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 oxidative stress and inflammatory signaling of healthy neonates and mothers.
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).
The mothers of girls featured a higher total antioxidant 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 receptor 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.
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 extrauterine aerobic environment requires an efficient 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 efficient antioxidant 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 inflammation during the delivery. Parturition has been identified as a source of proinflammatory mediators such as metabolites of arachidonic acid (prostaglandin E2 (PGE2)) and cytokines, including tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6). These mediators are potent stimulators for the production of ROS and in turn free radicals recruit inflammatory signalers in a vicious circle (2).
There are known sex specific differences 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 differences 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 mitochondrial respiratory chain can give rise to much more reactive radicals, resulting in random oxidation of all classes of cellular 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 effect on eicosanoids and glutathione, biological markers linked with the cellular red-ox state (9). Finally, cell death can be different in both magnitude and duration between male and female rats, supporting the notion that divergent pathways of cell death occur between genders (10), and differences in hormones production between genders has been correlated with development differences 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 efficacy of the antioxidant defense systems result in oxidative stress, which occurs in many pathologic processes and contributes significantly to disease mechanisms (2). It is reasonable to suggest that oxidative stress would be the key link between an 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 knowledge 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 knowledge gained on this issue is still very limited in certain aspects. Gender effects on the oxidative stress and inflammatory status have been addressed in clinical studies only to a limited extent and often with controversial results and virtually no data on the early life stage; therefore, our aim was to determine whether any gender-related difference exists concerning oxidative stress, inflammatory signaling, and biochemical parameters of healthy neonates and their mothers to understand the gender-dependent homeostatic redox mechanisms during the delivery.
The delivery involves diverse modifications in the plasmatic biomarkers. It is noteworthy the effect of gender and parturition 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 after 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 after delivery in the mothers of boys and girls (P < 0.01). In the neonates, catalase (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 after delivery (P < 0.01), decreased in the mother of girls compared with the mother of boys after delivery (P < 0.01), and in the neonates decreased in umbilical artery compared with umbilical vein (P < 0.05 for boys and girls). Membrane hydroperoxides increased in the mother of boys and girls after delivery (P < 0.01), decreased in umbilical artery of girls compared with umbilical vein (P < 0.01), and also decreased in umbilical artery of girls compared with boys (P < 0.01) (Figure 1). Total antioxidant status (TAS) decreased in the mother of boys after delivery (P < 0.01) and increased in the mother of girls compared with the mother of boys after 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 inflammatory 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-inflammatory 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 after delivery in the mother of girls (P < 0.05). With regard to the neonates, IL-6 increased in umbilical artery of boys compared with artery (P < 0.05) and decreased in umbilical 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) (Table 3). Finally, PGE2 levels were higher in the mothers of a boy compared with the mothers of a girl after the delivery (P < 0.01) (Figure 3).
Many aspects about oxidative stress and inflammation during parturition are still not totally clear, being necessary a more complete view of these processes both in the mother and the new born. The objective of this study, which was designed to determine whether any gender-related difference exists concerning oxidative stress, inflammatory signaling, and biochemical parameters, to understand the gender-dependent homeostatic redox mechanisms during the delivery, which will influence the postnatal pathologies that will suffer 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 oxidative 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 inflammatory signaling takes place, and in addition to have in consideration 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 effects of bilirubin even higher than those shown for vitamin E (13). The higher levels of bilirubin in the mother of girls after the childbirth indicate an antioxidant advantage in this situation of great oxidative aggression. In addition, the results show a major placental 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 demonstrate their influence 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 conducted 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 differences between umbilical vein and artery in girls. A higher production of oxytocin reduces the oxidative stress during the childbirth, having a key role as anti-inflammatory 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 beneficial influence on fetal development, because as our results shown, there is an uptake by the fetus, resulting in lower values of total cholesterol and phospholipids after 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 after childbirth, and a lower oxidative damage in umbilical artery compared with vein, findings in agreement with earlier reports (2). In relation to gender influence, a lower oxidative damage is found in the mothers of girls after childbirth and in umbilical artery of girls, with regard to the oxidative damage showed by the boys. These differences associated with the gender coincide with those found by other authors, although in adult subjects (4), but, to date, these differences have not been assessed in mothers and their neonates. These differences 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 findings of this study are that healthy female neonates in most cases have significantly higher TAS and higher levels of antioxidant enzymes, suggesting a better protective effect against oxidative 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 generation of ROS and/or reduced activity of antioxidants. Cellular respiration in the mitochondria is the dominant source of ROS. Therefore, 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 influences the degree of oxidative aggression suffered, being in our opinion, of great interest if we consider the high number of neonatal pathologies linked to the oxidative stress (11). In this sense, males and females suffer differing levels of oxidative insult during the adulthood, and the resultant damage may therefore be sufficient to explain the residual sex-specific lifespan difference 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 suffer 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 oxidative damage; therefore, the mothers of a boy during the delivery experience a decrease in plasmatic TAS, because of its reduction in the process of neutralization free radicals generated during the labor, compensating the higher oxidative damage.
With regard to the inflammatory signaling, cytokines are powerful mediators of cell growth and regulators of immunological and inflammatory reactions, and they play an important 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 inflammation during the delivery. Parturition has been identified as a source of proinflammatory mediators such as metabolites of arachidonic acid (PGE2) and cytokines, including TNF-α, and IL-6. These mediators are potent stimulators for the production of ROS and in turn free radicals recruit inflammatory signalers in a vicious circle.
In this study, anti-inflammatory 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 umbilical artery of boys. Our findings showing sex differences in the inflammatory response are consistent with earlier observations 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 differences (in adults) in plasma inflammatory cytokines levels in health (29) as well as in disease (30). Several factors have been implicated as possible causes for these differences. The most important factors thought to account for these differences include a difference in the proportion of fat tissue and its distribution (29), and the level of sex hormones (31). Some studies have found that after 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 after 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-inflammatory cytokines to balance the inflammation process; therefore, we recorded higher values of sTNF-RII in girls than in boys, fact that contributes to reduce the detrimental proinflammatory effects of TNF-α. This fact prevents the direct action of TNF-α with its proinflamatory receptors (sTNFR-I) (35). Moreover, sTNFR-II stimulation has revealed activation of the immunosuppressive IL-10 pathway and inhibits significantly the effects of several proinflammatory cytokines (36). In this sense, increased inflammatory signaling or abnormal activity in systems that implicates cytokines would be associated with several features of autism in the postnatal life (15). Sexually dimorphic effects of inflammatory mediators, including actions that extend beyond the nervous system to influence metabolic or immune reactions, also might be critical links to uncovering the mechanisms underlying the causes and effects of autism and depressions (15); therefore, the better inflammatory 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 specific differences in oxidative stress or inflammatory signaling, but all of them were conducted in adult humans (with scarce information about neonates and their mothers). This study were carried out for the first time to assess the gender effects on the oxidative stress and inflammatory status in healthy mothers and their neonates during the delivery and demonstrated that the in vivo biomarkers of oxidative stress and inflammation 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 overexpression of inflammatory 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 findings suggest an association between gender, oxidative stress, and inflammatory signaling, leading to a renewed interest in the neonate’s sex as a potential risk factor to several functional alterations with important repercussion for the neonate lifespan and the mother during the peripartum.
Fifty-six mothers with normal gestational course and spontaneous 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. These mothers gave birth to 27 boys and 29 girls. The inclusion criteria were no presence of disease, singleton gestation, normal course of pregnancy, term gestation with cephalic presentation, 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 first and fifth 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 normal in all the cases. No abnormalities were detected during labor and deliveries were spontaneous. The maternal–fetal ejection period lasted 45.2 ± 5.5 min, in all the subjects. The study was approved by the Bioethical Committee on Research Involving Human Subjects at the University Hospital “Virgen de las Nieves” in Granada, and consent was obtained from the parents after the nature and purpose of the study had been explained to them and were fully understood.
Maternal blood samples were obtained from the antecubital vein at two different 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 after cord clamping, to assess what substances are transferred 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 refrigerated centrifuge (Beckman, Fullerton, CA) to collect plasma and separate it from red blood cell pellets. Plasma samples were immediately frozen and stored at –80°C until analysis of TAS, total cholesterol, bilirubin, and phospholipids, as well as inflammatory cytokines. According to the method of Hanahan and Ekholm (37) erythrocyte cytosolic and membrane fractions were prepared by differential centrifugation with hypotonic hemolysis and successive differential centrifugations. Finally, the fraction obtained was aliquoted, snap-frozen in liquid nitrogen, and stored at –80°C until analysis.
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 phospholipids were measured employing phospholipids CHO–POD. Enzymatic colorimetric assay kit (Spinreact). All assays were performed according to the manufacturer’s guidelines.
To determine plasma TAS levels, freshly thawed batches of plasma were analyzed using TAS Randox kit (Randox laboratories, Crumlin, UK). The assay involves brief incubation of 2,2′-azinobis-di(3-ethylbenzthiazoline sulfonate) with peroxidase (metmyoglobin) and hydrogen peroxide, resulting in the generation of 2,2′-azinobis-di(3-ethylbenzthiazoline sulfonate) + radical cations. Results were expressed in mmol/l of Trolox equivalents. The reference range for human blood plasma is given by the manufacturer as 1.30–1.77 mmol/l. The linearity 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. This 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 dinucleotide phosphate, oxidized (NADP +) and reduced forms (NADPH) present in the cuvette. The subsequent oxidation of NADPH to nicotinamide adenine dinucleotide phosphate, oxidized (NADP+) was monitored spectrophotometrically (Thermo Spectronic, Rochester, NY) at 340 nm. Cumen hydroperoxide was used as substrate.
CAT activity was determined according to Aebi method (39), monitoring at 240 nm spectrophotometrically (Thermo Spectronic) the H2O2 decomposition form by catalytic activity of CAT. The activity was calculated from the first-order rate constant K (/s).
SOD activity was assayed according to the method of Crapo et al. (40). This method is based on the inhibition in the reduction of cytochrome c by SOD, measured spectrophotometrically (Thermo Spectronic) at 550 nm wavelength. One unit of the SOD activity is defined 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). The peroxide concentration is determined by reaction of the biological peroxides and a subsequent color reaction using TMB (3,3′,5,5′-tetramethylbenzidine) as substrate. The 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, Thermo Scientific, Rockford, IL). This kit is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence 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).
TNF-α, IL-6, and sTNF-RII plasma levels were determined using Biosource kits (Biosource Europe, Nivelles, Belgium), PGE2 was measured using a R&D kit (R&D Systems Europe, Abingdon, UK). The TNF-α, IL-6, and PGE2 are solid phase Enzyme Amplified Sensitivity Immunoassays performed on microtiter plate. In these assays, monoclonal antibodies (MAbs) blend directly against distinct TNF-α, IL-6, and PGE2 epitopes and subsequently with a secondary antibody, horseradish peroxidase-labeled-antibody MAb2 is then added. The plate was then read at wavelength between 450 and 490 nm on a microplate reader (Bio-tek).
The 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 horseradish peroxidase. After 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. The plate is then read at 450 nm wavelength on a Bio-tek microplate reader (Bio-tek). The intensity of this colored product is directly proportional to the concentration of sTNF-RII.
Results are reported as mean values ± SEM. Conformity to a normal distribution was examined using the Kolmogorov–Smirnov test. To assess differences between mothers (before labor vs. after labor) and the neonates (umbilical cord vein vs. artery), on each gender a paired Student’s t-test was performed, and to assess statistically significant differences between genders in mothers (before labor and after labor) and in the neonates (umbilical cord vein and artery), an unpaired Student’s t-test was performed. The level of significance was set at P < 0.05. SPSS version 20.0, 2011 (SPSS, Chicago, IL) software was used for data treatment and statistical analysis.
The authors have no conflicts of interest and no financial relationships relevant to this article to disclose. Category of study: Basic Science. No financial assistance was received to support this study.
The authors are grateful to Jesús Florido Navío and Luis Navarrete López-Cozar for their continuous support and help during the study.
About this article
Journal of Neuro-Oncology (2018)