers, which was associated with a restoration of hepatic cytochrome P450 3A2 levels
to control values. Treatment using metformin could not prevent advanced puberty,
but we observed that the number of antral follicles, follicular cysts and multi-oocyte
follicles returned to control values in the female offspring of obese mothers treated
with metformin. We also observed an increase in the levels of norepinephrine and the
norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol in the ovaries, indicating
increased sympathetic activity in female offspring induced by an obesogenic uterine
environment. We found that this effect was prevented by metformin administration.
From the results of this study, we concluded that metformin administration to obese
mothers during pregnancy and nursing partially prevents ovarian dysfunction in female
Journal of Endocrinology
offspring during adulthood. (2018) 239, 325–338

Introduction
Maternal obesity is associated with various endocrine
and metabolic disorders in offspring, including obesity
(Paliy  et  al. 2014), hepatic steatosis (Oben  et  al. 2010,
Mouralidarane et al. 2013), diabetes mellitus (Catalano &
de Mouzon 2015) and cardiovascular disease (Taylor et al.
2014, Roberts  et  al. 2015). In rodents, maternal obesity
induced by high-fat diet (HFD) is also associated with
reproductive dysfunction in the offspring, such as early
onset of puberty, impaired follicular development,
altered estrous cyclicity and altered sex hormone profile

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(Sloboda  et  al. 2009, Connor  et  al. 2012, Cheong  et  al.
2014, Ambrosetti et al. 2016). These features in the female
offspring of obese rats resemble the phenotype induced
by estrogen exposure during early life. Administration of
estrogenic compounds during the neonatal-to-infantile
period leads to early onset of puberty, formation of ovarian
follicular cysts and decreased follicular development in
female offspring (Rosa et al. 2003, Sotomayor-Zarate et al.
2008, Cruz et al. 2012). Consistent with these studies, we
observed increased serum estradiol levels in the offspring
of rats fed an HFD before and during pregnancy and during
nursing (Ambrosetti et al. 2016), where we demonstrated
that female offspring of obese rats had increased serum
estradiol levels from postnatal day (PND) 1 until early
adulthood (PND 60) and that these elevated serum
estradiol levels were probably due to decreased cytochrome
P450 3A2 (CYP3A2) levels in the liver, reducing the
liver’s capacity to metabolize serum estradiol, instead of
increasing ovarian production (Ambrosetti et al. 2016). In
addition, several studies showed that female offspring of
obese rats develop hepatic steatosis, altering liver function
(Oben et al. 2010, Mouralidarane et al. 2013, Ambrosetti
et  al. 2016). Therefore, an increase in serum estradiol
levels due to hepatic dysfunction in female offspring of
obese rats leads to long-term reproductive dysfunction.
Female rats exposed to high serum estradiol levels
during early life show increased levels of norepinephrine
(NE) in the ovaries, indicating an increased sympathetic
tone related to the formation of follicular cysts during
adulthood (Rosa et  al. 2003, Sotomayor-Zarate et  al.
2008, Cruz et  al. 2012). Surgical denervation of the
superior ovarian nerve, which carries sympathetic fibers
to the ovary, reduces follicular cysts, improves ovarian
follicular development and partially restores estrous
cyclicity (Rosa et al. 2003, Sotomayor-Zarate et al. 2008).
Serum estradiol can regulate tyrosine hydroxylase
expression (Maharjan et al. 2005), the rate-limiting step in
catecholamine synthesis, thus increasing NE levels. Serum
estradiol increases the expression of neurotrophic peptide
nerve growth factor (NGF), which could lead to higher
sympathetic fiber density in the ovary (Lara  et  al. 2000,
Dissen  et  al. 2009, Valladares  et  al. 2017). Persistently
increased levels of serum estradiol during the neonatal-
to-infantile-sensitive period (Cruz et  al. 2012) in female
offspring of obese mothers could increase NE levels and
alter ovarian morphology and function.
Administration of metformin (a biguanide used to
treat insulin resistance and diabetes mellitus 2) to obese
pregnant rats reportedly reduces fetal liver inflammation
(Harris  et  al. 2016), preventing liver alterations
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Prenatal metformin improves 239:3 326
ovarian function
developing in offspring growing in an obesogenic uterine
environment.
Therefore, we hypothesize that metformin
administration to obese pregnant rats fed an HFD during
pregnancy and nursing prevents the development of early
liver dysfunction and increase in serum estradiol levels,
thus preventing ovarian sympathetic hyperactivation and
improving ovarian morphology and function in female
offspring during adulthood. To test this hypothesis, we
evaluated the effects of metformin administration to
obese rats during pregnancy and nursing on their female
offspring’s liver CYP3A2 expression, serum estradiol levels,
ovarian sympathetic activity and follicular development
during adulthood.
Materials and methods
Experimental design
Thirty female virgin Sprague–Dawley rats were weighed at
least three times a week and their estrous cycle followed
by taking vaginal smears daily for 3  weeks before the
experiment (Fig.  1). Only rats with regular cycles were
selected (N = 27). Selected rats were weight-matched and
randomly divided into two groups: group 1 (controls;
N = 8) was fed a control diet (CD), and group 2 (N = 19) was
fed an HFD. After 3 weeks, the HFD-fed rats were weight-
matched and randomly divided again into two groups:
HFD (N = 10) and HFD plus metformin (HFD + MET; N = 9)
(Table 1). After 4 weeks, on the afternoon of the proestrus
stage, all female rats were placed with male rats with
proven fertility. The next morning, successful mating was
confirmed with sperm presence in the vaginal smears.
Female rats that did not mate were again placed with
male rats with proven fertility in the subsequent proestrus
stage but for only one more attempt. If the female rats
did not become pregnant after the second attempt, they
were classified as ‘unsuccessful pregnancies’ and excluded
from the study (N = 11). Pregnant rats were considered
the experimental unit. The final number of pregnant rats
was seven for the control group, five for the HFD group
and four for the HFD + MET group. The control group was
continuously fed a standard diet (27% of kilocalories in
protein, 60% of kilocalories in carbohydrates and 13% of
kilocalories in fat; LabDiet) for 1 month before pregnancy,
during pregnancy and during nursing. The HFD and
HFD + MET groups were fed an HFD (20% of kilocalories in
protein, 20% of kilocalories in carbohydrates and 60% of
kilocalories in fat; Research Diets Inc.) for 1 month before
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Figure 1
Experimental design. Adult virgin rats were divided into three groups: one group received a CD and the other two groups received an HFD. The HFD was
administered for 1 month before pregnancy, during pregnancy and until weaning. One of the two groups fed the HFD received metformin in tap water,
starting from 1 week before mating, during pregnancy and until weaning. The offspring of all groups were fed a CD from weaning until the end of the
experiments (PND 60). Superior boxes represent mothers, and inferior boxes represent offspring. The dashed fill represents the time of HFD consumption
alone, while the gray fill represents the simultaneous consumption of HFD and metformin.

pregnancy, during pregnancy and during nursing. The retroperitoneal white adipose tissue (rpWAT) and trunk
HFD + MET group was administered metformin dissolved blood were collected. The blood was centrifuged at 850 × g
in drinking water from week 3 of the experiment (1 week for 10 min to obtain serum, which was stored at −80°C.
before mating), during pregnancy and during nursing. All the collected tissues were stored at −80°C for further
Drinking water was measured each day to calculate and analyses.
follow the exact doses of metformin intake. The actual
dose was 160–200 mg/kg/day. After weaning, the male Control of weight and estrous cycle
offspring were killed and not considered in this study.
The body weights of all rats were recorded daily until
The female offspring of the three groups (control group,
the end of the experiment. The external genitalia were
N = 39; HFD group, N = 27; HFD + MET group, N = 14) were
observed daily from PND 25 onward to determine the
separated from their mothers and divided into groups of
day of vaginal opening. The estrous cycle stages were
4–5 rats per cage. The offspring were fed a CD from weaning
determined by taking vaginal smears from the vaginal
until killing. In some cases (in adult rats), we performed
opening until killing. The percentage of cyclicity was
the experiments on two offspring from the same mother,
calculated using the following formula: (No. cycles/(No.
wherein we averaged the values and considered the mean
days since vaginal opening/4))  × 100, modified from
to create graphs and perform statistical analyses.
Fernandois  et  al. (2012). This means that the cyclicity
All procedures were approved by the Institutional
percentage would be 100% if a rat had 4-day continuous
Committee for Bioethics and Animal Care, University of
cycles from vaginal opening until killing.
Valparaíso (CIBICA-UV, N°007-2013).

Histology and morphometric analyses of the ovaries

Euthanasia and tissue collection
All rats were killed by cervical decapitation at PND 14 or at
first estrus after PND 57 (PND 60 ± 3); adult rats were killed
after 6 h of fasting. At the time of killing, the ovaries, liver,
At the time of killing of the adult offspring, 18 right
ovaries (6 from each experimental group) were immersed
in Bouin’s fixative. After fixation, the ovaries were
embedded in paraffin, cut into 6 µm sections and stained

Table 1  Body weight and fertility of obese and metformin-treated dams.

Control HF HF + MET

Mothers body weight before diet (g) 222.8 ± 5.436 225.6 ± 5.995
Mothers body weight after 1 month of diet (g) 247.8 ± 6.654 274.6 ± 10.05
Number of pups 13.5 ± 0.500 11.6 ± 0.678
224.4 ± 5.826 Control vs HF P = 0.7319 (ns)
Control vs HF + MET P = 0.8430 (ns)
HF vs HF + MET P = 0.8880 (ns)
274.4 ± 7.710 Control vs HF P = 0.0322 (*)
Control vs HF + MET P = 0.0375 (*)
HF vs HF + MET P = 0.9896 (ns)
9.00 ± 2.16 Control vs HF P = 0.0467 (*)
Control vs HF + MET P = 0.0151 (*)
HF vs HF + MET P = 0.1413 (ns)

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with hematoxylin and eosin. Morphometric analysis of
the follicles was performed, as previously reported (Cruz
et  al. 2012). We counted the total number of healthy
antral follicles, atretic antral follicles, corpora lutea,
follicular cysts and multi-oocyte follicles (MOFs). Antral
follicles were counted when the nuclei of the oocytes
were visible. The antral follicles were classified as atretic
antral follicles if they had more than 5% of cells with
pyknotic nuclei in the largest cross section and if they
exhibited granulose shrinkage and occasional breakdown
of the oocyte germinal vesicle. Follicular structures with
more than one oocyte were classified as MOFs. Follicular
structures were classified as follicular cysts if they met the
following conditions: lack of an oocyte, absence of atresia
criteria and a large antral cavity.
Plasma hormone and glucose determination
We determined serum estradiol, estriol, insulin and
leptin levels by enzyme-linked immunoassays according
to the manufacturer’s instructions: serum estradiol
(11-ESTHU-E01, ALPCO Diagnostics, NH, USA), estriol
(20-FE3HU-01, ALPCO Diagnostics), leptin (EZRL-
83K, EMD Millipore) and insulin (EZRMI-13K, Merck
Millipore). Intra- and interassay variations were <5% for
serum estradiol, <4.7% for estriol, <6% for insulin and
<3% for leptin. The minimum detectable concentrations
were 10 pg/mL for serum estradiol, 0.075 ng/mL for estriol,
0.2 ng/mL for insulin and 0.05 ng/mL for leptin.
Serum glucose was determined by an enzymatic
method (11504, Biosystem, Barcelona, Spain). The intra-
and interassay coefficients of variation were <2.7%.
Western blot analyses
Hepatic CYP3A2 levels were detected by western blot
at PNDs 14 and 60. For this, 15 mg of liver tissue were
homogenized in radioimmunoprecipitation assay (RIPA)
buffer: 50 mM Tris–HCl, pH 7.4; 1% NP-40; 0.1% SDS;
150 mM NaCl; 2 mM EDTA; 50 mM NaF and 1× protease
inhibitor cocktail (Roche). Then, 30 µg of total protein
were separated by SDS-PAGE in 10% polyacrylamide gel
under reducing conditions. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as an internal
control. The separated proteins were transferred to a
nitrocellulose membrane, blocked with 5% milk for 1 h
and probed with either rabbit polyclonal anti-CYP3A2
antibody (AB1276 Merck Millipore; 1:250, overnight
incubation) or rabbit polyclonal anti-GAPDH antibody
(G9545, Sigma-Aldrich; 1:40,000, 1-h incubation).
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The antibody complexes were detected using goat antirabbit
immunoglobulin G (IgG) Fc (horseradish peroxidase
(HRP)) (ab97200, concentration  = 1:10,000; Abcam).
We used the EZ-ECL Enhanced Chemiluminescence
Detection Kit (Biological Industries, Israel) for complex
detection; chemiluminescence was captured using the
EpiChemi3 Darkroom system (UVP Inc., CA, USA). We
analyzed the results by measuring the pixel intensities of
bands using the semiquantification tool of the Image-Pro
Plus 6.0 program (Media Cybernetics, Inc., Rockville, MD,
USA). All western blots were performed at least thrice for
each liver sample in independent blots. The coefficient of
variation of the CYP3A2:GAPDH ratio for each individual
sample was ≤25% among the independent blots.
Norepinephrine and
3-methoxy-4-hydroxyphenylglycol levels
We quantified NE and its metabolite 3-methoxy-4-
hydroxyphenylglycol (MHPG) in the ovaries using high-
performance liquid chromatography (HPLC) coupled
with electrochemical detection by the EICOM ECD-700S
electrochemical detector (Amuza Neuroscience, San Diego,
CA, USA). In brief, we weighed the tissue samples using an
analytical balance and homogenized them manually in a
glass–glass homogenizer in 400 µL of 0.2 M perchloric acid
(PCA). The samples were then centrifuged at 16,000 × g for
15 min at 4°C. The supernatant was collected and stored at
−80°C until testing. Before the assay, we filtered an aliquot
of 200 μL of the supernatant in 13 mm disposable PVDF
membrane filters with 0.22 μm pores (Millex-GV, Merck
Millipore Ltda). Then, 20 μL of the filtrate were injected
into the JASCO PU-2089s plus HPLC system coupled with
the JASCO LC-NetII/analog-to-digital converter digitizer
(Tokyo, Japan); a Kromasil 100-3.5-C18 column (AkzoNobel
N.V., Amsterdam, Netherlands) was used. To integrate
the chromatograms, we used the JASCO ChromPass
Chromatography Data System v1.7.403.1 software (Tokyo,
Japan). The mobile phase consisted of 0.1 M NaH2PO4,
0.14 mM octyl sulfate, 0.02% EDTA and 1.5% acetonitrile
(pH 2.6). The flow rate was set at 1 mL/min, and the
electrochemical potential of the amperometric detector
was set at +750  mV to simultaneously detect NE and
MHPG. The retention time under these conditions was
5 min for NE and 10.5 min for MHPG.
Statistical analysis
All analyses were performed considering at least one
rat per litter as a representative experimental unit.
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When two or more siblings were used for analysis, we
considered the average of the values as a single data
value. All data are expressed as mean ± standard error
of mean (s.e.m.). One-way ANOVA (or two-way ANOVA
for body weight curve analysis) followed by Fisher’s
least significant difference test was used to compare the
results between the control, HFD and HFD + MET groups.
Statistical analyses were carried out with the GraphPad
Prism v6.0 software (GraphPad Software), and P < 0.05 was
considered statistically significant in all tests.
Results
Effects of HFD + MET before and during pregnancy
and during nursing on the mothers’ body weight
and fertility
Before beginning the experiments, the female rats were
randomly distributed in each experimental group; no
differences were observed in body weight among groups
(Table 1). As shown in Table 1, we found that after 1 month
of being fed an HFD, the rats showed a considerable weight
gain that metformin administration (administered 1 week
before mating and 3  weeks after onset of the HFD) did
not prevent. Regarding fertility, the HFD and HFD + MET
groups showed a fewer number of offspring (Table  1).
No differences in the female:male ratio was detected (data
not shown).
Effects of HFD + MET before and during pregnancy
and during nursing on the female offspring’s
metabolic and reproductive parameters
We analyzed the body weight of female offspring daily
from birth (PND 1) until PND 60. We found that the
HFD and HFD + MET offspring were heavier than the
control offspring from PND 1 until PND 60 (Fig.  2A).
Surprisingly, the offspring of the HFD + MET offspring
were heavier than the HFD offspring from PND 1 until
~PND 50 (P < 0.05 until PND50 for HFD vs HFD + MET)
being magnified this difference prepubertally (Fig.  2B,
C and D), but this difference disappeared at PND 60
(Fig. 2E). Interestingly, as shown in Table 2, at PND 60,
the rpWAT weight increased in both HFD and HFD + MET
offspring compared to control offspring, being more in
HFD + MET offspring than in HFD offspring. Finally, as
shown in Fig. 3, HFD offspring presented early onset of
puberty, evidenced as early vaginal opening (Fig. 3A and
B) and an early first estrus (Fig. 3C), an effect that was not
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prevented by metformin administration to the mothers
during pregnancy and nursing. With regard to the
estrous cycle, there were no differences in the number
or percentage of estrous cycles among groups. However,
we found an increase in permanency in the estrus stage
and a decrease in permanency in the diestrus stage in
HFD offspring compared to HFD  + 
MET and control
offspring (Table 3).
With regard to hormonal metabolic markers, we
found that leptin levels increased at PND 14 in HFD
offspring compared to control offspring, an effect that
was not prevented by metformin administration to the
mothers (HFD vs HFD  + 
MET, P = 0.5614; Table  2). As
shown in Table 2, at PND 60, leptin levels in HFD offspring
were equal to control offspring. Surprisingly, HFD + MET
offspring showed increased leptin levels compared to HFD
and control offspring. Insulin levels were increased in HFD
offspring compared to control offspring at PND 60 and
only slightly increased in HFD + MET offspring compared
to control offspring (controls vs HFD + MET, P = 0.0649).
Effects of HFD + MET before and during pregnancy
and during nursing on the female offspring’s serum
estradiol levels and hepatic CYP3A2 expression
We found that serum estradiol levels were higher at PND
14 in HFD offspring compared to control offspring, but
no differences were observed at PND 60 (Fig. 4A and B).
In addition, we found that metformin administered to
the mothers prevented an increase in serum estradiol at
PND 14 in the offspring (Fig. 4A). CYP3A2 expression in
the liver at PNDs 14 and 60 was lower in HFD offspring
than in control offspring of obese rats. However,
metformin treatment to the mothers prevented the
hepatic reduction of CYP3A2 induced by an HFD (Fig. 4G
and H). To assess serum estradiol biotransformation by
CYP3A2, we measured the plasma levels of serum estriol,
a metabolite from direct and one-step serum estradiol
biotransformation through hepatic CYP3A2. Figures  4C
and D show that serum estriol did not decrease at PNDs 14
and 60 in HFD offspring compared to control offspring.
However, when the serum estriol:serum estradiol ratio
was analyzed (as an indicator of serum estradiol clearance
by CYP3A2 metabolization), HFD offspring presented a
decrease compared to control offspring (controls vs HFD,
P = 0.047; HFD + MET vs HFD, P = 0.064). At PND 60, the
serum estriol:serum estradiol ratio showed only a slight,
but not significant, tendency to decrease (controls vs
HFD, P = 0.0889; HFD vs HFD + MET, P = 0.0715).
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Figure 2
Offspring body weight. (A) Body weight curve from birth until PND 60 and (B, C, D and E) average body weight at PNDs 1, 7, 14 and 60. Data are shown
as mean ± s.e.m. **P < 0.01 and ****P < 0.0001 for Control vs HFD; #P < 0.05, and ###P < 0.001 for HFD vs HFD + MET; ++P < 0.01 and ++++P < 0.0001 for Control vs
HFD + MET. Control, N = 7; HFD, N = 5; HFD + MET, N = 4.

Ovarian sympathetic activity in female offspring of
obese mothers and effects of metformin
administration during pregnancy and nursing
We measured NE and MHPG levels in the ovaries of
female offspring as an index of sympathetic activity
(Heal  et  al. 1989). Figure  5A shows that the ovarian NE
levels increased in HFD offspring, while they returned
to control values when the mothers were treated with
metformin. The same pattern was observed for MHPG
(Fig. 5B), indicating a decrease in NE release from ovaries
in the HFD + MET offspring compared to HFD offspring.
Ovarian follicular development in female offspring
of obese mothers and effects of metformin
administration during pregnancy and nursing
Morphological analysis of the ovaries at PND 60 was
conducted. Figure  6A shows a decrease in healthy
antral follicles in the female offspring of obese mothers
compared to control offspring. In addition, when healthy
antral follicles were analyzed by size (Fig. 6B), we observed
a decrease in the smallest antral follicles (<200 µm) in the
ovaries of HFD offspring compared to control offspring.
However, this effect was not prevented by administration
of metformin to the mothers during pregnancy and
nursing. With regard to atretic antral follicles, we found a
decrease in HFD offspring compared to control offspring
(Fig.  6C). Finally, we observed no significant changes
in the number of corpora lutea (Fig.  6D). However, two
abnormal structures, MOFs and follicular cysts, showed
an increase in the ovaries of HFD offspring (Fig.  6E and
F, respectively). Some of these MOFs were similar to
oocyte nests, which are usually observed in neonates
before follicular assembly (we found four oocyte nests in
three different HFD offspring ovaries from three different
litters). Figure  7 shows an oocyte nest–like structure
(Fig. 7A and B), a secondary MOF (Fig. 7C), an antral MOF
(Fig.  7D) and follicular cysts (Fig.  7E). All these effects
(decrease in healthy and atretic antral follicles, increase in
follicular cysts and increase in MOFs) were prevented in
adult offspring when metformin was administered to the
mothers during pregnancy and nursing.

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Table 2  Body weight and metabolic parameters in offspring of obese dams treated with metformin.

Control HF HF + MET

Liver weight PND14 (g)
Liver weight PND60 (g)
rpWAT PND60 (mg)
Leptin PND14 (ng/mL)
Leptin PND60 (ng/mL)
Insulin PND60 (ng/mL)
Glycemia PND14 (mmol/L)
Glycemia PND60 (mmol/L)
0.6947 ± 0.02512 0.8892 ± 0.05811 0.8314 ± 0.1046 Control vs HF P = 0.0290 (*)
Control vs HF + MET P = 0.1522 (ns)
HF vs HF + MET P = 0.5269 (ns)
7.477 ± 0.11783 8.044 ± 0.1921 8.013 ± 0.02574 Control vs HF P = 0.0283 (*)
Control vs HF + MET P = 0.0473 (*)
HF vs HF + MET P = 0.9040 (ns)
524 ± 44.22 818.7 ± 39.59 1003 ± 87.61 Control vs HF P = 0.0015 (**)
Control vs HF + MET P < 0.0001(****)
HF vs HF + MET P = 0.0477 (*)
1.573 ± 0.2589 4.117 ± 0.7462 3.498 ± 1.066 Control vs HF P = 0.024 (*)
Control vs HF + MET P = 0.08895 (ns)
HF vs HF + MET P = 0.5614 (ns)
1.293 ± 0.2092 1.385 ± 0.08292 2.051 ± 0.157 Control vs HF P = 0.6958 (ns)
Control vs HF + MET P = 0.0096 (**)
HF vs HF + MET P = 0.0232 (*)
1.030 ± 0.1158 1.553 ± 0.2441 1.601 ± 0.3055 Control vs HF P = 0.0473(*)
Control vs HF + MET P = 0.0649 (ns)
HF vs HF + MET P = 0.4520 (ns)
5.438 ± 0.1603 5.718 ± 0.2380 5.363 ± 0.1626 Control vs HF P = 0.3173 (ns)
Control vs HF + MET P = 0.7949 (ns)
HF vs HF + MET P = 0.2358 (ns)
5.249 ± 0.2349 4.635 ± 0.1637 5.239 ± 0.2242 Control vs HF P = 0.0836 (ns)
Control vs HF + MET P = 0.9761 (ns)
HF vs HF + MET P = 0.1258 (ns)

Discussion Maternal obesity and maternal exposure to an HFD in

Maternal obesity causes offspring to suffer from various
metabolic and reproductive disorders. We previously
demonstrated that female offspring of rats exposed to an
HFD for 1 month before pregnancy, during pregnancy and
during nursing have high serum estradiol levels during
early life (Ambrosetti et al. 2016), which is associated with
reproductive alterations such as early puberty, decreased
follicular development and polycystic ovaries (Ambrosetti
et al. 2016, Lin et al. 2017). We postulated that offspring
of these rats have a hepatic dysfunction that leads to low
hepatic serum estradiol metabolism through the enzyme
CYP3A2. This low hepatic serum estradiol metabolism
could cause high serum estradiol levels (Ambrosetti et al.
2016). In this study, we treated pregnant rats fed an HFD
with the drug metformin during pregnancy and nursing in
order to improve their metabolic profile and hence prevent
ovarian and hormonal dysfunctions in the offspring.
rats causes inflammation, insulin resistance and increased
accumulation of triglycerides in the fetal liver (Harris et al.
2016). This hepatic triglyceride accumulation is probably
due to a hypercaloric fetal microenvironment caused by
maternal insulin resistance, which causes high levels of
glucose and free fatty acids to transfer from maternal
blood to the fetal liver (Jansson & Powell 2007). The drug
metformin could improve insulin sensitivity in pregnant
rats and decrease the availability and hence the transfer
of these substances into fetal blood, thereby preventing
postnatal hepatic dysfunction. The results of this study
showed that administering metformin to obese pregnant
rats during pregnancy and nursing prevents a decrease in
CYP3A2 levels in female offspring and prevents an increase
in serum estradiol levels at PND 14, as we expected.
However, although metformin restored serum estradiol
levels to control values in the female offspring, it did
not prevent early onset of puberty (measured as vaginal

Figure 3
Puberty onset in offspring of mothers fed a CD,
an HFD or HFD + MET. (A) Mean day of age of
vaginal opening, (B) percentage of rats reaching
vaginal opening through time and (C) mean day
of age of first estrus. Data are shown as
mean ± s.e.m. *P < 0.05 for Control vs HFD; +P < 0.05
and ++P < 0.01 for Control vs HFD + MET. Control,
N = 7; HFD, N = 5; HFD + MET, N = 4.

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Table 3  Estrous cyclicity in offspring of controls, HF and HF + Met rats.
Control
Number of cycles 3.643 ± 0.2256
Percentage of cyclicity 65.16 ± 3.648
Percentage of permanency Proestrus 21.13 ± 2.023
in each estrous stage
Estrus 23.36 ± 1.512
Diestrus 59.56 ± 2.892
opening). This result suggested that maternal exposure to
an HFD during pregnancy and nursing activates another
mechanism that leads to precocious activation of the
hypothalamic–pituitary–ovarian axis. Another reason for
precocious puberty could be the increase in leptin levels,
which previous studies have demonstrated as a causal
factor for accelerating the onset of puberty (Ahima et al.
1997, Shalitin & Phillip 2003). In this study, we observed
that metformin administration to obese mothers did
not reduce leptin levels to control values in the female
offspring at PND 14; in fact, the levels increased by
PND 60. Therefore, increased leptin levels might induce
precocious puberty in the female offspring of rats fed an
HFD during pregnancy and nursing.
In this study, we did not find a significant decrease in
serum estradiol levels at PND 60, as previously reported
(Ambrosetti et al. 2016), even when the levels of CYP3A2
were still low and the plasma serum estriol:serum
estradiol ratio showed a tendency to decrease (HFD vs
controls, P = 0.077; HFD vs HFD + MET, P = 0.067). We
believe that the mature hypothalamic feedback might be
compensating for an increase in serum estradiol levels due
to hepatic dysfunction during adulthood, causing plasma
serum estradiol levels to be similar to control values or
slightly increased, as shown by Ambrosetti et  al. (2016).
However, before puberty, with an immature reproductive
hypothalamic axis (Picut et al. 2015), this increase in serum
estradiol levels is not compensated by the hypothalamic
feedback, which is demonstrated by the magnitude of
serum estradiol increase.
On the other hand, despite being fed a CD after
weaning, the HFD  + 
MET offspring were heavier than
the control offspring during adulthood. In addition, the
HFD + MET offspring had an increased body weight at
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HF HF + MET
3.48 ± 0.1855 3.35 ± 0.2021 Control vs HF P = 0.5911 (ns)
Control vs HF + MET P = 0.3717 (ns)
HF vs HF + MET P = 0.7074 (ns)
63.18 ± 3.66 57.73 ± 3.633 Control vs HF P = 0.7044 (ns)
Control vs HF + MET P = 0.1963 (ns)
HF vs HF + MET P = 0.3673 (ns)
19.62 ± 1.536 19.4 ± 0.991 Control vs HF P = 0.6237 (ns)
Control vs HF + MET P = 0.5995 (ns)
HF vs HF + MET P = 0.9501 (ns)
31.1 ± 2.824 22.6 ± 0.960 Control vs HF P = 0.0152 (*)
Control vs HF + MET P = 0.8178
HF vs HF + MET P = 0.0197 (*)
48.14 ± 2.821 60.6 ± 2.056 Control vs HF P = 0.0006 (***)
Control vs HF + MET P = 0.7511
HF vs HF + MET P = 0.0010 (***)
PNDs 1, 7 and 14, and until ~PND 50 compared to control
and HFD offspring. Moreover, although we observed no
differences in body weight between HFD and HFD + MET
offspring at PND 60, the offspring of rats treated with
metformin during pregnancy and nursing showed an
increased rpWAT weight, which was also reflected in
the leptin levels. This result showed that metformin
administration to rats during pregnancy and nursing
amplifies the effects of an HFD on body weight and fat
accumulation in the offspring.
Interestingly, a recent study on humans showed that
children of women with PCOS who were on metformin
during pregnancy had a higher BMI from age 6  months
until 4 years and an increased prevalence of obesity at age
4 years compared to children of mothers from the placebo
group (Engen Hanem et al. 2018). The reason could be the
transfer of metformin into the fetus through the placenta
(Kovo  et  al. 2008, Salomaki  et  al. 2013). In this regard,
the effects of metformin on the fetal liver are probably
beneficial, as demonstrated by other studies (Salomaki
et al. 2013, 2014, Harris et al. 2016); however, increasing
insulin sensitivity in adipose tissue of the fetus could
increase the storage of fat, favoring postnatal obesity.
In this study, we proposed that the phenotype
observed in adult female offspring of rats exposed to an
HFD before pregnancy, during pregnancy and during
nursing resembles that observed in women with PCOS, an
endocrine and metabolic disorder affecting a large number
of women of reproductive age. The offspring of obese
mothers have a higher prevalence of nonalcoholic fatty
liver disease, insulin resistance and hypertension, as is
usually seen in women with PCOS (Glintborg et al. 2016,
Li et al. 2016, Orio et al. 2016). However, several authors
have proposed a developmental origin of PCOS because of
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Figure 4
Hepatic serum estradiol and CYP3A2 levels. (A)
Serum estradiol and (C) serum estriol at PND 14;
N = 4 for group. (B) Serum estradiol and (D) serum
estriol at PND 60; N = 5 for control and HFD and
N = 4 for HFD + MET. (E) Hepatic CYP3A2 level at
PND 14; N = 4 for each experimental group. (F)
Hepatic CYP3A2 level at PND 60; N = 5 for control
and HFD and N = 4 for HFD + MET. (G)
Representative image of CYP3A2 western blot at
PND 14. (H) Representative image of CYP3A2
western blot at PND 60. Data are shown as
mean ± s.e.m. *P < 0.05 for control vs HFD; #P < 0.05
for HFD vs HFD + MET.

prenatal or early postnatal exposure to estrogenic or

Figure 5
Sympathetic activity in the ovary. (A) Total ovarian NE levels per ovary at
PND 60 and (B) total ovarian MHPG levels per ovary at PND 60. Control,
N = 7; HFD, N = 5; HFD + MET, N = 4. Data are shown as mean ± s.e.m.
**P < 0.01 for Control vs HFD; #P < 0.05, and ##P < 0.01 for HFD vs
HFD + MET.
androgenic compounds. Early exposure to estrogenic
compounds decreases antral follicles and increases
follicular cysts (Rosa et  al. 2003, Sotomayor-Zarate et  al.
2008, Padmanabhan & Veiga-Lopez 2011, Cruz et  al.
2012). We think that the increase in endogenous serum
estradiol levels we observed in our study is similar to the
endocrine disruption caused by estrogenic compounds. In
addition, we and other researchers demonstrated that the
adult female offspring of rats exposed to an HFD during
pregnancy and nursing have a low number of antral
follicles and a high number of follicular cysts (Cheong
et al. 2014, Ambrosetti et al. 2016). The decrease in healthy

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and atretic antral follicles observed in this study was also
observed by Ambrosetti et al. (2016) at PND 60, which is a
short time after puberty and indicates low formation and
growing of follicles. In addition, Ambrosetti et al. (2016)
and Tsoulis et  al. (2016) showed a decrease in healthy
antral follicles and an increase in atretic antral follicles at
PND 120, a long time after puberty. Because gonadotropin
follicle-stimulating hormone (FSH), through action on its
follicle-stimulating hormone receptor, is essential for both
the formation/growth of follicles and the prevention of
atresia (Hirshfield & Midgley 1978, Chun et al. 1996), it is
possible that FSH sensitivity of follicles could be altered in
HFD offspring. Tsoulis et al. (2016) showed an increase in
anti-Mullerian hormone (AMH) expression in antral
follicles. This finding supports the observed ovarian
morphological changes, since AMH decreases follicular
Figure 7
MOF and follicular cyst histology. (A and B) Oocyte nest–like structure, (C) secondary MOF, (D) healthy antral MOF and (E) follicular cyst. All the images
are of ovaries from the HFD offspring.
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Figure 6
Ovarian follicular development. The counting of
ovarian follicles at different stages is shown.
Black bars: offspring of Control rats; white bars:
offspring of HFD rats; gray bars: offspring of
HFD + MET rats. (A) Healthy antral follicles, (B) size
distribution of healthy antral follicles, (C) atretic
antral follicles, (D) corpora lutea, (E) MOFs, and
(F) follicular cysts. N = 4 for Control, HFD and
HFD + MET. Data are shown as mean ± s.e.m.
*P < 0.05, **P < 0.01 and ***P < 0.001 for Control
vs HFD; #P < 0.05 for HFD vs HFD + MET in each
graph.
sensitivity to FSH, blocking the formation of follicles and
increasing the rate of atresia (Visser et al. 2007, Pellatt et al.
2011). Similarly, when neonatal rats are exposed to serum
estradiol valerate, there is an increase in AMH in the antral
follicles at PND 60, indicating that the serum estradiol
increase during this period (as was observed in our study)
alters the genetic expression of AMH in follicles during
adulthood (Martinez-Pinto  et  al. 2018). Reinforcing this
finding is another feature we observed in our study and
was also observed by Connor et al. (2012): an increase in
permanency in the estrus stage, meaning slow follicular
formation and growth. However, in this study, we found
that metformin administration to mothers during
pregnancy and nursing prevents the decrease in antral
follicles and the increase in follicular cysts observed in
HFD offspring. We think that metformin’s effect of
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preventing an increase in serum estradiol levels in female
offspring of obese mothers during the first few days of life
ultimately prevents a decrease in antral follicles and an
increase in follicular cysts, as discussed later. Another
feature of animals exposed to estrogenic compounds is
long-term permanence of MOFs in the ovary
(Jefferson et al. 2002, Kipp et al. 2007). In this study, we
observed an increase in MOFs in female offspring of obese
mothers that was prevented by administration of
metformin to the mothers. A possible explanation for the
appearance of MOFs is the effect of estrogenic compounds
in preventing or delaying follicular assembly (Kezele &
Skinner 2003, Pepling  et  al. 2010) and then altering the
formation of primordial follicles with individual oocytes.
This follicular assembly in rats starts before birth but ends
around PND 4 (Pepling et al. 2010, Pepling 2012). In this
study, we did not measure serum estradiol levels within
this time window, but in a previous study, we demonstrated
that endogenous serum estradiol levels increase at PNDs 1
and 7 in female offspring of obese mothers (Ambrosetti
et al. 2016). Therefore, we hypothesized that an increase
in serum estradiol levels during early postnatal life and
even before birth might disrupt follicular assembly and
increase the probability of MOF generation, which we
observed in adult female rats. Moreover, upon histological
evaluation, some of the MOFs we found were similar to
oocyte nests, reinforcing the idea of aberrant follicular
assembly. Another effect of exposure to estrogenic
compounds in early life is a long-term increase in the
activity of the sympathetic nervous system in the ovary.
For example, early exposure to serum estradiol valerate
increases NE levels in the ovary during adulthood (Rosa
et al. 2003, Sotomayor-Zarate et al. 2008, Cruz et al. 2012).
In this study, we demonstrated that female offspring of
obese mothers have increased NE levels in the ovary.
Therefore, increased endogenous serum estradiol levels
during the neonatal-to-prepubertal period could be the
cause of increased NE levels in the ovary during adulthood.
This increase in NE levels was prevented by metformin
administration to pregnant and nursing mothers, which
also prevented an increase in serum estradiol levels at
PND 14 in female offspring. The mechanisms explaining
this increase in NE levels are not clear, but one plausible
explanation is the ability of estrogenic compounds to
increase NGF expression (Sotomayor-Zarate et  al. 2008),
which is strongly related to a higher density of nerve
fibers. An additional factor is the neonatal-to-prepuberal
hyperleptinemia occurring in offspring, as demonstrated
by us and other researchers (Ambrosetti et  al. 2016,
Zambrano  et  al. 2016, Tellechea  et  al. 2017).
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ovarian function
The higher leptin levels during early development cause
an increase in sympathetic outflow to several tissues, such
as adipose tissue, the heart and kidneys (Rahmouni 2010,
Samuelsson et al. 2010, Taylor et al. 2014). Conversely, a
decrease in NE levels in plasma and peripheral tissues,
particularly brown and white adipose tissue, was observed
in obese and leptin-deficient (ob/ob) mice (Knehans &
Romsos 1983, 1984) and obese and leptin receptor-
deficient (fa/fa) rats (York  et  al. 1985, Rosenthal  et  al.
1996). On the other hand, hyperleptinemic diabetic
(db/db) mice, which have a nonfunctional ObRb leptin
receptor, have increased NE levels in the ovary and uterus
(Garris 2004). Therefore, we hypothesized that an increase
in leptin levels during early development might increase
the sympathetic tone of the ovary during adulthood, as in
other organs, but this idea needs to be investigated in the
future. Interestingly, women with PCOS have a higher
density of sympathetic fibers in the ovary and an increase
in overall sympathetic activity (Heider  et  al. 2001,
Lansdown & Rees 2012, Wojtkiewicz  et  al. 2014). This
increased sympathetic nerve activity is usually determined
by measuring NE metabolite levels (see Table  1 in
Lansdown & Rees 2012). In this study, we measured the
NE metabolite MHPG, which is formed from the enzymatic
reaction through monoamine oxidase and catechol-o-
methyl transferase (COMT). Since COMT has an
extracellular distribution, MHPG is an indicator of NE
release (Lookingland  et  al. 1991). We found increased
MHPG levels in the HFD offspring compared to control
offspring, reflecting an increase in NE release. Interestingly,
administration of metformin to obese mothers prevented
the increase in MHPG levels in the ovaries of offspring,
suggesting that metformin decreases sympathetic activity
in the ovaries during adulthood, probably by preventing
an increase in serum estradiol levels during early life.
We conclude that female offspring of mothers
exposed to an HFD before pregnancy, during pregnancy,
and during nursing have a PCOS-like reproductive
phenotype, including metabolic and reproductive
dysfunctions. The administration of metformin to
mothers fed an HFD during pregnancy and nursing
partially prevents the PCOS-like reproductive phenotype
but amplifies some metabolic impairments, such as fat
accumulation and increased body weight, in the offspring.
Finally, metformin can improve hepatic function in
the newborn, mitigating reproductive dysfunction by
improving the metabolism of serum estradiol during
the most vulnerable period (PNDs 1–14) when serum
estradiol disruption can occur during postnatal ovarian
development in rats (Cruz et al. 2012).
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Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Fondecyt grant number 11130707 and DIUV-CL
N° 01/2006 to Gonzalo Cruz.
Acknowledgements
This work was supported by Fondecyt Initiation Grant 11130707-CONICYT.
Additional funds were provided by the Center for Neurobiology and
Cerebral Plasticity (CNPC) – Universidad de Valparaíso. The authors thank
Tania Cerda from the University of Valparaíso for technical support.
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Received in final form 21 August 2018

Accepted 10 September 2018
Accepted Preprint published online 10 September 2018

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https://doi.org/10.1530/JOE-18-0352 Published by Bioscientifica Ltd.
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