Systems Biology in Reproductive Medicine

ISSN: 1939-6368 (Print) 1939-6376 (Online) Journal homepage: https://www.tandfonline.com/loi/iaan20

Experimental diabetes impairs maternal

reproductive performance in pregnant Wistar rats
and their offspring

Leticia Bequer, Tahiry Gómez, José L. Molina, Alain Álvarez, Claudia Chaviano
& Sonia Clapés

To cite this article: Leticia Bequer, Tahiry Gómez, José L. Molina, Alain Álvarez, Claudia Chaviano
& Sonia Clapés (2018) Experimental diabetes impairs maternal reproductive performance in
pregnant Wistar rats and their offspring, Systems Biology in Reproductive Medicine, 64:1, 60-70,
DOI: 10.1080/19396368.2017.1395928

To link to this article: https://doi.org/10.1080/19396368.2017.1395928

Published online: 20 Nov 2017.

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SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE
2018, VOL. 64, NO. 1, 60–70
https://doi.org/10.1080/19396368.2017.1395928

RESEARCH ARTICLE

Experimental diabetes impairs maternal reproductive performance in pregnant

Wistar rats and their offspring
Leticia Bequera, Tahiry Gómeza, José L. Molinaa, Alain Álvareza, Claudia Chavianoa, and Sonia Clapésb
a
Biomedical Research Center, Medical College of Villa Clara, Cuba; bMedical College of Havana, Cuba

ABSTRACT ARTICLE HISTORY

The aim of this study was to determine the effect of mild hyperglycemia on metabolism during Received 27 March 2017
pregnancy, the maternal reproductive performance, and the characteristics of the offspring in Revised 4 October 2017
neonatal mild diabetic-induced Wistar rats. The experimental diabetes model was generated by Accepted 5 October 2017
neonatal streptozotocin administration (100 mg of streptozotocin/Kg bw/sc) in female Wistar rats. KEYWORDS
At adulthood, the control and diabetic group were mated. At the 20th day of gestation, a Diabetes mellitus;
maternal and fetal blood sample were collected for biochemical measurement. The maternal experimental;
livers, fetal livers, and placenta were removed for oxidative stress measurements. Maternal hyperglycemia; pregnancy;
reproductive outcomes and fetal and placental morphometric measurements were analyzed. rats; streptozocin; Wistar
The fetuses were classified as small, appropriate, and large for pregnancy age, and examined
for the presence of external anomalies. The diabetic group showed mild hyperglycemia, altered
glucose tolerance, increased total cholesterol, triglycerides, and hemoglobin A1c during preg-
nancy. At the 20th day of gestation the diabetic mothers presented increased reabsorptions and
embryonic losses before and after implantation, reduced corpora lutea number, litter size,
implantation sites, live fetuses, and decreased efficiency of implantation rate. Similarly, the off-
spring showed reduced fetal, craniofacial, and placental dimensions, in addition to a higher
proportion of small fetuses for pregnancy age. Mild hyperglycemia during pregnancy did not
generate marked oxidative stress in the mother, and in fetal liver and placenta decreased
antioxidant activity was evident by significant consumption of reduced glutathione. Mild diabetes
led to a negative impact on maternal reproductive performance and characteristics of the off-
spring. This experimental model reproduced maternal and fetal outcomes of pregnant rats
presenting controlled diabetes.
Abbreviations: bw: body weight; sc: subcutaneous; DM: diabetes mellitus; STZ: streptozotocin;
OGTT: oral glucose tolerance test; ITT: insulin tolerance test; GSH: glutathione; MDA: malondial-
dehyde; AOPPs: advanced oxidation protein products; TBARs: thiobarbituric acid reaction; SPA:
small for pregancy age; APA: appropriate for pregnancy age; LPG: large for pregnancy age; ROS:
reactive oxygen species

Introduction Several epidemiological and experimental studies

have demonstrated that infants born from mothers
Diabetes mellitus (DM) is a disorder of carbohydrate,
with pregestational diabetes are more likely to have
fat, and protein metabolism, which is characterized
malformations than those born from mothers without
by absolute or relative lack of insulin, resulting in
diabetes [Baack et al. 2014; Han et al. 2015]. These
hyperglycemia [American-Diabetes-Association 2017;
malformations involve brain and neural tube defects,
Saini et al. 2013]. Diabetes in pregnancy is character-
spinal cord, caudal regression syndrome, congenital
ized by frequent disturbances in both development
heart defects and major vessel defects, skeletal dyspla-
and fetal growth and it is the principal cause of birth
sia, and anomalies in kidneys and gut [Baack et al.
defects and perinatal mortality. Mothers with dia-
2014; Han et al. 2015; Loeken 2008].
betes are more susceptible to miscarriages. Classic
The mechanisms of complications in diabetic preg-
consequences of diabetic pregnancy encompass
nancy remain largely unknown, although a number of
alterations in maternal reproductive performance,
hypotheses have been suggested during the last decade
changes in fetal growth, spontaneous abortions, and
based on clinical and experimental findings. It is recog-
perinatal death [Baack et al. 2014; Damasceno et al.
nized that good metabolic control of the maternal dis-
2014; Hajj et al. 2014].
ease is considered to diminish the risk of fetal

CONTACT Leticia Bequer leticiacbm@infomed.sld.cu Biomedical Research Center, Medical College of Villa Clara, B, No. 35A, e/2 y 3 Rpto, Virginia,
Santa Clara, Villa Clara, Cuba. CP: 50200.
© 2017 Assistance Publique-Hôpitaux de Paris
 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 61

anomalies. Therefore, hyperglycemia is hypothesized to Results

be the most important teratogen in addition to the
In the present study, diabetes was induced by STZ on
excesses of ketones, fatty acids, triglycerides, and vari-
the second day following the birth of 15 female pups,
ably glycerol [Baack et al. 2014; Han et al. 2015]. There
and 10 additional pups were inoculated with only
is strong evidence that hyperglycemia during gestation
citrate buffer as controls. All experimental adult rats
results in the generation of reactive oxygen species
were mated with non-diabetic males and only 11 dia-
(ROS), which induces a state of oxidative stress in the
betic adult rats (Diabetic group: DG) and 9 non-dia-
intrauterine milieu. This condition can induce damage
betic rats (Control group: CG) presented a positive
to the embryo, the placenta, the fetus, and the offspring
vaginal smear. The fertility rate was 73% in the DG
[Damasceno et al. 2014; Jawerbaum and White 2010].
and 90% in the CG. Throughout pregnancy, an increase
Because of logical limitations involving human preg-
in body weight in both groups was detected. However,
nancy studies, it is necessary to use animal models to
maternal weight gain in diabetic rats (94.18 ± 7.064 g)
research anomalies during early gestation, congenital
was significantly lower (p<0.05) compared with the
malformations, and placental and fetal alterations. The
control rats (121.03 ± 5.162 g). The blood glucose levels
method most often used to produce experimental dia-
during pregnancy are shown in Figure 1A. Maternal
betes is chemical induction by streptozotocin (STZ)
blood glucose levels in diabetic rats were higher com-
[Jawerbaum and White 2010]. Thus, we hypothesized
pared with non-diabetic rats at 0 and 7 days. However,
that a particular neonatal STZ-induced diabetic rat
at 14 and 20 days of pregnancy glucose values were
model would provoke a diabetogenic state that repli-
similar in both groups. The oral glucose tolerance test
cates the clinical representation of type 2 DM in
(OGTT) showed an increase in blood glucose levels for
humans. The aim of this study was to determine the
diabetic rats at 0, 30, 60, and 120 minutes compared
effect of mild hyperglycemia on metabolism during
with control rats (Figure 1B). The insulin tolerance test
pregnancy, the maternal reproductive performance,
(ITT) blood glucose levels were significantly higher in
and characteristics of the offspring in mild diabetic-
DG at two time points (0 and 30 minutes) compared
induced neonatal Wistar rats.
with the CG (Figure 1C).

Figure 1. Metabolic profile during pregnancy in experimental groups. (A) Glycemic levels during pregnancy: In the morning of 0, 7,
14, and 20 days of gestation the blood glucose levels (mmol/L) were measured to CG (n=9) and DG (n= 11) after fasting for 12 hours.
(B) Oral glucose tolerance test (OGTT) in experimental groups: At the seventh day of gestation, glucose solution (2 g glucose/kg) was
administered to CG (n=9) and DG (n= 11) after fasting for 12 hours. The glucose levels (mmol/L) were examined before glucose-
loading and immediately at 30, 60, and 120 minutes after glucose-loading. (C) Insulin tolerance test (ITT) in experimental groups: At
the fourteenth day of gestation, insulin solution (Rapid Insulin 3.33 U/mL) was inoculated subcutaneously in the dorsal area to CG
(n=9) and DG (n= 11) in non-fasting conditions. The glucose levels (mmol/L) were examined before insulin injection and
immediately at 30, 60 and 120 minutes after insulin injection. Values are expressed as Means ± SEM. Mann-Whitney test was
used to determine differences between groups. Superscripts (*) indicate significance differences (p<0.05). DG: Diabetic group, CG:
Control group.
62 L. BEQUER ET AL.

Serum biochemical and metabolic parameters from
mothers at gestation day 20 are shown in Table 1. The
serum concentrations of total cholesterol, triglycerides,
VLDL, creatinine, AST, and HbA1c presented a signif-
icant increase in the DG compared with the CG. The
metabolic parameters insulin, blood glucose, HOMA-
IR, and HOMA- cell β were not modified in DG respect
to CG due to the effect of mild hyperglycemia.
Maternal reproductive and offspring performance
Diabetic rats had a significantly reduced uterus weight,
litter size, number of implantation sites and live fetuses
at term, and efficiency of implantation compared with
control rats. The number of corpora lutea, dead fetuses,
malformed fetuses, and viability index did not show
differences between the experimental groups. The DG
showed a higher rate of reabsorption and percentage of
losses pre and post-implantation, compared with the
CG (Table 2).
We obtained 118 fetuses and placentas from 9 con-
trol rats and 111 from 11 diabetic rats. Differences in
size of the fetus from the diabetic mother compared
with the control were observed. The diabetic offspring
showed a significant reduction in fetal weight, fetal
dimensions (except diameter between the eyeballs),
and the weight and size of the placenta. The fetal
blood glucose levels did not differ between the experi-
mental groups (DG: 8.08 ± 0.420 mmo/L vs. CG: 7.53 ±
0.343 mmo/L) (Table 3).
There were no external anomalies in CG, except
generalized edema in three dead fetuses (2.7 % of 111
fetuses). Four fetuses from diabetic mothers showed
generalized edema coinciding with the dead fetuses
(3.39 % of 118 fetuses examined) and a single fetus
presenting gastroschisis (0.84%), acrania (0.84%), and
macroglossia (0.84%) (Figure 2A). All live fetuses had
harmonic axial symmetry evidenced by craniofacial
dimensions and external observation. As previously
noted, fetuses born from diabetic mothers had growth
retardation. Therefore, they showed significant

Table 1. Maternal serum biochemical and metabolic parameters determined at day 20 of gestation.
DG CG
Serum biochemical parameters Creatinine (μmol/L) 27.25 ± 2.152 27.62 ± 2.251
Total Cholesterol (mmol/L) 2.41 ± 0.079 * 1.90 ± 0.142
Triglycerides (mmol/L) 2.39 ± 0.347 * 1.47 ± 0.190
VLDL (mmol/L) 1.09 ± 0.157 * 0.66 ± 0.086
ALT (U/L) 63.75 ± 15.027 46.62 ± 6.849
AST (U/L) 87.66 ± 10.340 * 72.26 ± 7.539
Metabolic parameters Insulin (μU/mL) 10.74 ± 0.760 12.24 ± 0.921
Blood glucose (mmol/L) 5.27 ± 0.632 4.90 ± 0.277
HOMA-IR 2.62 ± 0.512 2.73 ± 0.308
HOMA-cell β 155.63 ± 24.320 176.50 ± 23.609
HbA1c (%) 4.82 ± 0.582 * 3.37 ± 0.175
Values are expressed as Means ± SEM. Mann-Whitney test was used to determine differences between groups. Superscripts (*) indicate significance
differences (p<0.05). DG: Diabetic group, CG: Control group.

Table 2. Maternal reproductive performance in experimental groups.

DG (n=11) CG (n=9)
Uterus Weigth (g) N 511.21 614.44
Means ± SEM 51.12 ± 6.243* 68.27 ± 3.839
Litter Size N 115 118
Means ± SEM 10.45 ± 1.245* 13.11 ± 0.715
Corpora Lutea N 156 130
Means ± SEM 14.18 ± 0.861 14.44 ± 0.851
Implantation Sites N 117 121
Means ± SEM 10.63 ± 1.259* 13.44 ± 0.668
Reabsorptions N 6 3
Means ± SEM 0.54 ± 0.365* 0.33 ± 0.166
Live Fetuses at term N 107 114
Means ± SEM 9.72 ± 1.199* 12.66 ± 0.726
Dead Fetuses N 4 4
Means ± SEM 0.36 ± 0.203 0.44 ± 0.175
Malformed Fetuses N 1 0
Means ± SEM 0.09 ± 0.090 0.00 ± 0.000
Efficiency of Implantation (%) 76.94 * 93.78
Pre-Implantation Loss (%) 23.05 * 6.21
Post-Implantation Loss (%) 7.63 * 5.97
Viability Index 0.92 0.94
Values are expressed as Means ± SEM. Mann-Whitney test was used to determine differences between groups. Superscripts (*) indicate significance
differences (p<0.05). DG: Diabetic group, CG: Control group.
 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 63

Table 3. Morphometric characteristics of the offspring of diabetic rats and controls.

Fetal and placental characteristics Group N Means ± SEM
Fetal dimensions Weight (g) DG 111 3.29 ± 0.043*
CG 118 3.64 ± 0.031
Crown-rump length (cm) DG 111 3.50 ± 0.019*
CG 118 3.67 ± 0.014
Tail length (cm) DG 91 1.24 ± 0.011*
CG 118 1.29 ± 0.010
Craniofacial dimensions Anteroposterior diameter (cm) DG 91 1.44 ± 0.011*
CG 118 1.49 ± 0.008
Biparietal diameter (cm) DG 91 0.84 ± 0.009*
CG 118 0.89 ± 0.007
Diameter between the eyeballs (cm) DG 91 0.54 ± 0.009
CG 118 0.56 ± 0.007
Diameter between nasal pore and pinna right (cm) DG 91 1.34 ± 0.010*
CG 118 1.38 ± 0.008
Diameter between nasal pore and pinna left (cm) DG 91 1.34 ± 0.010*
CG 118 1.38 ± 0.008
Placental dimensions Larger diameter (cm) DG 111 1.36 ± 0.013
CG 118 1.37 ± 0.010
Smaller diameter (cm) DG 111 1.16 ± 0.010*
CG 118 1.20 ± 0.008
Thickness (cm) DG 111 0.40 ± 0.009
CG 118 0.41 ± 0.007
Weight (g) DG 111 0.45 ± 0.009*
CG 118 0.47 ± 0.008
Placental Index DG 111 0.14 ± 0.003*
CG 118 0.13 ± 0.002
Values are expressed as Means ± SEM. Mann-Whitney test was used to determine differences between groups. Superscripts (*) indicate significance
differences (p<0.05). DG: Diabetic group, CG: Control group.

differences with the classification pattern established
according to the weight of the fetuses from CG. A
total of 91.50% of the offspring of healthy rats showed
fetuses with appropriate weight for pregnancy age
(APA). The remaining fetuses were equally distributed
in small for pregnancy age (SPA) and large for preg-
nancy age (LPA). The DG had a significant increase in
SPA fetuses, and decrement of APA and LPA fetuses
compared with CG (Figure 2B).
Oxidative stress status in mother and offspring
Oxidative stress determinations (antioxidant activity
and markers of damage to lipids and proteins) were
made in maternal liver, three samples of pooled fetal
livers, and placenta. In maternal and fetal liver from
DG, concentrations of glutathione (GSH), malondial-
dehyde (MDA), and advanced oxidation protein pro-
ducts (PAOP) showed no alteration. In placental tissue,
mild diabetes caused a decrease in GSH and modifica-
tions to protein damage (Figure 3). DNA fragmentation
products were observed in the livers of diabetic
mothers and were significantly higher than in the
liver of healthy mothers (DG: 5.67 ± 0.382 µM vs.
CG: 4.04 ± 0.612 µM, p<0.05).
Discussion
Pregestational DM is one of the most common com-
plications in pregnancy with serious consequences for
the mother and her offspring. In this study we estab-
lished an animal model to determine the effect of mild
hyperglycemia on metabolism during pregnancy, the
maternal reproductive performance, and characteristics
of the offspring.
The chemical induction with STZ in the neonatal
period to generate adult rats with mild hyperglycemia
has been used by several research teams with clinical
results similar to human diabetic syndrome [Bequer
et al. 2016; Bequer et al. 2014; Jawerbaum and White
2010; Kiss et al. 2013; Lukačínová et al. 2013; Srinivasan
and Ramarao 2007]. In this study, the CG and DG were
mated at day 90 and studied following confirmation of
pregnancy. In the mild hyperglycemic group, neonatal
STZ treatment created a range of damage to beta cells,
leading to a variable range of insulin insufficiency and
hyperglycemia in adulthood. For that reason, within the
first 7 days of pregnancy glycemia in diabetic rats was
higher than the controls. In the rest of the measure-
ments (days 14 and 20) blood glucose was similar to
that observed in healthy rats. Testing glucose tolerance
and insulin on day 7 and 14 of pregnancy yielded
abnormal results reproducing the clinical state of gesta-
tional diabetes that occurs in human gestation. Some
authors [Lessi et al. 2010; Sinzato et al. 2012] found
similar results in pregnant rats that were inoculated at
birth with a single dose of STZ. In the hyperglycemic
group, the glycemic control during pregnancy was
similar to that usually present during pregnancy in
women with well controlled diabetes. Usually women
64 L. BEQUER ET AL.

Figure 2. Morphological outcome in offspring of experimental group during pregnancy. (A) External anomalies in fetuses of diabetic
mothers: The fetuses of CG (n=118) and DG (n= 111) were examined macroscopically to identify external malformations. (B) Fetal
weight classification of the offspring of diabetic rats and controls: Fetuses were classified as small for pregnancy age (SPA) when
weight was less than percentile 5; appropriate for pregnancy age (APA) when weight was between percentiles 5 and 95; and large
for pregnancy age (LPA) when weight was greater than percentile 95. Chi Square Test was used to determine differences between
groups. Superscripts (*) indicate significance differences (p<0.05). DG: Diabetic group, CG: Control group.

with previously diagnosed type 2 DM reach the preg-
nancy with medical treatment and therefore with most
of the parameters within normal ranges.
Total cholesterol and triglycerides were quantified at
the end of the pregnancy and were significantly higher
in the DG group. Similar results were obtained for AST,
however ALT and creatinine were normal. Normal
pregnancy is an anabolic state in which significant
hormonal changes occur and is considered a
diabetogenic state or progressive resistance to insulin
effect due to changes in the pattern of insulin secretion
and sensitivity to its changes.
Analogous to the experimental model, human preg-
nancy increases serum triglycerides, however, cholesterol,
glycerol, and fatty acids also increase. These adjustments
in nutrient metabolism, in addition to changes in the
anatomy and physiology of the mother (distribution and
volume of adipose tissue), support fetal growth and
 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 65

Figure 3. Oxidative stress in experimental groups. Oxidative stress determinations were made in homogenized maternal liver (500
mg), three samples of pooled fetal livers (200 mg), and placenta (400 mg). Fetus selection was carried out randomly in a proportion
of approximately 50% depending on the total of the litter. (A) Advanced oxidation protein products (AOPPs). (B) Malondialdehyde
levels (MDA). (C) Reduced glutathione (GSH). Values are expressed as Means ± SEM. Mann-Whitney test was used to determine
differences between groups. Superscripts (*) indicate significance differences (p<0.05). DG: Diabetic group; CG: Control group.

development while maintaining maternal homeostasis in
preparation for lactation [King 2000]. The excretion of
the nitrogenous compounds (e.g., ammonia, creatinine,
and uric acid) might also increase during human preg-
nancy although serum creatinine was not modified in DG
compared with CG. Dyslipidemia and alterations in renal
serum profile are reported in studies in non pregnant rats
with moderate hyperglycemia [Gómez et al. 2014; Sinzato
et al. 2009].
The results of maternal performance of this study
corroborate the results of others [Damasceno et al.
2013; Sinzato et al. 2012; Spadotto et al. 2012] con-
firming that this experimental model is a useful tool
in the study of diabetes complicated pregnancy. The
reduction in the number of corpora lutea, parallels a
reduction in the number of oocytes liberated during
the ovulation. The hyperglycemic distribution of the
intrauterine milieu is coincident with a reduction in
mature fertilized, implanted oocytes and therefore
implantation sites, live fetuses at term, uterus weight,
and litter size in diabetic mothers as compared to a
healthy state. Due to these alterations there was a
significant increase in loss rates pre and post-
implantation in the DG. However, the embryos that
were implanted did successfully develop, as there
were no differences in the number of dead fetuses.
In the experiment, only one malformed fetus with
gastroschisis, acrania, and macroglossia was found. The
mother had the highest blood glucose value of the DG
at day 7 of pregnancy in addition to 8% of HbA1c at
the end of gestation. Our findings recapitulate prior
results in rodents and humans [Castori 2013;
Damasceno et al. 2012; Zhao and Reece 2013].
The consequences of diabetes during pregnancy are
not limited to birth defects. Restricted embryofetal
vitality in maternal human diabetes may have different
clinical presentations, including early spontaneous
abortion, late (i.e., second-trimester) spontaneous abor-
tion, and stillbirths/neonatal deaths [Castori 2013;
Soutelo and Faraj 2010]. In this experiment the DG
group showed a significant increase in the number of
reabsorptions compared with the CG group. As
expected, the higher rate of reabsorption and embryo-
nic death rate might have contributed to reducing the
uterus weight and litter size in DG. Similar results have
been reported in diabetic pregnancies in experimental
models [Damasceno et al. 2013; Sinzato et al. 2012;
Spadotto et al. 2012].
The mild hyperglycemia that occured in early preg-
nancy (mainly between days 7 and 9 of gestation) was
sufficient to develop changes in the stage of embryo-
genesis and cause embryonic death. However, in severe
diabetes, high hyperglycemia leads to inadequate devel-
opment of offspring, reapsortions, and embryonic and
fetal malformations [Damasceno et al. 2014; Jawerbaum
and White 2010]. All in vivo investigations in rodents
lend strong support to the fuel mediated teratogenesis
hypothesis, specifically glucose. Fecundity potential is
lower in DG, as shown that during approximately 3
estrus cycles fewer diabetic rats mating with healthy
66 L. BEQUER ET AL.
males had confirmed pregnancies. Studies of women
with diabetes showed reproductive abnormalities such
as delayed menarche and increased incidence of men-
strual cycle irregularities and delayed ovulation
[Jonasson et al. 2007].
The offspring of DG did not have significant
impaired glycemia compared with the CG, however,
there was an increased presence of small fetuses.
Morphometric measurements in all fetuses corroborate
the presence of fetuses with intrauterine growth restric-
tion (IUGR) because at gestation day 20, fetal and
placental dimensions decreased in diabetic compared
with healthy offspring. Our results are similar to those
found by others [Corvino et al. 2017; Saito et al. 2013;
Spadotto et al. 2012; Volpato et al. 2014] in models of
severe and mild diabetes, confirming this experimental
design can also be used for the study of offspring with
IUGR.
In human diabetic pregnancy, macrosomic fetuses
are common, but, in experimental models often fetuses
are obtained with growth restrictions [Volpato et al.
2014]. Fetal growth is a complex process that depends
on the genotype and epigenotype of the fetus, maternal
nutrition, the availability of nutrients and oxygen to the
fetus, intrauterine insults, and a variety of growth factor
and proteins of maternal, fetal, and placental origin.
When the maternal environment is perturbed during
pregnancy by oxidative stress, hypoxia, or hyperglyce-
mia, impaired fetal development will result in a
retarded fetal growth [Heshmat 2017]. Kervran and
colleagues [1978] when studying the offspring of rats
with mild hyperglycemia during pregnancy, suggested
that the differences between the clinical findings in
humans and the experimental results using rats were
due to the short pregnancy time in the rat and differ-
ences in the percentages of adipose tissue in rat fetuses
and human offspring and the greater weight gain in the
human species. In severe experimental diabetes, high
fetal frequency with IUGR and increased placental
weight is reported. This is interpreted as a compensa-
tory mechanism to maximize maternal–fetal exchange
and nutrient supply to the developing fetus [Volpato
et al. 2014]. Indeed, in most human pregnancies, pla-
cental weights are also elevated in cases of LGA births.
These data suggest that metabolic control in early preg-
nancy is an important determinant for fetoplacental
growth throughout gestation. As we have shown, the
maternal hyperglycemic milieu was not harmful
enough to alter the placental functional unit.
However, the placental indexes of the DG were affected
by the fetal and placental weights, confirming placental
incapacity as nutrition and oxygenation organ of the
fetus in development.
This study explored some aspects of redox status in
maternal and fetal liver, and placenta. In the literature
there are not many experiments that involve the study of
the oxidative status in 3 fundamental organs in mothers
and offspring at the same time. Previous reports docu-
mented elevated oxidative stress markers and disturbed
redox environment during diabetic gestation in both
maternal and fetal tissue [Lappas et al. 2011]. However,
in this study in the liver of the diabetic mother, a slight but
not significant trend to increased lipid peroxidation and
protein oxidation in addition to decreased GSH concen-
tration was observed. However, a significantly higher
levels of DNA fragmentation due to oxidation was noted.
It is known that the circulating levels of maternal
free radicals and anti-oxidants are altered in severe
diabetic pregnancies. Maternal measures of lipid perox-
idation, protein oxidation, and antioxidant activity in
serum and plasma are increased in diabetic pregnant
women compared with normal glucose tolerant preg-
nant women suggesting that oxidative stress is evident
in patients with poor glycemic control [El-Bassiouni
et al. 2016; Lappas et al. 2011].
In placenta and fetal liver in diabetic and control
offspring the concentration of GSH was significantly
decreased. The concentration of MDA was slightly
increased and the accumulation of AOPPs was reduced.
In a similar study, Kamel and colleagues [2014] found
that oxidative stress was prenatally associated with dis-
turbed redox status of the essential metabolic organs
(pancreas and liver) as indicated by depleted GSH and
increased GSSG as reflected in the decrease in the ratio
of GSH/GSSG. Moreover, in the placenta, similar
results were found indicating that in both organs oxi-
dative state manifests equally.
Structural and functional placental abnormalities are
present in diabetic pregnancies. These alterations are
evident both in the experimental models of chemical
diabetes and in human placentas [White et al. 2002].
Placenta is the major exchange surface between mother
and fetus and plays an essential role in fetal develop-
ment. It has several roles such as: secretion of hor-
mones, interface to allow exchange of metabolites,
oxygen, and nutrients between maternal and fetal cir-
culation, as well as modulating the maternal immune
system and fetal growth [Saad et al. 2016; Yates et al.
2017]. Studies in human and in animal models has
shown that placenta generated antioxidant enzymes
can be either up-regulated, to compensate the oxidative
dysbalance, or down-regulated, overwhelmed by the
increased ROS. These changes are dependent on the
developmental stage and are generated in response to
the gradual increase in ROS, which is more pronounced
at term [Lappas et al. 2011].

In the current study, the GSH was determined only,
therefore research is limited by absence of information
about the performance of antioxidant enzymes such as
SOD, catalase, and others. However, the GSH provides
data on the antioxidant defense that may be important
for understanding the mechanisms involved in main-
taining the oxidative balance.
In contrast to other studies, our study did not
show significant lipid peroxidation in the liver of
fetuses and diabetic mothers in association with pla-
centa. However, diabetic offspring show a higher
MDA concentration. White and colleagues [2002]
demonstrated that in the STZ-neonatal model placen-
tal lipoperoxidation seems to increase throughout
gestation in both control and STZ-induced diabetic
rats (the increase is higher in diabetic pregnancy).
The associated, increase in ROS levels, by a high
production of lipoperoxides in diabetic rats, is
favored by a diminished antioxidant capacity in
these animals. Other authors report similar changes
during diabetic pregnancy [El-Bassiouni et al. 2016].
The accumulation of plasma AOPPs is found in
human and experimental diabetes as well as other
conditions [Huang et al. 2013]. However, contrary
to expectations, we observed a reduced accumulation
of AOPPs in liver and placental samples from fetuses
of DG as compared to the CG. Previous studies have
noted the importance of ROS as intermediate com-
pounds with unpaired electrons, having, therefore, a
very short lifetime. Hence, they are not transferred to
the developing embryo or fetus. ROS is the conse-
quence of hyperglycemic intrauterine milieu
[Ericsson et al. 2007]. A probable explanation to the
significant consumption of GSH and the absence of
damage to lipids and proteins in fetal liver and in the
placenta of diabetic offspring is the action of GSH on
suitable substrates. The oxidative and degradative
actions of free radicals on macromolecules does not
allow the increase in oxidative stress.
The results obtained in this study suggest that the
neonatal STZ-induced diabetic rat model presents a less
severe non-insulin dependence that is compatible with
pregnancy in the absence of insulin treatment. This is
similar to type 2 diabetes with good glycemic control.
Perhaps the hyperglycemic milieu of exposed mothers,
fetuses, and placentas is not sufficient to generate
marked oxidative stress if the oxidative damage path-
ways are not completely activated. Biochemical studies
are required to resolve this issue and elucidate the
mechanisms involved.
In the current study, neonatal STZ-induced mild dia-
betic dams showed mild hyperglycemia, altered glucose
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 67
tolerance and increased total cholesterol, triglycerides,
and HbA1c during pregnancy. At day 20 of gestation,
the mild diabetic mothers and their offspring presented
impaired reproductive performance and fetal and pla-
cental dimensions, in addition to a higher proportion of
small for pregnancy age pups. Mild hyperglycemia dur-
ing pregnancy did not generate marked oxidative stress
in the mother. In fetal liver and placenta, the decreased
antioxidant activity is evident by significant consump-
tion of GSH. This experimental model reproduced
maternal and fetal outcomes of pregnant rats presenting
controlled diabetes and may prove useful to identify
mechanisms involved and to test new intervention stra-
tegies in human diabetic pregnancy.
Materials and methods
Animals and experimental design
The experimental design used in this study was approved
by the Committee on Ethics from Biomedical Research
Center of Medical College in Villa Clara, Cuba, and
followed the recommendations for animal experimenta-
tion of the National Institutes of Health.
Female offspring from rats suitable for reproduc-
tion, obtained from the Center for Laboratory
Animal Production (CENPALAB) in Cuba, were ran-
domly distributed into two experimental groups.
Mild experimental diabetes was induced in 15 pups
2 days after birth by subcutaneous injection of 100
mg/kg of body weight of STZ dissolved in 0.1 M
citrate buffer (pH4.5). The animals in the control
group received only citrate buffer in similar condi-
tions (n=10). All pups remained with their mothers
during the lactation period.
Nighty day-old diabetic and non-diabetic female rats
were mated with non-diabetic males during approxi-
mately 3 estral cycles (15 d). Non-mated female rats in
this period were considered infertile and excluded from
the experiment. The morning when spermatozoa were
found in the vaginal smear was designated gestational
day 0. Nonpregnant rats were excluded from the
experiment. The fertility in both groups was calculated
as 100 x number of rats with positive vaginal smear/
number of mated female rats. All pregnant female rats
were maintained in individual cages and the maternal
weight gain was measured during pregnancy. During
the study, the rats were maintained in an experimental
room under controlled conditions of temperature (22 ±
2◦C), humidity (40 ± 10%), and a 12-h light/dark cycle
with ad libitum access to commercial diet and tap
water.
68 L. BEQUER ET AL.
Rats at 20th day of gestation were anesthetized with
sodium thiopental (50 mg/kg) and exsanguinated for
collecting maternal blood for biochemistry determina-
tions. Then, laparotomy was executed to remove the
uterine horns and the ovaries for the morphometric
and biochemistry studies. Mother and fetal livers, and
placentas were utilized in oxidative stress assays.
Biochemical profile determination
In the morning of 0, 7, 14, and 20 d of pregnancy, after
12 h of fasting, the blood glucose levels were measured
in all experimental animals. OGTT and ITT were per-
formed in both groups in the seventh and fourteen day
of pregnancy, respectively, according to the modified
methodology of Kiss et al. [2012]. In all cases, the
glucose measurements were performed using a gluc-
ometer and biosensors (SUMA) and blood samples
were obtained from a cut tip tail.
At day 20 of gestation, mother´s serum was imme-
diately separated after blood collected in anticoagulant-
free test tubes by centrifugation at 3,000 rpm for 20
min at 4°C, and kept at -40°C until used in analysis of
lipid, renal, and hepatic profile. Serum concentrations
of total cholesterol, triglycerides, very low-density lipo-
protein cholesterol (VLDL), creatinine, ALT, and AST
were determined by Clinical Chemistry Analyzer
(HITACHI) with HELFA-Diagnostics kits. Whole
blood was used to perform the hemoglobin A1c
(HbA1c) measurement, a quantitative turbidimetric
test for the quantification of glycated hemoglobin,
which is measured by absorption at 600nm using latex
agglutination inhibition rate assay (CPM-Scientifica-
Tecnologie-Biomediche, Rome, Italy).
Insulin concentration in serum was determined by
immunoradiometric assay (IRMA kit Insulin RK-
400CT, in Radiometer SRN1C-02). Insulin resistance
and B cell function indexes from glucose/insulin pair
were assessed. The homeostasis model determination
Homeostasis-Model-Assessment (HOMA) was used
according to Matthews et al. [1985] in humans and
validated by Cacho et al. [2008] in different species of
rats.
Maternal reproductive and offspring performance
During laparotomy, the uterine horns were removed for
the morphometric analysis of the litter. The ovaries were
also removed to quantify the corpora lutea in stereomicro-
scope. Besides the number of implantation sites, resorp-
tions, and live and dead fetuses, fetal malformations were
counted to calculate the litter size, the rate of efficiency of
implantation, pre-implantation loss, post-implantation
loss, and viability index according to Saito et al. [2010].
The morphometric analysis included sexing and fetal
dimensions (weight, measurements of crown-rump,
and tail length), craniofacial dimensions (presence of
bones of the skull, biparietal diameter, anteroposterior
diameter, diameter between the eyeballs, diameter
between nasal pore, and right and left pinna), and
placental dimensions (larger and smaller diameter,
thickness, and weight). The placental index was calcu-
lated as placental weight/fetal weight. External malfor-
mations of the fetuses were recorded with a camera
(Cannon) for posterior macroscopic study.
The macroscopic study of craniofacial structures
included the existence and type of facial clefts (maxillary,
mandibular, or both), presence of exophthalmos on each
side, the absence of whiskers on each side, normal
implantation or low in both ears, malformations of the
pinna (anotia, microtia, or dysmorphia), presence and
position of facial appendages and nasal deformities
(depressed nasal bridge, increased hump, anteverted
nares), and presence and position of structures in oral
cavity (lips, tongue, and palatine). The external analysis
also included the observation of anterior and posterior
extremities (absence or excess fingers, position and size
of limbs), thoracic region, abdominal and dorsal (pre-
sence of bleeding, hematoma, or defect of neural tube
closure), and tail (size and shape).
Fetal blood glucose was measured immediately after
fetuses were removed from the uterine horns. Blood
drop for quantification was obtained from a small
puncture in the femoral vein of the fetus, for which
glucometer and biosensors (SUMA) were used. The
fetuses were classified according to the percentile values
of fetal weights of the CG as small (SPA), appropriate
(APA), and large for pregnancy age (LPG) based on the
modified methodology of Soulimane-Moktari and col-
leagues [Soulimane-Moktari et al. 2005].
Oxidative stress status in mother and offspring
Oxidative stress determinations were made in 500 mg
of maternal liver, 200 mg of 3 samples of pooled fetal
livers, and 400 mg of placenta. Fetuse selection for this
study was carried out randomly in a proportion of
approximately 50% depending on the total of the litter.
Tissue samples were homogenized and supernatants
were collected and stored at -40°C until used for the
determinations of reduced GSH, MDA levels, and
AOPPs according to Gómez et al. [2014]. The DNA
from of precipitate maternal liver homogenate was
extracted following the methodology of Bounce. The
fragmentation products of deoxyribose present in the
DNA solution was determined by the thiobarbituric
acid reaction (TBARs) [Rashid et al. 1999].

Statistical analysis
The results were presented as mean and standard error
of the mean (SEM) and the comparisons between
groups were performed using Mann-Whitney test.
The chi-square test was used for comparison of propor-
tions. Statistical significance was considered as p<0.05.
Acknowledgments
The authors are grateful to Orelvis Portal (Departamento de
Biología, Universidad Central “Marta Abreu” de Las Villas,
Cuba) and Yaqueline Cárdenas (Departamento de Ingles,
Universidad de Ciencias Médicas de Villa Clara, Cuba) for
critical comments on the manuscript.
Declaration of interest
This research did not receive any specific grant from any
funding agency in the public, commercial, or not-for-profit
sector. The authors declare that there is no conflicts of inter-
est that could be perceived as prejudicing to the impartiality
of the research reported.
Notes on contributors
Designed the study, performed the experiments, analyzed the
data, and wrote the paper: LB, TG; Performed experiments
and procedures with experimental animals. Analyzed the data
and critical review of the manuscript: JLM, AA; Performed
biochemical determinations, analyzed the data and critical
review of the manuscript: CC; Conception and design of
research study and critical review of the manuscript: SC.
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