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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 4  |  Issue : 2  |  Page : 32-38

Vitamin E reduces placental oxidative stress and improves pregnancy outcome in pregnant rats at high altitude


1 Department of Physiology, College of Medicine, King Khalid University, KSA
2 Department of Physiology, College of Medicine, Sudan International University, Khartoum, Sudan
3 Department of Physiology, College of Medicine, King Khalid University, KSA, Saudi Arabia

Date of Web Publication13-Aug-2020

Correspondence Address:
MBBS, PhD Mohammed E M. Khalid
Department of Physiology, College of Medicine, King Khalid University, P.O. Box 641, Abha
KSA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1658-743X.292036

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  Abstract 


Objectives: To explore antioxidant (vitamin E) effects during pregnancy at high altitude (3000 m) on pregnancy outcomes. Methodology: Thirty-six pregnant Wistar rats, born and maintained at high altitude, were divided into three groups: Vitamin E-treated (vitamin E dissolved in olive oil), vehicle-treated (olive oil), and control (no treatment) groups. The mean hemoglobin concentration and hematocrit were estimated and used as indices for maternal hypoxia. The number of pups and their birth weight were determined. Placental oxidant conditions were assessed using placental malondialdehyde (MDA) levels and placental antioxidant enzyme activity was determined by superoxide dismutase (SOD) and glutathione peroxidase (GPX). Placentae were then examined histologically, and the average percentage of villi with syncytial knots, cytotrophoblastic cells, and fetal capillaries were used as indices of placental hypoxia. Results: Vitamin E-treated pregnant rats had less maternal and placental hypoxia than control and vehicle-treated groups. The vitamin E-treated group showed a higher number of newborn pups than control and vehicle-treated groups. Conclusion: Vitamin E administration to pregnant rats at high altitude may improve birth outcome by improving both maternal and placental hypoxia. Controlled trial studies to investigate the effect of vitamin E therapy on pregnancy outcomes in pregnant women at high altitude are recommended.

Keywords: high altitude, pregnancy, vitamin E, oxidative stress, birth outcome


How to cite this article:
M. Khalid ME, Ahmed HS, Osman OM, AL Hashem FH. Vitamin E reduces placental oxidative stress and improves pregnancy outcome in pregnant rats at high altitude. King Khalid Univ J Health Sci 2019;4:32-8

How to cite this URL:
M. Khalid ME, Ahmed HS, Osman OM, AL Hashem FH. Vitamin E reduces placental oxidative stress and improves pregnancy outcome in pregnant rats at high altitude. King Khalid Univ J Health Sci [serial online] 2019 [cited 2021 Mar 7];4:32-8. Available from: https://www.kkujhs.org/text.asp?2019/4/2/32/292036




  Introduction Top


Imbalance between antioxidant defenses and reactive oxygen species production can result in oxidative stress.[1] Even during a normal pregnancy, the placenta is exposed to some degree of oxidative stress. Hypobaric hypoxia at high altitude could increase placental oxidative stress, leading to oxidative damage to lipids, proteins, and DNA.[2],[3] Other researchers argued that chronic high-altitude hypoxia reduces oxidative stress in the placenta.[4] An increase in placental oxidants or a decrease in placental antioxidant activity was found to be associated with an adverse pregnancy outcome, including early pregnancy loss,[5] fetal growth retardation[6], and low birth weight.[7]

The mechanism of the adverse birth outcome by placental oxidative stress was thought to result from an alteration in the placental structure and function, which ultimately altered fetal growth and development.[8] The low birth weight characteristics of high altitude newborns compared with low altitude ones[9] and the higher incidence of unexplained preterm birth at high altitude compared with low elevation were found to be strongly associated with morphological changes in placentae of pregnant women in high altitude areas in southern Saudi Arabia.[10] These morphological changes appear to be secondary to maternal hypoxia, and they were caused by high altitude hypoxia.

This study explored the effects of antioxidant therapy, vitamin E, on maternal hypoxia resulting from high altitude hypoxia in pregnant rats that were born and maintained at high altitude (3000 m) and its effect on pregnancy outcome.


  Materials and Methods Top


This study was conducted on 36 adult (90-day-old) pregnant female Wistar rats (200–250 g) that were born and maintained in the animal house at King Khalid University at 22°C on a 12 hour light/dark cycle with free access to a semisynthetic control diet and water. All of the experimental protocols were performed by researchers and were approved by the Ethics Committee at King Khalid University. The experiments were conducted according to the National Institutes of Health guide for the care and use of laboratory animals.

Pregnancy in rats was confirmed by analyzing vaginal smears after mating for 2 days. The day on which sperms appeared in the vaginal smears was designated as day 0 of gestation. The rats were then divided into three equal groups: control, vehicle-treated, and vitamin E-treated groups. The control group received nothing, while the vehicle-treated group received an intraperitoneal injection of 0.25 mL of olive oil on alternating days throughout the pregnancy. The vitamin E-treated group was intraperitoneally injected with 100 mg/kg of vitamin E dissolved in olive oil (0.25 mL) on alternate days throughout the pregnancy. For each rat in the three groups, venous blood samples were obtained from the tail at weeks 1 and 2, and on the day of sacrifice to determine the hemoglobin concentration and hematocrit value using the Colter method. The mean hemoglobin concentrations and hematocrit values were used as indices of maternal hypoxia. On day 20, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (2 mg/100 g body weight). Using a pair of scissors, a U-shaped incision was made in the abdomen to expose the abdominal cavity. The uterus was opened and the conceptuses (fetuses, fetal membranes, and placentae) were dissected and the number of pups was counted. The individual pup weights were determined using a Sartorius precision balance, which recorded to the nearest 0.5 g. The mean number of pups and the mean birth weight for each group were then computed. Placental tissue (1 g) was obtained and homogenized in 4 mL of physiological buffer solution using an electric ground glass homogenizer. The homogenized tissue was then centrifuged at 4°C. The supernatant was taken and stored frozen at -40°C until the placental superoxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) levels were determined. The first two were used as markers for placental antioxidant enzyme activity and the last one was used as a marker for placental oxidants. Placentae were examined histologically using sections stained with hematoxylin–eosin. The mean percentage of villi with syncytial knots, cytotrophoblastic cells, and fetal capillaries was determined for each rat in the three groups, and these results were used as indices for placental hypoxia.

Statistical analysis

IBM SPSS Statistics for Windows version 20 (IBM Corp., Armonk, NY, USA) was used for the standard statistical analysis. Values are expressed as the mean ± standard deviation or percentages. A one-way ANOVA was used to compare between the three means. To test for a correlation between two variables, Spearman’s correlation test was used. P<0.05 was considered to be significant.


  Results Top


[Table 1] shows the maternal characteristics (weight and age), indices of maternal hypoxia (hemoglobin concentration and hematocrit value), indices of placental oxidative stress (SOD, GPX, and MDA), and birth outcome (number and birth weight of pups). The mean age and weight were not significantly different among the three groups. Hemoglobin concentrations and hematocrit values were significantly lower in the vitamin E-treated group than in the control and vehicle-treated groups (P<0.03 for all). No significant differences in hematocrit values and hemoglobin concentrations were found between the vehicle-treated and control groups.
Table 1: Maternal characteristics, indices of maternal hypoxia, indices of placental oxidative stress and birth outcome

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The SOD and GPX levels were significantly higher in vitamin E-treated rats than in control (P<0.001) and vehicle-treated (P<0.001) rats. SOD and GPX levels showed no significant differences between the control and vehicle-treated groups. The MDA level was significantly higher in the control and vehicle-treated groups than in the vitamin E-treated group (P<0.001 for both). The MDA level showed no significant differences between the control and vehicle-treated groups.

The incidence of syncytial knots and cytotrophoblastic cells was higher in the control group than in the vitamin E-treated group (P<0.04 and P<0.002, respectively). They were also higher in the vehicle-treated group than in the vitamin E-treated group (P<0.04 and P<0.001, respectively). No significant differences were observed in incidences of syncytial knots and cytotrophoblastic cells between the control group and the vehicle-treated group. Both the control group and the vehicle-treated group showed significantly higher numbers of fetal capillaries than the vitamin E-treated group (P<0.001 for both). No significant difference was found in the number of fetal capillaries between the control and vehicle-treated groups.

Vitamin E-treated rats had a significantly higher number of pups compared to the control (P<0.03) and the vehicle-treated (P<0.04) groups, while there was no significant difference observed in the number of pups between the vehicle-treated group and the control group. No significant differences were found in the mean birth weights between the three groups.

[Table 2] shows the correlations between placental oxidative stress markers (SOD, GPX, and MDA) and the indices of maternal hypoxia, indices of placental hypoxia, and birth outcome. There were significant positive correlations between indices of maternal hypoxia and between SOD and GPX levels, while there were significant negative correlations between indices of maternal hypoxia and MDA level [Table 2]. SOD and GPX levels showed significant negative correlations with incidences of syncytial knots, cytotrophoblastic cells, and the number of fetal capillaries, while the MDA level showed an opposite trend. The SOD and GPX levels showed significant positive correlations with the number of pups, while the MDA showed significant negative correlations with the number of pups. SOD, GPX, and MDA did not show significant correlations with the individual pup birth weight.
Table 2: Correlations between placental oxidative stress markers and the indices of maternal hypoxia, indices of placental hypoxia, and birth outcome

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The incidence of syncytial knots and cytotrophoblastic cells and the number of fetal capillaries showed significant negative correlations with the mean number of pups and insignificant correlations with the birth weight of individual pups [Table 3].
Table 3: Correlations between placental indices and fetal characteristics

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Figure 1: Photomicrograph of H&E staining of placental tissue from (A) control, (B) vehicle-treated, and (C) vitamin E-treated rats. Groups with syncytial knots (bold arrows), cytotrophoblastic cells (small arrows), and fetal capillaries (big arrows). H&E-stained section

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  Discussion Top


Vitamin E was chosen as an antioxidant in this study because its administration was shown to be safe during the entire pregnancy[11]. Vitamin E can also prevent chronic diseases, especially those that are believed to have an oxidative stress component, while other antioxidants such as vitamins B and C do not have this ability.[12]

Hypobaric hypoxia is known to induce the erythropoietin gene, increasing hemoglobin concentrations and hematocrit values.[13] This study showed that vitamin E administration to pregnant rats at high altitude reduces maternal hypoxia. Our findings agreed with other studies in the field[14]. The possible mechanism by which vitamin E reduces the maternal hemoglobin concentration and hematocrit value is that vitamin E may have reduced hypoxia-induced factor 1 (HIF1) gene expression that stimulates erythropoietin secretion.[15]

The observed reduction in the placental oxidative stress markers seems to be secondary to the improvement in maternal hypoxia. The indices of maternal hypoxia (hemoglobin concentrations and hematocrit values) showed significant negative correlations with the placental antioxidants SOD and GPX, and significant positive correlations with the placental oxidant MDA. These findings agree with previously published work in the field.[16] The indices for placental hypoxia (incidence of syncytial knots and cytotrophoblastic cells, and the number of fetal capillaries in terminal chorionic villi of the placentae) were significantly lower in the vitamin E-treated group than in the control group as shown in [Table 1].

The markers for placental antioxidant enzyme activity (SOD and GPX levels), which were higher in the vitamin E-treated group than in the vehicle and control groups, showed negative and significant correlations with the indices for placental hypoxia, whereas the placental oxidant MDA showed a positive correlation with the indices of placental hypoxia. Such relationships may explain the effect of vitamin E reducing placental oxidants and increasing placental antioxidants, which resulted in a decrease in placental hypoxic changes in pregnant rats at high altitude. No data are available on the effect of vitamin E on placental morphology at high altitude. However, studies performed in other conditions of placental hypoxia such as preeclampsia and gestational diabetes revealed that vitamin E reduced trophoblast proliferation[17] and angiogenesis that was caused by oxidative stress.[18]

A less hypoxic placenta resulting from vitamin E administration improved birth outcomes by significantly increasing the number of pups, thereby preventing fetal loss. These results are consistent with other studies. Abdullah and Ibrahim[19] reported a reduction in the number of pups in albino rats that were exposed to oxidative stress. Similarly, the administration of vitamin E to pregnant ewes during late pregnancy was found to reduce the number of stillbirths and increase the number of lambs that were born.[20] However, the improvement in the placental condition was not significantly reflected in the birth weight of the pups, although it did increase in the vitamin E-treated group compared with the control groups. The lack of the significant increase in birth weight of newborn pups may be because of the short duration of pregnancy in rats (3 weeks) compared to humans (40 weeks) or because of an insufficient dose of vitamin E.

One limitation of this study was that only measuring the oxidative stress activity at birth might not provide a complete picture of maternal oxidative stress during pregnancy because oxidative stress markers may change significantly during pregnancy. However, these marker levels may reasonably cover a critical period of rapid fetal growth at full term.[21]


  Conclusion Top


Administration of vitamin E to pregnant rats at high altitude improved birth outcomes, as shown by the significant increase in the number of newborn pups. A controlled trial to investigate the effect of vitamin E therapy on pregnancy outcomes in pregnant women at high altitude is required.

Conflicts of Interest: None



 
  References Top

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2.
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Jensen GM, Moore LG. The effect of high altitude and other risk factors on birth weight: independent or interactive effects? Am J Public Health 1997;87(6):1003- 7.  Back to cited text no. 9
    
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Khalid MEM, Ahmed HS, Osman OM, Al Hashem FH. The relationship of birth weight, body shape and body composition at birth to altitude in Saudi Arabia. Int J Morphol 2016;34(3): 1109-1116.  Back to cited text no. 10
    
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Boskovic R, Gargaun L, Oren D, Djulus J, Koren G. Pregnancy outcome following high doses of Vitamin E supplementation. Reprod Toxicol 2005;20(1):85-88.  Back to cited text no. 11
    
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Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willet WC. Vitamin E consumption and risk of coronary disease in women. N Engl J Med 1993;328(20): 1444-1449.  Back to cited text no. 12
    
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Gonzales GF, Steenland K, Tapia V. Maternal hemoglobin level and fetal outcome at low and high altitudes. Am J Physiol Regul Integr Comp Physiol 2009;297(5):R1477-R1485.  Back to cited text no. 13
    
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Richter HG, Camm EJ, Modi BN, Naeem F, Cross CM, Cindrova-Davies T, Spasic-Boskovic O, Dunster C, Mudway IS, Kelly FJ, et al. Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats. J Physiol 2012;590(6):1377-87.  Back to cited text no. 14
    
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Bell EL, Chandel NS. Mitochondrial oxygen sensing: regulation of hypoxia-inducible factor by mitochondrial generated reactive oxygen species. Essays Biochem 2007;43(Aug):17-27.  Back to cited text no. 15
    
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Chappell LC, Seed PT, Kelly FJ, Briley A, Hunt BJ, Charnock-Jones DS, Mallet A, Poston L. Vitamin C and E supplementation in women at risk of preeclampsia is associated with changes in indices of oxidative stress and placental function. Am J Obstet Gynecol 2002;187(3):777-84.  Back to cited text no. 16
    
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Schwendemann W, Hou X, Brost B, Jiang SW. Vitamin E blocks ROS-mediated syncytin expression and trophoblast differentiation. Am J Obstet Gynecol 2007;197(6): S114.  Back to cited text no. 17
    
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Poston L, Igosheva N, Mistry HD, Seed PT, Shennan AH, Rana S, Karumanchi SA, Chappell LC. Role of oxidative stress and antioxidant supplementation in pregnancy disorders. Am J Clin Nutr 2011;94(6 Suppl):1980S-1985S.  Back to cited text no. 18
    
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Abdullah QH, Ibrahim SA. Impact of oxidative stress on pregnancy outcome in albino rats. Iraqi J Vet Sci 2012;26(2):93-100.  Back to cited text no. 19
    
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Dønnem I, Randby AT, Hektoen L, Avdem F, Meling S, Våge ÅØ, Ådnøy T, Steinheim G, Waage S. Effect of vitamin E supplementation to ewes in late pregnancy on the rate of stillborn lambs. Small Rumin Res 2015;125(Aug): 154-162.  Back to cited text no. 20
    
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Jaakkola JJK, Jaakkola N, Zahlsen K. Fetal growth and length of gestation in relation to prenatal exposure to environmental tobacco smoke assessed by hair nicotine concentration. Environ Health Perspect 2001;109(6):557- 561.  Back to cited text no. 21
    


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