Gender specific differences in oxidative stress and inflammatory signaling in healthy term neonates and their mothers

Abstract

Background:
Gender is a crucial determinant of life span, but little is known about gender differences in free radical homeostasis and inflammatory signalling. The aim of the study was to determine gender-related differences concerning oxidative stress and inflammatory signalling of healthy neonates and mothers.

Methods:
56 mothers with normal gestational course and spontaneous delivery were selected. Blood samples were collected from the mother (at the beginning of delivery and start of expulsive period) and from neonate (from umbilical cord vein and artery).

Results:
The mothers of girls featured a higher total antioxidant status (TAS) and lower plasma hydroperoxides than the mother of boys. Regarding the neonates, the girls featured a higher TAS and lower plasma membrane hydroperoxides in umbilical cord artery together with higher catalase (CAT), Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities. Lower levels of interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-a) and prostaglandin E2 (PGE-2) were observed in the mothers of girls and higher level of soluble tumour necrosis factor receptor II (sTNF-RII). In the neonates, lower levels of IL-6 and TNF-a were observed in umbilical artery and higher sTNF-RII in umbilical cord vein and artery of girls.

Conclusion:
An association between gender, oxidative stress and inflammation signalling exists, leading to a renewed interest in the neonate’s sex as a potential risk factor to several alterations.

Full text

Copyright © 2016 International Pediatric Research Foundation, Inc.
Articles
Basic Science Investigation
nature publishing group
BACKGROUND: Gender is a crucial determinant of life span,
but little is known about gender differences in free radical
homeostasis and inflammatory signaling. The aim of the study
was to determine gender-related differences concerning oxi-
dative stress and inflammatory signaling of healthy neonates
and mothers.
METHODS: Fifty-six mothers with normal gestational course
and spontaneous delivery were selected. Blood samples were
collected from the mother (at the beginning of delivery and
start of expulsive period) and from neonate (from umbilical
cord vein and artery).
RESULTS: The mothers of girls featured a higher total anti-
oxidant status and lower plasma hydroperoxides than the
mother of boys. Regarding the neonates, the girls featured a
higher total antioxidant status and lower plasma membrane
hydroperoxides in umbilical cord artery together with higher
catalase, glutathione peroxidase, and superoxide dismutase
activities. Lower levels of interleukin 6, tumor necrosis factor
alpha, and prostaglandin E2 were observed in the mothers of
girls and higher level of soluble tumor necrosis factor recep-
tor II. In the neonates, lower levels of interleukin 6 and tumor
necrosis factor alpha were observed in umbilical artery and
higher soluble tumor necrosis factor receptor II in umbilical
cord vein and artery of girls.
CONCLUSION: An association between gender, oxidative
stress, and inflammation signaling exists, leading to a renewed
interest in the neonate’s sex as a potential risk factor to several
alterations.
Birth is, in itself, a hyperoxic challenge and this new extra-
uterine aerobic environment requires an ecient cellular
system to produce energy, which also produces an important
amount of free radicals. To protect against this source of free
radicals and against others sources that show an increased
activity during birth, the organism have an ecient anti-
oxidant system (1,2). However, when reactive oxygen species
(ROS) faced with an inadequate antioxidant defense, these
molecules disrupt cell integrity and cause tissue injury (2).
Another important factor contributing to the increase in
ROS production is the evoked inammation during the deliv-
ery. Parturition has been identied as a source of proinam-
matory mediators such as metabolites of arachidonic acid
(prostaglandin E2 (PGE2)) and cytokines, including tumor
necrosis factor α (TNF-α), and interleukin 6 (IL-6). ese
mediators are potent stimulators for the production of ROS
and in turn free radicals recruit inammatory signalers in a
vicious circle (2).
ere are known sex specic dierences in fetal growth and
fetal and neonatal morbidity and mortality (3), and gender is
also a crucial determinant of life span, but little is known about
gender dierences in free radical homeostasis (4).
In addition, mitochondria from female generate half the
amount of superoxide radicals than those of the males (5,6).
Superoxide radicals generated adventitiously by the mitochon-
drial respiratory chain can give rise to much more reactive
radicals, resulting in random oxidation of all classes of cellu-
lar macromolecules (7). In addition, using a human umbilical
vein model, some authors (8) reported that the infusion of the
organic peroxide tertbutylhydroperoxide produced a gender-
related eect on eicosanoids and glutathione, biological mark-
ers linked with the cellular red-ox state (9). Finally, cell death
can be dierent in both magnitude and duration between male
and female rats, supporting the notion that divergent pathways
of cell death occur between genders (10), and dierences in
hormones production between genders has been correlated
with development dierences in brain structure or chemistry
and sexual dimorphism in neurological disorders (4).
It has been established that an excessive and/or sustained
increase in free radical production associated with diminished
ecacy of the antioxidant defense systems result in oxidative
stress, which occurs in many pathologic processes and contrib-
utes signicantly to disease mechanisms (2). It is reasonable to
suggest that oxidative stress would be the key link between an
Received 9 March 2016; accepted 23 March 2016; advance online publication 22 June 2016. doi:10.1038/pr.2016.112
1Department of Physiology, University of Granada, Granada, Spain; 2Institute of Nutrition and Food Technology “José Mataix Verdú”, University of Granada, Granada, Spain;
3Department of Biochemistry and Molecular Biology II, University of Granada, Spain; 4Department Obstetrics and Gynecology, School of Medicine, University of Granada, Spain;
5Service of Obstetrics and Gynecology, University Hospital San Cecilio, Granada, Spain. Correspondence: Julio José Ochoa Herrera (jjoh@ugr.es)
Gender specic dierences in oxidative stress and
inammatory signaling in healthy term neonates and their
mothers
JavierDiaz-Castro1,2, MarioPulido-Moran2,3, JorgeMoreno-Fernandez1,2, NaroaKajarabille1,2, CatalinadePaco4,5,
MariaGarrido-Sanchez4,5, SoniaPrados4,5 and Julio J.Ochoa1,2
Volume 80 | Number 4 | October 2016 Pediatric RESEARCH 595

Copyright © 2016 International Pediatric Research Foundation, Inc.
Articles Diaz-Castro et al.
adverse prenatal environment and increased morbidity later in
life. In fact, adverse fetal growth is frequently associated with a
number of oxidative insults and several postnatal pathologies
such as chronic obstructive lung disease, retinopathy, with an
oxidative etiology (11).
Taking into account the above-mentioned points, the knowl-
edge of the antioxidant/oxidative status in normal pregnancy
may help to deepen in the physiopathological mechanisms
and treatment of diseases associated with pregnancy. However,
despite the importance of the mentioned aspects, the knowl-
edge gained on this issue is still very limited in certain aspects.
Gender eects on the oxidative stress and inammatory sta-
tus have been addressed in clinical studies only to a limited
extent and oen with controversial results and virtually no
data on the early life stage; therefore, our aim was to determine
whether any gender-related dierence exists concerning oxi-
dative stress, inammatory signaling, and biochemical param-
eters of healthy neonates and their mothers to understand the
gender-dependent homeostatic redox mechanisms during the
delivery.
RESULTS
e delivery involves diverse modications in the plasmatic
biomarkers. It is noteworthy the eect of gender and partu-
rition on the plasmatic lipids studied. Total cholesterol was
higher in the mother of boys before delivery (P < 0.05) and in
umbilical vein of boys (P < 0.01). Phospholipids were higher
in the mother of girls before delivery (P < 0.05). With respect
to bilirubin, we observed an increase in bilirubin levels in the
mother of girls aer delivery (P < 0.05) and a decrease in its
concentrations in the umbilical artery of the girls compared
with the vein (P < 0.01), with lower values than in the other
groups. Triglycerides were higher in the umbilical artery of
girls compared with the umbilical artery of boys (P < 0.01)
(Table 1).
With regard to the enzymatic antioxidant system of the
mothers and their neonates (Table 2 ), the results show that
glutathione peroxidase (GPx) activity decreased aer delivery
in the mothers of boys and girls (P < 0.01). In the neonates, cat-
alase (CAT) activity decreased in umbilical artery of boys (P <
0.05) and increased in umbilical artery of girls compared with
the boys (P < 0.05). GPx increased in umbilical artery and vein
of girls compared with boys (P < 0.01). Superoxide dismutase
(SOD) activity decreased in umbilical artery of girls compared
with vein (P < 0.01) and increased in umbilical artery of girls
compared with umbilical artery of boys (P < 0.01).
Plasma hydroperoxides increased in the mother of boys aer
delivery (P < 0.01), decreased in the mother of girls compared
with the mother of boys aer delivery (P < 0.01), and in the
neonates decreased in umbilical artery compared with umbili-
cal vein (P < 0.05 for boys and girls). Membrane hydroperox-
ides increased in the mother of boys and girls aer delivery
(P < 0.01), decreased in umbilical artery of girls compared
with umbilical vein (P < 0.01), and also decreased in umbili-
cal artery of girls compared with boys (P < 0.01) (Figure 1).
Total antioxidant status (TAS) decreased in the mother of boys
aer delivery (P < 0.01) and increased in the mother of girls
compared with the mother of boys aer delivery (P < 0.05).
In addition, TAS increased in umbilical artery compared with
umbilical vein in both genders (P < 0.05) and increased in
umbilical artery of girls compared with umbilical artery of
boys (P < 0.05) (Figure 2).
On the other hand, parturition leads to an overexpression
of inammatory cytokines such as IL-6 and TNF-α in mother
of boys and girls (P < 0.01 for IL-6 and P < 0.001 for TNF-
α). Anti-inammatory cytokine soluble receptor II of TNF-α
(sTNF-RII) increased in the mother of girls before delivery
compared with the mother of boys (P < 0.01) and decreased
aer delivery in the mother of girls (P < 0.05). With regard
to the neonates, IL-6 increased in umbilical artery of boys
Table 1. Biochemical parameters of mothers and their neonates
Mothers
Mother of boy Mother of girls
Before delivery After delivery Before delivery After delivery
Total bilirubin (µmol/l) 46.97 ± 5.65 44.14 ± 3.38 45.92 ± 5.64a54.89 ± 4.03
Total cholesterol (mg/dl) 256.64 ± 7.10a232.40 ± 9.72 257.01 ± 8.33 252.26 ± 9.69
Phospholipids (mg/dl) 191.74 ± 5.18 183.48 ± 4.31 200.59 ± 6.55a181.83 ± 6.22
Triglycerides (mg/dl) 195.26 ± 13.84 189.06 ± 13.72 200.93 ± 12.38 182.37 ± 10.91
Neonates
Boys Girls
Umbilical vein Umbilical artery Umbilical vein Umbilical artery
Total bilirubin (µmol/l) 28.44 ± 2.55 27.38 ± 2.10 31.85 ± 2.18a24.64 ± 1.70
Total cholesterol (mg/dl) 66.99 ± 2.05a,b 58.49 ± 2.41 62.50 ± 1.49 62.07 ± 1.62
Phospholipids (mg/dl) 97.80 ± 4.68 89.33 ± 3.66 96.54 ± 3.71 89.31 ± 4.02
Triglycerides (mg/dl) 41.72 ± 2.46 40.57 ± 2.69b45.35 ± 3.12 48.26 ± 3.20
Results are expressed as mean ± SEM.
aMeans were different from the same group after delivery (in the mothers) and different from unmbilical artery (in the nenonate) (P < 0.05). bMeans were different from the
corresponding group of girls (before delivery, after delivery, umbilical artery, umbilical vein) (P < 0.05).
596 Pediatric RESEARCH Volume 80 | Number 4 | October 2016

Copyright © 2016 International Pediatric Research Foundation, Inc.
Gender dierences in term neonates Articles
compared with artery (P < 0.05) and decreased in umbili-
cal artery of girls compared with boys (P < 0.05). TNF-α
decreased in umbilical artery of girls compared with umbilical
artery of boys (P < 0.01). sTNF-RII increased in umbilical vein
(P < 0.05) and umbilical artery of girls compared with boys
(P < 0.01). In addition, sTNF-RII increased in umbilical artery
compared with vein in the girls (P < 0.05) (Tabl e 3 ). Finally,
PGE2 levels were higher in the mothers of a boy compared with
the mothers of a girl aer the delivery (P < 0.01) (Figure 3).
DISCUSSION
Many aspects about oxidative stress and inammation during
parturition are still not totally clear, being necessary a more
complete view of these processes both in the mother and the
new born. e objective of this study, which was designed to
determine whether any gender-related dierence exists con-
cerning oxidative stress, inammatory signaling, and bio-
chemical parameters, to understand the gender-dependent
homeostatic redox mechanisms during the delivery, which
will inuence the postnatal pathologies that will suer the
neonates in their lifespan, because there is a crosslink between
an oxidative stress and several postnatal pathologies such as
chronic obstructive lung disease, retinopathy, with an oxida-
tive etiology (11,12).
In our case, our main aim was to focus on the moment of the
delivery, when a major output of free radicals and inamma-
tory signaling takes place, and in addition to have in consider-
ation the role of the placental barrier, blood samples of mothers
were taken from the antecubital vein, at the beginning of the
cervix dilatation and immediately before the maternal–fetal
ejection in the mother, and also blood samples were collected
from the umbilical vein and arteries of the neonates. Taking a
sample from each blood type we can assess what substances
are transferred to the fetus from the mother and to the mother
from the fetus, showing the role of placental barrier.
Many studies have showed antioxidant eects of bilirubin
even higher than those shown for vitamin E (13). e higher
levels of bilirubin in the mother of girls aer the childbirth
indicate an antioxidant advantage in this situation of great
Table 2. Antioxidant enzymes activities of mothers and their neonates
Mothers
Mother of boy Mother of girls
Before delivery After delivery Before delivery After delivery
CAT cytosol erythrocyte (K/seg·mg) 0.41 ± 0.03 0.42 ± 0.02 0.38 ± 0.02 0.38 ± 0.03
GPx cytosol erythrocyte (U/mg) 54.38 ± 3.74a65.71 ± 4.18 56.05 ± 3.04a64.11 ± 3.38
SOD cytosol erythrocyte (U/mg) 214.51 ± 12.60 227.74 ± 12.45 214.22 ± 11.61 215.32 ± 10.83
Neonates
Boys Girls
Umbilical vein Umbilical artery Umbilical vein Umbilical artery
CAT cytosol erythrocyte (K/seg·mg) 0.29 ± 0.01*a 0.26 ± 0.01b0.31 ± 0.01 0.29 ± 0.01
GPx cytosol erythrocyte (U/mg) 30.22 ± 0.93b30.97 ± 1.25b33.82 ± 1.16 34.58 ± 1.54
SOD cytosol erythrocyte (U/mg) 228.55 ± 7.69 221.58 ± 9.20b223.18 ± 8.56a248.12 ± 10.64
Results are expressed as mean ± SEM.
CAT, catalase; GPX, glutathione peroxidase; SOD, superdoxide dismutase.
aMeans were different from the same group after delivery (in the mothers) and different from umbilical artery (in the nenonate) (P < 0.05). bMeans were different from the
corresponding group of girls (before delivery, after delivery, umbilical artery, and umbilical vein) (P < 0.05).
Figure 1. Plasma (a) and erythrocyte membrane (b)hydroperoxides of
mothers and their neonates (dark bar for boys and clear bar for girls).
Results are expressed as mean ± SEM.aMeans were dierent from the same
group after delivery (in the mothers) and dierent from umbilical artery
(in the nenonate) (P < 0.05). bMeans were dierent from the corresponding
group of girls (before delivery, after delivery, umbilical artery, umbilical
vein) (P < 0.05).
0.25
0.20
0.15
Plasma hydroperoxides (nmol/mg)
Membrane hydroperoxides (nmol/mg)
0.10
0.05
0.00
30
25
20
15
10
5
0
Before
delivery
After
delivery
*
**
Umbilical
vein
†
*
*
*
†
Umbilical
artery
NeonatesMothers
Before
delivery
After
delivery
Umbilical
vein
Umbilical
artery
NeonatesMothers
a
b
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Copyright © 2016 International Pediatric Research Foundation, Inc.
Articles Diaz-Castro et al.
oxidative aggression. In addition, the results show a major pla-
cental transfer of bilirubin to the girls, giving place to a major
protection to the evoked oxidative stress. With respect to its
origin, there are many factors that have been studied to dem-
onstrate their inuence on bilirubin levels, being one of the
most important factors the oxytocin, which increase during
parturition (14). It is well known that oxytocin expression is
usually higher in females (15). In this sense, a recent study con-
ducted by Silva et al. (2014) (16) indicated that oxytocin levels
were higher in the mothers of girls and the reduced duration
of labor; therefore, we can assume that the mothers of girls
increased bilirubin transfer to the umbilical vein, explaining
the dierences between umbilical vein and artery in girls. A
higher production of oxytocin reduces the oxidative stress dur-
ing the childbirth, having a key role as anti-inammatory and
conditioning the development of neuronal pathologies in the
mother (postpartum depression) and in the neonate (autism
disorders) (17).
Increases in serum lipids are common during the second half
of pregnancy (18) and could be related, at least in part, with
pregnancy hormones and the stress of delivery (19). Anyway,
this maternal hyperlipidemia could have a benecial inuence
on fetal development, because as our results shown, there is
an uptake by the fetus, resulting in lower values of total cho-
lesterol and phospholipids aer delivery, probably due to high
necessity of these molecules for the neonate and the increased
maternal–fetal transfer (18).
In general, a higher oxidative aggression is observed in the
mothers aer childbirth, and a lower oxidative damage in
umbilical artery compared with vein, ndings in agreement
with earlier reports (2). In relation to gender inuence, a lower
oxidative damage is found in the mothers of girls aer child-
birth and in umbilical artery of girls, with regard to the oxida-
tive damage showed by the boys. ese dierences associated
with the gender coincide with those found by other authors,
although in adult subjects (4), but, to date, these dierences
have not been assessed in mothers and their neonates. ese
dierences can be because of a lower oxidative damage or free
radicals output both in the mother and in the neonate and they
are associated to the female gender or to a high antioxidant
defense. With regard to the antioxidant system, the major nd-
ings of this study are that healthy female neonates in most cases
have signicantly higher TAS and higher levels of antioxidant
enzymes, suggesting a better protective eect against oxidative
Figure 2. Plasma total antioxidant capacity (TAS) of mothers and their
neonates (dark bar for boys and clear bar for girls). Results are expressed
as mean ± SEM. aMeans were dierent from the same group after delivery
(in the mothers) and dierent from umbilical artery (in the nenonate)
(P < 0.05). bMeans were dierent from the corresponding group of girls
(before delivery, after delivery, umbilical artery, umbilical vein) (P < 0.05).
TAS (nmol/mg)
30
25
20
15
10
5
0
*
*
*
†
†
Before
delivery
After
delivery
Umbilical
vein
Umbilical
artery
NeonatesMothers
Table 3. Inflammatory parameters of mothers and their neonates
Mothers
Mother of boy Mother of girls
Before
delivery
After
delivery
Before
delivery
After
delivery
IL-6 (pg/ml) 7.28 ± 0.52a,b 9.30 ± 0.44b5.93 ± 0.45a8.18 ± 0.45
TNF-α
(pg/ml)
17.12 ± 1.62a,b 26.20 ± 2.84 12.21 ± 1.56a27.49 ± 1.51
sTNF-RII
(ng/ml)
5.80 ± 0.42b5.93 ± 0.38 7.14 ± 0.38a6.19 ± 0.19
Neonates
Boys Girls
Umbilical
vein
Umbilical
artery
Umbilical
vein
Umbilical
artery
IL-6 (pg/ml) 2.48 ± 0.18a3.12 ± 0.19b2.28 ± 0.14 2.56 ± 0.13
TNF-α
(pg/ml)
21.76 ± 1.06 22.90 ± 0.84b18.79 ± 0.77 19.19 ± 0.86
sTNF-RII
(ng/ml)
11.21 ± 0.40b11.15 ± 0.39b12.28 ± 0.34a13.47 ± 0.35
IL-6, interleukin 6; TNF-α, tumor necrosis factor alpha; sTNF-RII, soluble receptor of
tumor necrosis factor II.
Results are expressed as mean ± SEM.
aMeans were different from the same group after delivery (in the mothers) and different
from umbilical artery (in the nenonate) (P < 0.05). bMeans were different from the
corresponding group of girls (before delivery, after delivery, umbilical artery, and
umbilical vein) (P < 0.05).
Figure 3. Plasma prostaglandin E2 (PGE2) concentration of mothers
and their neonates (dark bar for boys and clear bar for girls). Results are
expressed as mean ± SEM. aMeans were dierent from the same group
after delivery (in the mothers) and dierent from umbilical artery (in the
nenonate) (P < 0.05). bMeans were dierent from the corresponding group
of girls (before delivery, after delivery, umbilical artery, umbilical vein) (P
< 0.05).
1,000
**
900
Plasma PGE-2 (pg/ml)
800
700
600
500
400
300
200
100
0
†
†
Before
delivery
After
delivery
Umbilical
vein
Umbilical
artery
NeonatesMothers
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Gender dierences in term neonates Articles
damage in the girls compared with the boys. Earlier studies have
reported that human erythrocyte GPx activity is higher in adult
females compared with males (20). Erythrocyte GPx activity is
positively correlated with serum estrogen and with estrogens.
Estradiol upregulates the expression of SOD and GPx activating
Mitogen-activated protein (MAP) kinases and nuclear factor-
kappa-light-chain-enhancer of activated B cells (NF-κB) (21),
pathways that lead to the upregulation of SOD and GPx gene
expression (4). Some authors report that the expression of SOD
is approximately double in females than in adult males (22). In a
similar way, GPx expression and activity is markedly increased
in females when compared with males, increase that can be
attributed to estrogens. Antioxidant properties of estrogen may
also contribute to lower oxidative stress in females (23).
As mentioned earlier, another aspect to be taken into account
is the free radicals output. In this sense, greater oxidative stress
in men could be due, at least in part, to an increased genera-
tion of ROS and/or reduced activity of antioxidants. Cellular
respiration in the mitochondria is the dominant source of
ROS. erefore, a higher baseline metabolic rate in males than
in females (24) might contribute to a higher level of oxidative
stress in the male neonates.
Data of this study reveal that the gender of the neonate
inuences the degree of oxidative aggression suered, being
in our opinion, of great interest if we consider the high num-
ber of neonatal pathologies linked to the oxidative stress (11).
Inthis sense, males and females suer diering levels of oxi-
dative insult during the adulthood, and the resultant damage
may therefore be sucient to explain the residual sex-specic
lifespan dierence between genders. Indeed there is mounting
evidence to suggest that male humans express lower levels of
protective enzymes such as SOD and CAT than females and
consequently suer higher levels of oxidative damage (21,25).
With regard to the mothers, the antioxidant system results
are in agreement with the information featured by the oxida-
tive damage; therefore, the mothers of a boy during the delivery
experience a decrease in plasmatic TAS, because of its reduc-
tion in the process of neutralization free radicals generated
during the labor, compensating the higher oxidative damage.
With regard to the inammatory signaling, cytokines are
powerful mediators of cell growth and regulators of immuno-
logical and inammatory reactions, and they play an impor-
tant role in pregnancy (26), facts that result in the formation
of ROS (27). Another important factor contributing to the
increase in ROS production is the evoked inammation dur-
ing the delivery. Parturition has been identied as a source of
proinammatory mediators such as metabolites of arachidonic
acid (PGE2) and cytokines, including TNF-α, and IL-6. ese
mediators are potent stimulators for the production of ROS
and in turn free radicals recruit inammatory signalers in a
vicious circle.
In this study, anti-inammatory cytokine sTNF-RII
increased in the mother of girls before delivery compared with
the mother of boys. With regard to the neonates, IL-6 increased
in umbilical artery of boys compared with vein and decreased
in umbilical artery of girls compared with boys, while TNF-α
decreased in umbilical artery of girls compared with umbili-
cal artery of boys. Our ndings showing sex dierences in the
inammatory response are consistent with earlier observa-
tions indicating that female cultured cells are more resistant
than male to oxidant-induced cell death (28). Several lines of
evidence suggest the presence of gender dierences (in adults)
in plasma inammatory cytokines levels in health (29) as well
as in disease (30). Several factors have been implicated as pos-
sible causes for these dierences. e most important factors
thought to account for these dierences include a dierence
in the proportion of fat tissue and its distribution (29), and
the level of sex hormones (31). Some studies have found that
aer the onset of labor there were high concentrations of IL-6
(32) and TNF-α (33), results in agreement with our results in
the mothers aer delivery. Part of this IL-6 seems to be from
placenta which also releases TNF-α (34). However, the female
neonates are able to produce anti-inammatory cytokines
to balance the inammation process; therefore, we recorded
higher values of sTNF-RII in girls than in boys, fact that con-
tributes to reduce the detrimental proinammatory eects of
TNF-α. is fact prevents the direct action of TNF-α with its
proinamatory receptors (sTNFR-I) (35). Moreover, sTNFR-II
stimulation has revealed activation of the immunosuppressive
IL-10 pathway and inhibits signicantly the eects of sev-
eral proinammatory cytokines (36). In this sense, increased
inammatory signaling or abnormal activity in systems that
implicates cytokines would be associated with several features
of autism in the postnatal life (15). Sexually dimorphic eects
of inammatory mediators, including actions that extend
beyond the nervous system to inuence metabolic or immune
reactions, also might be critical links to uncovering the mecha-
nisms underlying the causes and eects of autism and depres-
sions (15); therefore, the better inammatory state in the girls
would explain the lower incidence of these pathologies in the
postnatal life.
In conclusion, to date, several studies have been conducted
about the sex specic dierences in oxidative stress or inam-
matory signaling, but all of them were conducted in adult
humans (with scarce information about neonates and their
mothers). is study were carried out for the rst time to
assess the gender eects on the oxidative stress and inam-
matory status in healthy mothers and their neonates during
the delivery and demonstrated that the in vivo biomarkers
of oxidative stress and inammation signaling were greater
in healthy male than in female neonates, indicating that the
girls can face better the evoked oxidative damage than the boys
during the delivery. With regard to the antioxidant system of
the neonates, the results show that the girls have a higher TAS,
CAT, SOD, and GPx activities than the boys and an overex-
pression of inammatory cytokines, such as TNF-α, in boys
compared with the girls; however, sTNF-RII was higher in
the girls compared with the boys. With regard to the mothers,
IL-6 and TNF-α were higher in the mothers of boys before the
delivery, whereas sTNF-RII was lower in the mothers of boys.
All these ndings suggest an association between gender, oxi-
dative stress, and inammatory signaling, leading to a renewed
Volume 80 | Number 4 | October 2016 Pediatric RESEARCH 599

Copyright © 2016 International Pediatric Research Foundation, Inc.
Articles Diaz-Castro et al.
interest in the neonate’s sex as a potential risk factor to sev-
eral functional alterations with important repercussion for the
neonate lifespan and the mother during the peripartum.
METHODS
Study Population
Fiy-six mothers with normal gestational course and spontane-
ous onset of labor followed by normal delivery were enrolled in the
study. Mean age was 29.9 ± 0.64 y, and mean gestational age was
39.3 ± 0.2 wk. ese mothers gave birth to 27 boys and 29 girls. e
inclusion criteria were no presence of disease, singleton gestation,
normal course of pregnancy, term gestation with cephalic presenta-
tion, body mass index of 18–30 kg/m2 at the start of pregnancy, weight
gain of 8–12 kg since pregnancy onset, gestational age at delivery of
37–42 wk, spontaneous vaginal delivery, new born with appropriate
weight for gestational age, new born with Apgar index ≥ 7 at rst
and h minutes of life, and normal monitoring results. Progress of
delivery was determined by vaginal examinations every one to two
according to clinical conditions. Uterine contractions and fetal heart
rate were constantly monitored by cardiotocography and were nor-
mal in all the cases. No abnormalities were detected during labor
and deliveries were spontaneous. e maternal–fetal ejection period
lasted 45.2 ± 5.5 min, in all the subjects. e study was approved by
the Bioethical Committee on Research Involving Human Subjects at
the University Hospital “Virgen de las Nieves” in Granada, and con-
sent was obtained from the parents aer the nature and purpose of the
study had been explained to them and were fully understood.
Blood Sampling
Maternal blood samples were obtained from the antecubital vein at
two dierent times: at the beginning of the active phase of labor and
at the start of expulsion when the fetus was at station +2. From the
umbilical cord, blood samples were collected from vein and artery,
immediately aer cord clamping, to assess what substances are trans-
ferred from mother to fetus and vice versa. Blood was immediately
centrifuged at 1,750g for 10 min at 4 °C in a Beckman GS-6R refrig-
erated centrifuge (Beckman, Fullerton, CA) to collect plasma and
separate it from red blood cell pellets. Plasma samples were imme-
diately frozen and stored at –80°C until analysis of TAS, total choles-
terol, bilirubin, and phospholipids, as well as inammatory cytokines.
According to the method of Hanahan and Ekholm (37) erythrocyte
cytosolic and membrane fractions were prepared by dierential cen-
trifugation with hypotonic hemolysis and successive dierential cen-
trifugations. Finally, the fraction obtained was aliquoted, snap-frozen
in liquid nitrogen, and stored at –80°C until analysis.
Biochemical Measures
Total bilirubin was determined employing the Bilirubin Total and
Direct dimethylsulfoxide, colorimetric assay kit (Spinreact, Gerona,
Spain), total cholesterol by using cholesterol CHOD–POD liquid
(Spinreact). Triglycerides levels were evaluated with triglycerides
GPO–POD. Enzymatic colorimetric assay kit (Spinreact) and phos-
pholipids were measured employing phospholipids CHO–POD.
Enzymatic colorimetric assay kit (Spinreact). All assays were per-
formed according to the manufacturer’s guidelines.
Oxidative Stress
To determine plasma TAS levels, freshly thawed batches of plasma were
analyzed using TAS Randox kit (Randox laboratories, Crumlin, UK).
e assay involves brief incubation of 2,2′-azinobis-di(3-ethylbenz-
thiazoline sulfonate) with peroxidase (metmyoglobin) and hydrogen
peroxide, resulting in the generation of 2,2′-azinobis-di(3-ethylben-
zthiazoline sulfonate) + radical cations. Results were expressed in
mmol/l of Trolox equivalents. e reference range for human blood
plasma is given by the manufacturer as 1.30–1.77 mmol/l. e linear-
ity of calibration extends to 2.5 mmol/l of Trolox. Measurements in
duplicate were used to determine intra-assay variability.
GPx activity was measured by Flohé and Günzler (38) method. is
is based on the immediate generation of oxidized glutathione (GSSG)
during the reaction catalyzed by GPx. GSSG is continually reduced by
an excess of glutathione reductase and nicotinamide adenine dinucle-
otide phosphate, oxidized (NADP +) and reduced forms (NADPH)
present in the cuvette. e subsequent oxidation of NADPH to nico-
tinamide adenine dinucleotide phosphate, oxidized (NADP+) was
monitored spectrophotometrically (ermo Spectronic, Rochester,
NY) at 340 nm. Cumen hydroperoxide was used as substrate.
CAT activity was determined according to Aebi method (39), mon-
itoring at 240 nm spectrophotometrically (ermo Spectronic) the
H2O2 decomposition form by catalytic activity of CAT. e activity
was calculated from the rst-order rate constant K (/s).
SOD activity was assayed according to the method of Crapo etal.
(40). is method is based on the inhibition in the reduction of
cytochrome c by SOD, measured spectrophotometrically (ermo
Spectronic) at 550 nm wavelength. One unit of the SOD activity is
dened as the amount of enzyme required to produce 50% inhibition
of the rate of reduction of cytochrome c.
Plasma hydroperoxides were determined using the OxyStat kit
(Biomedica Gruppe, Vienna, Austria). e peroxide concentration
is determined by reaction of the biological peroxides and a subse-
quent color reaction using TMB (3,3′,5,5′-tetramethylbenzidine) as
substrate. e plate was measured at 450 nm wavelength on a Bio-
Tek microplate reader (Bio-Tek, Vermont). Erythrocyte membrane
hydroperoxides were estimated using a commercial kit (Pierce™
Quantitative Peroxide Assay Kits, ermo Scientic, Rockford, IL).
is kit is based on the principle of the rapid peroxide-mediated oxi-
dation of Fe2+ to Fe3+ under acidic conditions. e latter, in the pres-
ence of xylenol orange, forms a Fe3+-xylenol orange complex which
can be measured spectrophotometrically at 560 nm wavelength
(Perkin Elmer UV-VIS Lambda-16, Norwalk, CT).
Cytokine Measures
TNF-α, IL-6, and sTNF-RII plasma levels were determined using
Biosource kits (Biosource Europe, Nivelles, Belgium), PGE2 was mea-
sured using a R&D kit (R&D Systems Europe, Abingdon, UK). e
TNF-α, IL-6, and PGE2 are solid phase Enzyme Amplied Sensitivity
Immunoassays performed on microtiter plate. In these assays, mono-
clonal antibodies (MAbs) blend directly against distinct TNF-α,
IL-6, and PGE2 epitopes and subsequently with a secondary anti-
body, horseradish peroxidase-labeled-antibody MAb2 is then added.
e plate was then read at wavelength between 450 and 490 nm on a
microplate reader (Bio-tek).
e sTNF-RII kit is a solid phase sandwich Enzyme Linked-
Immune-Sorbent Assay. In this assay, an MAb blends directly against
sTNF-RII. sTNF-RII standards, controls, and unknown samples are
pipetted into the wells together with a MAb labeled with horserad-
ish peroxidase. Aer washing, the substrate solution is added, which
is acted upon by the bound enzyme to produce blue color. Finally,
the stop solution reagent is added, ending the reaction and turning
around yellow. e plate is then read at 450 nm wavelength on a Bio-
tek microplate reader (Bio-tek). e intensity of this colored product
is directly proportional to the concentration of sTNF-RII.
Statistical Analysis
Results are reported as mean values ± SEM. Conformity to a normal
distribution was examined using the Kolmogorov–Smirnov test. To
assess dierences between mothers (before labor vs. aer labor) and
the neonates (umbilical cord vein vs. artery), on each gender a paired
Student’s t-test was performed, and to assess statistically signicant
dierences between genders in mothers (before labor and aer labor)
and in the neonates (umbilical cord vein and artery), an unpaired
Student’s t-test was performed. e level of signicance was set at P <
0.05. SPSS version 20.0, 2011 (SPSS, Chicago, IL) soware was used
for data treatment and statistical analysis.
ACKNOWLEDGMENTS
The authors are grateful to Jesús Florido Navío and Luis Navarrete López-
Cozar for their continuous support and help during the study.
Disclosure: The authors have no conicts of interest and no nancial rela-
tionships relevant to this article to disclose. Category of study: Basic Science.
No nancial assistance was received to support this study.
600 Pediatric RESEARCH Volume 80 | Number 4 | October 2016

Copyright © 2016 International Pediatric Research Foundation, Inc.
Gender dierences in term neonates Articles
REfERENCES
1. Winklhofer-Roob BM. Oxygen free radicals and antioxidants in cystic
brosis: the concept of an oxidant-antioxidant imbalance. Acta Paediatr
Suppl 1994;83:49–57.
2. Díaz-CastroJ, FloridoJ, KajarabilleN, et al. A new approach to oxidative
stress and inammatory signaling during labour in healthy mothers and
neonates. Oxid Med Cell Longev 2015;2015:178536.
3. Clion VL. Review: Sex and the human placenta: mediating dierential
strategies of fetal growth and survival. Placenta 2010;31 Suppl:S33–9.
4. Vina J, GambiniJ, Lopez-Grueso R, Abdelaziz KM, Jove M, Borras C.
Females live longer than males: role of oxidative stress. Curr Pharm Des
2011;17:3959–65.
5. BalabanRS, NemotoS, FinkelT. Mitochondria, oxidants, and aging. Cell
2005;120:483–95.
6. BorrásC, SastreJ, García-SalaD, LloretA, PallardóFV, ViñaJ. Mitochon-
dria from females exhibit higher antioxidant gene expression and lower oxi-
dative damage than males. Free Radic Biol Med 2003;34:546–52.
7. deGreyAD. Free radicals in aging: causal complexity and its biomedical
implications. Free Radic Res 2006;40:1244–9.
8. Lavoie JC, Chessex P. Gender-related response to a tert-butyl hydroper-
oxide-induced oxidation in human neonatal tissue. Free Radic Biol Med
1994;16:307–13.
9. HempelSL, WesselsDA, SpectorAA. Eect of glutathione on endothelial pros-
tacyclin synthesis aer anoxia. Am J Physiol 1993;264(6 Pt 1):C1448–57.
10. NuñezJL, LauschkeDM, JuraskaJM. Cell death in the development of the
posterior cortex in male and female rats. J Comp Neurol 2001;436:32–41.
11. OchoaJJ, Contreras-ChovaF, MuñozS, et al. Fluidity and oxidative stress
in erythrocytes from very low birth weight infants during their rst 7 days
of life. Free Radic Res 2007;41:1035–40.
12. RajdlD, RacekJ, SteinerováA, et al. Markers of oxidative stress in diabetic
mothers and their infants during delivery. Physiol Res 2005;54:429–36.
13. ShekeebShahabM, KumarP, SharmaN, NarangA, PrasadR. Evaluation of
oxidant and antioxidant status in term neonates: a plausible protective role
of bilirubin. Mol Cell Biochem 2008;317:51–9.
14. OralE, GezerA, CagdasA, PakkalN. Oxytocin infusion in labor: the eect
dierent indications and the use of dierent diluents on neonatal bilirubin
levels. Arch Gynecol Obstet 2003;267:117–20.
15. CarterCS. Sex dierences in oxytocin and vasopressin: implications for
autism spectrum disorders? Behav Brain Res 2007;176:170–86.
16. SilvaD, ColvinL, HagemannE, BowerC. Environmental risk factors by
gender associated with attention-decit/hyperactivity disorder. Pediatrics
2014;133:e14–22.
17. LeeHJ, MacbethAH, PaganiJH, YoungWS3rd. Oxytocin: the great facili-
tator of life. Prog Neurobiol 2009;88:127–51.
18. Mazurkiewicz JC, Watts GF, Warburton FG, Slavin BM, Lowy C,
KoukkouE. Serum lipids, lipoproteins and apolipoproteins in pregnant
non-diabetic patients. J Clin Pathol 1994;47:728–31.
19. Herrera E. Lipid metabolism in pregnancy and its consequences in the
fetus and newborn. Endocrine 2002;19:43–55.
20. AndersenHR, NielsenJB, Nielsen F, GrandjeanP. Antioxidative enzyme
activities in human erythrocytes. Clin Chem 1997;43:562–8.
21. ViñaJ, BorrásC, GambiniJ, SastreJ, PallardóFV. Why females live longer
than males? Importance of the upregulation of longevity-associated genes
by oestrogenic compounds. FEBS Lett 2005;579:2541–5.
22. BoverisA, Cadenas E. Mitochondrial production of hydrogen peroxide
regulation by nitric oxide and the role of ubisemiquinone. IUBMB Life
2000;50:245–50.
23. SugiokaK, ShimosegawaY, NakanoM. Estrogens as natural antioxidants
of membrane phospholipid peroxidation. FEBS Lett 1987;210:37–9.
24. MeijerGA, WesterterpKR, SarisWH, tenHoorF. Sleeping metabolic rate
in relation to body composition and the menstrual cycle. Am J Clin Nutr
1992;55:637–40.
25. Tomás-Zapico C, Alvarez-GarcíaO, SierraV, et al. Oxidative damage in
the livers of senescence-accelerated mice: a gender-related response. Can J
Physiol Pharmacol 2006;84:213–20.
26. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine
interactions in the maternal-fetal relationship: is successful pregnancy a
TH2 phenomenon? Immunol Today 1993;14:353–6.
27. StípekS, MĕchurováA, CrkovskáJ, ZimaT, PláteníkJ. Lipid peroxidation
and superoxide dismutase activity in umbilical and maternal blood. Bio-
chem Mol Biol Int 1995;35:705–11.
28. LiuM, OyarzabalEA, YangR, MurphySJ, HurnPD. A novel method for
assessing sex-specic and genotype-specic response to injury in astrocyte
culture. J Neurosci Methods 2008;171:214–7.
29. Cartier A, Côté M, Lemieux I, et al. Sex dierences in inammatory
markers: what is the contribution of visceral adiposity? Am J Clin Nutr
2009;89:1307–14.
30. BecklakeMR, KaumannF. Gender dierences in airway behaviour over
the human life span. orax 1999;54:1119–38.
31. Ben-ZakenCohenS, ParéPD, ManSF, Sin DD. e growing burden of
chronic obstructive pulmonary disease and lung cancer in women: exam-
ining sex dierences in cigarette smoke metabolism. Am J Respir Crit Care
Med 2007;176:113–20.
32. GunnL, HardimanP, armaratnamS, LoweD, ChardT. Measurement
of interleukin-1 alpha and interleukin-6 in pregnancy-associated tissues.
Reprod Fertil Dev 1996;8:1069–73.
33. OpsjłnSL, WathenNC, TingulstadS, et al. Tumor necrosis factor, interleu-
kin-1, and interleukin-6 in normal human pregnancy. Am J Obstet Gyne-
col 1993;169(2 Pt 1):397–404.
34. SteinbornA, vonGallC, HildenbrandR, StutteHJ, KaufmannM. Identi-
cation of placental cytokine-producing cells in term and preterm labor.
Obstet Gynecol 1998;91:329–35.
35. Van Zee KJ, Kohno T, Fischer E, Rock CS, Moldawer LL, Lowry SF.
Tumor necrosis factor soluble receptors circulate during experimen-
tal and clinical inammation and can protect against excessive tumor
necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci USA 1992;
89:4845–9.
36. Gonzalez P, Burgaya F, AcarinL, Peluo H, Castellano B, Gonzalez B.
Interleukin-10 and interleukin-10 receptor-I are upregulated in glial cells
aer an excitotoxic injury to the postnatal rat brain. J Neuropathol Exp
Neurol 2009;68:391–403.
37. HanahanDJ, EkholmJE. e preparation of red cell ghosts (membranes).
Methods Enzymol 1974;31:168–72.
38. FlohéL, GünzlerWA. Assays of glutathione peroxidase. Methods Enzymol
1984;105:114–21.
39. AebiH. Catalase in vitro. Methods Enzymol 1984;105:121–6.
40. CrapoJD, McCordJM, FridovichI. Preparation and assay of superoxide
dismutases. Methods Enzymol 1978;53:382–93.
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