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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 We obtained 118 fetuses and placentas from 9 con-
mothers at gestation day 20 are shown in Table 1. The trol rats and 111 from 11 diabetic rats. Differences in
serum concentrations of total cholesterol, triglycerides, size of the fetus from the diabetic mother compared
VLDL, creatinine, AST, and HbA1c presented a signif- with the control were observed. The diabetic offspring
icant increase in the DG compared with the CG. The showed a significant reduction in fetal weight, fetal
metabolic parameters insulin, blood glucose, HOMA- dimensions (except diameter between the eyeballs),
IR, and HOMA- cell β were not modified in DG respect and the weight and size of the placenta. The fetal
to CG due to the effect of mild hyperglycemia. 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
Maternal reproductive and offspring performance
generalized edema in three dead fetuses (2.7 % of 111
Diabetic rats had a significantly reduced uterus weight, fetuses). Four fetuses from diabetic mothers showed
litter size, number of implantation sites and live fetuses generalized edema coinciding with the dead fetuses
at term, and efficiency of implantation compared with (3.39 % of 118 fetuses examined) and a single fetus
control rats. The number of corpora lutea, dead fetuses, presenting gastroschisis (0.84%), acrania (0.84%), and
malformed fetuses, and viability index did not show macroglossia (0.84%) (Figure 2A). All live fetuses had
differences between the experimental groups. The DG harmonic axial symmetry evidenced by craniofacial
showed a higher rate of reabsorption and percentage of dimensions and external observation. As previously
losses pre and post-implantation, compared with the noted, fetuses born from diabetic mothers had growth
CG (Table 2). 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 the mother and her offspring. In this study we estab-
according to the weight of the fetuses from CG. A lished an animal model to determine the effect of mild
total of 91.50% of the offspring of healthy rats showed hyperglycemia on metabolism during pregnancy, the
fetuses with appropriate weight for pregnancy age maternal reproductive performance, and characteristics
(APA). The remaining fetuses were equally distributed of the offspring.
in small for pregnancy age (SPA) and large for preg- The chemical induction with STZ in the neonatal
nancy age (LPA). The DG had a significant increase in period to generate adult rats with mild hyperglycemia
SPA fetuses, and decrement of APA and LPA fetuses has been used by several research teams with clinical
compared with CG (Figure 2B). 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
Oxidative stress status in mother and offspring
and Ramarao 2007]. In this study, the CG and DG were
Oxidative stress determinations (antioxidant activity mated at day 90 and studied following confirmation of
and markers of damage to lipids and proteins) were pregnancy. In the mild hyperglycemic group, neonatal
made in maternal liver, three samples of pooled fetal STZ treatment created a range of damage to beta cells,
livers, and placenta. In maternal and fetal liver from leading to a variable range of insulin insufficiency and
DG, concentrations of glutathione (GSH), malondial- hyperglycemia in adulthood. For that reason, within the
dehyde (MDA), and advanced oxidation protein pro- first 7 days of pregnancy glycemia in diabetic rats was
ducts (PAOP) showed no alteration. In placental tissue, higher than the controls. In the rest of the measure-
mild diabetes caused a decrease in GSH and modifica- ments (days 14 and 20) blood glucose was similar to
tions to protein damage (Figure 3). DNA fragmentation that observed in healthy rats. Testing glucose tolerance
products were observed in the livers of diabetic and insulin on day 7 and 14 of pregnancy yielded
mothers and were significantly higher than in the abnormal results reproducing the clinical state of gesta-
liver of healthy mothers (DG: 5.67 ± 0.382 µM vs. tional diabetes that occurs in human gestation. Some
CG: 4.04 ± 0.612 µM, p<0.05). 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
Discussion
group, the glycemic control during pregnancy was
Pregestational DM is one of the most common com- similar to that usually present during pregnancy in
plications in pregnancy with serious consequences for 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- diabetogenic state or progressive resistance to insulin
nancy with medical treatment and therefore with most effect due to changes in the pattern of insulin secretion
of the parameters within normal ranges. and sensitivity to its changes.
Total cholesterol and triglycerides were quantified at Analogous to the experimental model, human preg-
the end of the pregnancy and were significantly higher nancy increases serum triglycerides, however, cholesterol,
in the DG group. Similar results were obtained for AST, glycerol, and fatty acids also increase. These adjustments
however ALT and creatinine were normal. Normal in nutrient metabolism, in addition to changes in the
pregnancy is an anabolic state in which significant anatomy and physiology of the mother (distribution and
hormonal changes occur and is considered a 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 the end of gestation. Our findings recapitulate prior
preparation for lactation [King 2000]. The excretion of results in rodents and humans [Castori 2013;
the nitrogenous compounds (e.g., ammonia, creatinine, Damasceno et al. 2012; Zhao and Reece 2013].
and uric acid) might also increase during human preg- The consequences of diabetes during pregnancy are
nancy although serum creatinine was not modified in DG not limited to birth defects. Restricted embryofetal
compared with CG. Dyslipidemia and alterations in renal vitality in maternal human diabetes may have different
serum profile are reported in studies in non pregnant rats clinical presentations, including early spontaneous
with moderate hyperglycemia [Gómez et al. 2014; Sinzato abortion, late (i.e., second-trimester) spontaneous abor-
et al. 2009]. tion, and stillbirths/neonatal deaths [Castori 2013;
The results of maternal performance of this study Soutelo and Faraj 2010]. In this experiment the DG
corroborate the results of others [Damasceno et al. group showed a significant increase in the number of
2013; Sinzato et al. 2012; Spadotto et al. 2012] con- reabsorptions compared with the CG group. As
firming that this experimental model is a useful tool expected, the higher rate of reabsorption and embryo-
in the study of diabetes complicated pregnancy. The nic death rate might have contributed to reducing the
reduction in the number of corpora lutea, parallels a uterus weight and litter size in DG. Similar results have
reduction in the number of oocytes liberated during been reported in diabetic pregnancies in experimental
the ovulation. The hyperglycemic distribution of the models [Damasceno et al. 2013; Sinzato et al. 2012;
intrauterine milieu is coincident with a reduction in Spadotto et al. 2012].
mature fertilized, implanted oocytes and therefore The mild hyperglycemia that occured in early preg-
implantation sites, live fetuses at term, uterus weight, nancy (mainly between days 7 and 9 of gestation) was
and litter size in diabetic mothers as compared to a sufficient to develop changes in the stage of embryo-
healthy state. Due to these alterations there was a genesis and cause embryonic death. However, in severe
significant increase in loss rates pre and post- diabetes, high hyperglycemia leads to inadequate devel-
implantation in the DG. However, the embryos that opment of offspring, reapsortions, and embryonic and
were implanted did successfully develop, as there fetal malformations [Damasceno et al. 2014; Jawerbaum
were no differences in the number of dead fetuses. and White 2010]. All in vivo investigations in rodents
In the experiment, only one malformed fetus with lend strong support to the fuel mediated teratogenesis
gastroschisis, acrania, and macroglossia was found. The hypothesis, specifically glucose. Fecundity potential is
mother had the highest blood glucose value of the DG lower in DG, as shown that during approximately 3
at day 7 of pregnancy in addition to 8% of HbA1c at estrus cycles fewer diabetic rats mating with healthy
66 L. BEQUER ET AL.

males had confirmed pregnancies. Studies of women This study explored some aspects of redox status in
with diabetes showed reproductive abnormalities such maternal and fetal liver, and placenta. In the literature
as delayed menarche and increased incidence of men- there are not many experiments that involve the study of
strual cycle irregularities and delayed ovulation the oxidative status in 3 fundamental organs in mothers
[Jonasson et al. 2007]. and offspring at the same time. Previous reports docu-
The offspring of DG did not have significant mented elevated oxidative stress markers and disturbed
impaired glycemia compared with the CG, however, redox environment during diabetic gestation in both
there was an increased presence of small fetuses. maternal and fetal tissue [Lappas et al. 2011]. However,
Morphometric measurements in all fetuses corroborate in this study in the liver of the diabetic mother, a slight but
the presence of fetuses with intrauterine growth restric- not significant trend to increased lipid peroxidation and
tion (IUGR) because at gestation day 20, fetal and protein oxidation in addition to decreased GSH concen-
placental dimensions decreased in diabetic compared tration was observed. However, a significantly higher
with healthy offspring. Our results are similar to those levels of DNA fragmentation due to oxidation was noted.
found by others [Corvino et al. 2017; Saito et al. 2013; It is known that the circulating levels of maternal
Spadotto et al. 2012; Volpato et al. 2014] in models of free radicals and anti-oxidants are altered in severe
severe and mild diabetes, confirming this experimental diabetic pregnancies. Maternal measures of lipid perox-
design can also be used for the study of offspring with idation, protein oxidation, and antioxidant activity in
IUGR. serum and plasma are increased in diabetic pregnant
In human diabetic pregnancy, macrosomic fetuses women compared with normal glucose tolerant preg-
are common, but, in experimental models often fetuses nant women suggesting that oxidative stress is evident
are obtained with growth restrictions [Volpato et al. in patients with poor glycemic control [El-Bassiouni
2014]. Fetal growth is a complex process that depends et al. 2016; Lappas et al. 2011].
on the genotype and epigenotype of the fetus, maternal In placenta and fetal liver in diabetic and control
nutrition, the availability of nutrients and oxygen to the offspring the concentration of GSH was significantly
fetus, intrauterine insults, and a variety of growth factor decreased. The concentration of MDA was slightly
and proteins of maternal, fetal, and placental origin. increased and the accumulation of AOPPs was reduced.
When the maternal environment is perturbed during In a similar study, Kamel and colleagues [2014] found
pregnancy by oxidative stress, hypoxia, or hyperglyce- that oxidative stress was prenatally associated with dis-
mia, impaired fetal development will result in a turbed redox status of the essential metabolic organs
retarded fetal growth [Heshmat 2017]. Kervran and (pancreas and liver) as indicated by depleted GSH and
colleagues [1978] when studying the offspring of rats increased GSSG as reflected in the decrease in the ratio
with mild hyperglycemia during pregnancy, suggested of GSH/GSSG. Moreover, in the placenta, similar
that the differences between the clinical findings in results were found indicating that in both organs oxi-
humans and the experimental results using rats were dative state manifests equally.
due to the short pregnancy time in the rat and differ- Structural and functional placental abnormalities are
ences in the percentages of adipose tissue in rat fetuses present in diabetic pregnancies. These alterations are
and human offspring and the greater weight gain in the evident both in the experimental models of chemical
human species. In severe experimental diabetes, high diabetes and in human placentas [White et al. 2002].
fetal frequency with IUGR and increased placental Placenta is the major exchange surface between mother
weight is reported. This is interpreted as a compensa- and fetus and plays an essential role in fetal develop-
tory mechanism to maximize maternal–fetal exchange ment. It has several roles such as: secretion of hor-
and nutrient supply to the developing fetus [Volpato mones, interface to allow exchange of metabolites,
et al. 2014]. Indeed, in most human pregnancies, pla- oxygen, and nutrients between maternal and fetal cir-
cental weights are also elevated in cases of LGA births. culation, as well as modulating the maternal immune
These data suggest that metabolic control in early preg- system and fetal growth [Saad et al. 2016; Yates et al.
nancy is an important determinant for fetoplacental 2017]. Studies in human and in animal models has
growth throughout gestation. As we have shown, the shown that placenta generated antioxidant enzymes
maternal hyperglycemic milieu was not harmful can be either up-regulated, to compensate the oxidative
enough to alter the placental functional unit. dysbalance, or down-regulated, overwhelmed by the
However, the placental indexes of the DG were affected increased ROS. These changes are dependent on the
by the fetal and placental weights, confirming placental developmental stage and are generated in response to
incapacity as nutrition and oxygenation organ of the the gradual increase in ROS, which is more pronounced
fetus in development. at term [Lappas et al. 2011].
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 67

In the current study, the GSH was determined only, tolerance and increased total cholesterol, triglycerides,
therefore research is limited by absence of information and HbA1c during pregnancy. At day 20 of gestation,
about the performance of antioxidant enzymes such as the mild diabetic mothers and their offspring presented
SOD, catalase, and others. However, the GSH provides impaired reproductive performance and fetal and pla-
data on the antioxidant defense that may be important cental dimensions, in addition to a higher proportion of
for understanding the mechanisms involved in main- small for pregnancy age pups. Mild hyperglycemia dur-
taining the oxidative balance. ing pregnancy did not generate marked oxidative stress
In contrast to other studies, our study did not in the mother. In fetal liver and placenta, the decreased
show significant lipid peroxidation in the liver of antioxidant activity is evident by significant consump-
fetuses and diabetic mothers in association with pla- tion of GSH. This experimental model reproduced
centa. However, diabetic offspring show a higher maternal and fetal outcomes of pregnant rats presenting
MDA concentration. White and colleagues [2002] controlled diabetes and may prove useful to identify
demonstrated that in the STZ-neonatal model placen- mechanisms involved and to test new intervention stra-
tal lipoperoxidation seems to increase throughout tegies in human diabetic pregnancy.
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 Materials and methods
production of lipoperoxides in diabetic rats, is
Animals and experimental design
favored by a diminished antioxidant capacity in
these animals. Other authors report similar changes The experimental design used in this study was approved
during diabetic pregnancy [El-Bassiouni et al. 2016]. by the Committee on Ethics from Biomedical Research
The accumulation of plasma AOPPs is found in Center of Medical College in Villa Clara, Cuba, and
human and experimental diabetes as well as other followed the recommendations for animal experimenta-
conditions [Huang et al. 2013]. However, contrary tion of the National Institutes of Health.
to expectations, we observed a reduced accumulation Female offspring from rats suitable for reproduc-
of AOPPs in liver and placental samples from fetuses tion, obtained from the Center for Laboratory
of DG as compared to the CG. Previous studies have Animal Production (CENPALAB) in Cuba, were ran-
noted the importance of ROS as intermediate com- domly distributed into two experimental groups.
pounds with unpaired electrons, having, therefore, a Mild experimental diabetes was induced in 15 pups
very short lifetime. Hence, they are not transferred to 2 days after birth by subcutaneous injection of 100
the developing embryo or fetus. ROS is the conse- mg/kg of body weight of STZ dissolved in 0.1 M
quence of hyperglycemic intrauterine milieu citrate buffer (pH4.5). The animals in the control
[Ericsson et al. 2007]. A probable explanation to the group received only citrate buffer in similar condi-
significant consumption of GSH and the absence of tions (n=10). All pups remained with their mothers
damage to lipids and proteins in fetal liver and in the during the lactation period.
placenta of diabetic offspring is the action of GSH on Nighty day-old diabetic and non-diabetic female rats
suitable substrates. The oxidative and degradative were mated with non-diabetic males during approxi-
actions of free radicals on macromolecules does not mately 3 estral cycles (15 d). Non-mated female rats in
allow the increase in oxidative stress. this period were considered infertile and excluded from
The results obtained in this study suggest that the the experiment. The morning when spermatozoa were
neonatal STZ-induced diabetic rat model presents a less found in the vaginal smear was designated gestational
severe non-insulin dependence that is compatible with day 0. Nonpregnant rats were excluded from the
pregnancy in the absence of insulin treatment. This is experiment. The fertility in both groups was calculated
similar to type 2 diabetes with good glycemic control. as 100 x number of rats with positive vaginal smear/
Perhaps the hyperglycemic milieu of exposed mothers, number of mated female rats. All pregnant female rats
fetuses, and placentas is not sufficient to generate were maintained in individual cages and the maternal
marked oxidative stress if the oxidative damage path- weight gain was measured during pregnancy. During
ways are not completely activated. Biochemical studies the study, the rats were maintained in an experimental
are required to resolve this issue and elucidate the room under controlled conditions of temperature (22 ±
mechanisms involved. 2◦C), humidity (40 ± 10%), and a 12-h light/dark cycle
In the current study, neonatal STZ-induced mild dia- with ad libitum access to commercial diet and tap
betic dams showed mild hyperglycemia, altered glucose water.
68 L. BEQUER ET AL.

Rats at 20th day of gestation were anesthetized with The morphometric analysis included sexing and fetal
sodium thiopental (50 mg/kg) and exsanguinated for dimensions (weight, measurements of crown-rump,
collecting maternal blood for biochemistry determina- and tail length), craniofacial dimensions (presence of
tions. Then, laparotomy was executed to remove the bones of the skull, biparietal diameter, anteroposterior
uterine horns and the ovaries for the morphometric diameter, diameter between the eyeballs, diameter
and biochemistry studies. Mother and fetal livers, and between nasal pore, and right and left pinna), and
placentas were utilized in oxidative stress assays. placental dimensions (larger and smaller diameter,
thickness, and weight). The placental index was calcu-
lated as placental weight/fetal weight. External malfor-
Biochemical profile determination
mations of the fetuses were recorded with a camera
In the morning of 0, 7, 14, and 20 d of pregnancy, after (Cannon) for posterior macroscopic study.
12 h of fasting, the blood glucose levels were measured The macroscopic study of craniofacial structures
in all experimental animals. OGTT and ITT were per- included the existence and type of facial clefts (maxillary,
formed in both groups in the seventh and fourteen day mandibular, or both), presence of exophthalmos on each
of pregnancy, respectively, according to the modified side, the absence of whiskers on each side, normal
methodology of Kiss et al. [2012]. In all cases, the implantation or low in both ears, malformations of the
glucose measurements were performed using a gluc- pinna (anotia, microtia, or dysmorphia), presence and
ometer and biosensors (SUMA) and blood samples position of facial appendages and nasal deformities
were obtained from a cut tip tail. (depressed nasal bridge, increased hump, anteverted
At day 20 of gestation, mother´s serum was imme- nares), and presence and position of structures in oral
diately separated after blood collected in anticoagulant- cavity (lips, tongue, and palatine). The external analysis
free test tubes by centrifugation at 3,000 rpm for 20 also included the observation of anterior and posterior
min at 4°C, and kept at -40°C until used in analysis of extremities (absence or excess fingers, position and size
lipid, renal, and hepatic profile. Serum concentrations of limbs), thoracic region, abdominal and dorsal (pre-
of total cholesterol, triglycerides, very low-density lipo- sence of bleeding, hematoma, or defect of neural tube
protein cholesterol (VLDL), creatinine, ALT, and AST closure), and tail (size and shape).
were determined by Clinical Chemistry Analyzer Fetal blood glucose was measured immediately after
(HITACHI) with HELFA-Diagnostics kits. Whole fetuses were removed from the uterine horns. Blood
blood was used to perform the hemoglobin A1c drop for quantification was obtained from a small
(HbA1c) measurement, a quantitative turbidimetric puncture in the femoral vein of the fetus, for which
test for the quantification of glycated hemoglobin, glucometer and biosensors (SUMA) were used. The
which is measured by absorption at 600nm using latex fetuses were classified according to the percentile values
agglutination inhibition rate assay (CPM-Scientifica- of fetal weights of the CG as small (SPA), appropriate
Tecnologie-Biomediche, Rome, Italy). (APA), and large for pregnancy age (LPG) based on the
Insulin concentration in serum was determined by modified methodology of Soulimane-Moktari and col-
immunoradiometric assay (IRMA kit Insulin RK- leagues [Soulimane-Moktari et al. 2005].
400CT, in Radiometer SRN1C-02). Insulin resistance
and B cell function indexes from glucose/insulin pair
Oxidative stress status in mother and offspring
were assessed. The homeostasis model determination
Homeostasis-Model-Assessment (HOMA) was used Oxidative stress determinations were made in 500 mg
according to Matthews et al. [1985] in humans and of maternal liver, 200 mg of 3 samples of pooled fetal
validated by Cacho et al. [2008] in different species of livers, and 400 mg of placenta. Fetuse selection for this
rats. study was carried out randomly in a proportion of
approximately 50% depending on the total of the litter.
Maternal reproductive and offspring performance Tissue samples were homogenized and supernatants
During laparotomy, the uterine horns were removed for were collected and stored at -40°C until used for the
the morphometric analysis of the litter. The ovaries were determinations of reduced GSH, MDA levels, and
also removed to quantify the corpora lutea in stereomicro- AOPPs according to Gómez et al. [2014]. The DNA
scope. Besides the number of implantation sites, resorp- from of precipitate maternal liver homogenate was
tions, and live and dead fetuses, fetal malformations were extracted following the methodology of Bounce. The
counted to calculate the litter size, the rate of efficiency of fragmentation products of deoxyribose present in the
implantation, pre-implantation loss, post-implantation DNA solution was determined by the thiobarbituric
loss, and viability index according to Saito et al. [2010]. acid reaction (TBARs) [Rashid et al. 1999].
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 69

Statistical analysis Corvino, S.B., Damasceno, D.C., Sinzato, Y.K., Netto, A.O.,
Macedo, N.C., Zambrano, E., et al. (2017) Comparative
The results were presented as mean and standard error analysis of two different models of swimming applied to
of the mean (SEM) and the comparisons between pregnant rats born small for pregnant age. An Acad Bras
groups were performed using Mann-Whitney test. Cienc 89: 223–230.
The chi-square test was used for comparison of propor- Damasceno, D.C., Netto, A.O., Iessi, I.L., Gallego, F.Q.,
Corvino, S.B., Dallaqua, B., et al. (2014) Streptozotocin-
tions. Statistical significance was considered as p<0.05.
induced diabetes models: Pathophysiological mechanisms
and fetal outcomes. BioMed Res-Intern: 1–11.
Damasceno, D.C., Pontes, H., Vaz, G.F., Vasques-Silva, F.A.,
Acknowledgments Mattos, I., Vieira, M., et al. (2012) Diabetic rats exercised
The authors are grateful to Orelvis Portal (Departamento de prior to and during pregnancy: Maternal reproductive out-
Biología, Universidad Central “Marta Abreu” de Las Villas, come, biochemical profile, and frequency of fetal anoma-
Cuba) and Yaqueline Cárdenas (Departamento de Ingles, lies. Reprod Sci 20: 1–9.
Universidad de Ciencias Médicas de Villa Clara, Cuba) for Damasceno, D.C., Sinzato, Y.K., Bueno, A., Netto, A.O.,
critical comments on the manuscript. Dallaqua, B., Gallego, F.Q., et al. (2013) Mild diabetes models
and their maternal-fetal repercussions. J Diabetes Res: 1–9.
El-Bassiouni, E.A., Helmy, M.H., Rawash, N., El-Zoghby, S.
Declaration of interest M., El-Nabi, M., Rayah, A. (2016) Embryopathy in experi-
mental diabetic gestation: assessment of oxidative stress
This research did not receive any specific grant from any and antioxidant defence. Brit J Biomed Sci 62: 71–76.
funding agency in the public, commercial, or not-for-profit Ericsson, A., Saljo, K., Sjostrand, E., Jansson, N., Prasad, P.,
sector. The authors declare that there is no conflicts of inter- Powell, T., et al. (2007) Brief hyperglycaemia in the early
est that could be perceived as prejudicing to the impartiality pregnant rat increases fetal weight at term by stimulating
of the research reported. placental growth and affecting placental nutrient transport.
J Physiol 581: 1323–1332.
Gómez, T., Bequer, L., Sánchez, C., de la Barca, M., Muro, I.,
Notes on contributors Reyes, M.A., et al. (2014) Inducción neonatal de hiperglu-
cemias moderadas: indicadores metabólicos y de estrés
Designed the study, performed the experiments, analyzed the oxidativo en ratas adultas. Rev ALAD 4: 148–157.
data, and wrote the paper: LB, TG; Performed experiments Hajj, N., Schneider, E., Lehnen, H., Haaf, T. (2014)
and procedures with experimental animals. Analyzed the data Epigenetics and life-long consequences of an adverse
and critical review of the manuscript: JLM, AA; Performed nutritional and diabetic intrauterine environment.
biochemical determinations, analyzed the data and critical Reproduction 148: 111–120.
review of the manuscript: CC; Conception and design of Han, S., Wang, G., Jin, Y., Ma, Z., Jia, W., Wu, X., et al.
research study and critical review of the manuscript: SC. (2015) Investigating the mechanism of hyperglycemia-
induced fetal cardiac hypertrophy. PLoS ONE 10: 1–19.
Heshmat, S.H. (2017) Intrauterine Growth Restriction-A
References Review Article. Anatomy Physiol Biochem Int J 1: 1–5.
Huang, Q.T., Wang, S.S., Zhang, M., Huang, L.P., Tian, J.W.,
American-Diabetes-Association (2017) Classification and
Yu, Y.H., et al. (2013) Advanced oxidation protein pro-
Diagnosis of Diabetes. Diabetes Care 40: 11–24.
ducts enhances soluble Fms-like tyrosine kinase 1 expres-
Baack, M.L., Wang, C., Hu, S., Segar, J.L., Norris, A.W.
sion in trophoblasts: A possible link between oxidative
(2014) Hyperglycemia induces embryopathy, even in the
stress and preeclampsia. Placenta 34: 949–952.
absence of systemic maternal diabetes: An in vivo test of
Jawerbaum, A., White, V. (2010) Animal models in diabetes
the fuel mediated teratogenesis hypothesis. Reprod Toxicol
and pregnancy. Endocr Rev 31: 680–701.
46: 129–136.
Jonasson, J.M., Brismar, K., Sparén, P., Lambe, M.L., Nyrén,
Bequer, L., Gómez, T., Molina, J.L., Artiles, D., Bermúdez, R.,
O., Ostenson, C., et al. (2007) Fertility in women with type
Clapés, S. (2016) Acción diabetogénica de la estreptozoto-
1 diabetes. Diabetes Care 30: 2271–2276
cina en un modelo experimental de inducción neonatal.
Kamel, M.A., Helmy, M.H., Hanafi, M.Y., Mahmoud, S.A.,
Biomédica 26: 230–238.
Elfetooh, H.A. (2014) Effect of maternal diabetes on pre-
Bequer, L., Gómez, T., Molina, J.L., López, F., Gómez, C.L.,
and post-natal redox status of F1 rat offspring. Open J
Clapés, S. (2014) Inducción de hiperglicemias moderadas
Endocr Metab Dis 4: 111–127.
en ratas wistar por inoculación neonatal de estreptozoto-
Kervran, A., Guillaume, M., Jost, A. (1978) The endocrine
cina. ¿Inyección subcutánea o intraperitoneal? Rev Argent
pancreas of the fetus from diabetic pregnant rat.
Endocrinol Metab 51: 178–184.
Diabetologia 15: 387–393.
Cacho, J., Sevillano, J., de Castro, J., Herrera, E., Ramos, M.P.
King, J.C. (2000) Physiology of pregnancy and nutrient meta-
(2008) Validation of simple indexes to assess insulin sen-
bolism. Am J Clin Nutr 71: 1218S-1225S.
sitivity during pregnancy in Wistar and Sprague-Dawley
Kiss, A.C., Woodside, B., Felício, L.F., Anselmo-Franci, J.,
rats. Am J Physiol Endocrinol Metab 295: E1269–E1276.
Damasceno, D.C. (2012) Impact of maternal mild hyper-
Castori, M. (2013) Diabetic embryopathy: A developmental
glycemia on maternal care and offspring development and
perspective from fertilization to adulthood. Mol
behavior of Wistar rats. Physiol Behav 107: 292–300.
Syndromol 4: 74–86.
70 L. BEQUER ET AL.

Kiss, A.C., Woodside, B., Sinzato, Y.K., Bernardi, M.M., Repercussions of mild diabetes on pregnancy in
Kempinas, W.G., Anselmo-Franci, J.A., et al. (2013) Wistar rats and on the fetal development. Diabetol
Neonatally induced mild diabetes: influence on develop- Metab Syndr 2: 20–26.
ment, behavior and reproductive function of female Wistar Sinzato, Y.K., Lima, P.H., de Campos, K.E., Kiss, A.C., Rudge,
rats. Diabetol Metab Syndr 5: 2–10. M.V., Damasceno, D.C. (2009) Neonatally-induced dia-
Lappas, M., Hiden, U., Desoye, G., Froehlich, J., Hauguel-de betes: lipid profile outcomes and oxidative stress status in
Mouzon, S., Jawerbaum, A. (2011) The role of oxidative adult rats. Rev Assoc Med Bras 55: 384–388.
stress in the pathophysiology of gestational diabetes melli- Sinzato, Y.K., Volpato, G.T., Lessi, I.L., Bueno, A.,
tus. Antioxid Redox Signal 15: 3061–3100. Calderon Ide, M., Rudge, M.V., et al. (2012)
Lessi, I.L., Bueno, A., Sinzato, Y.K., Taylor, K.N., Rudge, M. Neonatally induced mild diabetes in rats and its effect
V., Damasceno, D.C. (2010) Evaluation of neonatally- on maternal, placental, and fetal parameters. Exp
induced mild diabetes in rats: Maternal and fetal repercus- Diabetes Res: 1–7.
sions. Diabetol Metab Syndr 2: 31–37. Soulimane-Moktari, N., Guermouche, B., Yessoufou, A.,
Loeken, M.R. (2008) Challenges in understanding diabetic Saker, M., Moutairou, K., Hichami, A., et al. (2005)
embryopathy. Diabetes 57: 3187–3188. Modulation of lipid metabolism by n−3 polyunsaturated
Lukačínová, A., Hubková, B., Rácz, O., Ništiar, F. (2013) Animal fatty acids in gestational diabetic rats and their macroso-
models for study of diabetes mellitus. In Diabetes Mellitus – mic offspring. Clinical Science 109: 287–295.
Insights and Perspectives ed Oguntibeju, O.O. In Tech, Soutelo, M.J., Faraj, G. (2010) Aborto recurrente y diabetes.
Croatia pp 229–253. Rev SAEGRE XVII 27–35.
Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Spadotto, R., Damasceno, D.C., Godinho, A.F., Porto, E.M.,
Treacher, D.F., Turner, R.C. (1985) Homeostasis model assess- Perobelli, J.E., De Grava, W. (2012) Reproductive physiol-
ment: insulin resistance and beta-cell function from fasting ogy, and physical and sexual development of female off-
plasma glucose and insulin concentrations in man. spring born to diabetic dams. Arq Bras Endocrinol Metab
Diabetologia 28: 412–419. 56: 96–103.
Rashid, R., Langfinger, D., Wagner, R., Schuchmann, Von Srinivasan, K., Ramarao, P. (2007) Animal models in type 2
Sonntag, C. (1999) Bleomycin versus OH-radical-induced mal- diabetes research: An overview. Indian J Med Res 125:
onaldehydic-product formation in DNA. Int J Radiat Biol 75: 451–472.
101–109. Volpato, G.T., Damasceno, D.C., Sinzato, Y.K., Ribeiro, V.M.,
Saad, M.I., Abdelkhalek, T.M., Saleh, M.T., Haiba, M.M., Rudge, M.V., Calderon, I.M. (2014) Oxidative stress status
Tawfik, S.H., Kamel, M.A. (2016) Maternal diabetes and placental implications in diabetic rats undergoing
impairs oxidative and inflammatory response in murine swimming exercise after embryonic implantation. Reprod
placenta. SpringerPlus 5: 532–541. Sci 22: 602–608
Saini, S., Kumari, S., Verma, S.K., Sharma, A.K. (2013) A White, V., Jawerbaum, A., Sinner, D., Pustovrh, C.,
review on different types of animal models for pharmaco- Capobianco, E., González, E. (2002) Oxidative stress
logical evaluation of antidiabetic drugs. Int J Pharm and altered prostanoid production in the placenta of
Phytopharmacol Res 3: 2–12. streptozotocin-induced diabetic rats. Reprod Fertil Dev
Saito, F.H., Damasceno, D.C., Dallaqua, B., Moreno, I., 14: 117–123.
Vieira, M., De Mattos, I., et al. (2013) Heat shock protein Yates, N., Crew, R.C., Wyrwoll, C.S. (2017) Vitamin D defi-
production and immunity and altered fetal development in ciency and impaired placental function: potential regula-
diabetic pregnant rats. Cell Stress Chaperon 18: 25–33. tion by glucocorticoids? Reproduction 153: 163–171.
Saito, F.H., Damasceno, D.C., Kempinas, W.G., Morceli, Zhao, Z., Reece, A. (2013) New concepts in diabetic embryo-
G., Sinzato, Y.K., Taylor, K.N., et al. (2010) pathy. Clin Lab Med 33: 207–233.