108 Original ISSN 2413-0516 J Contemp Med Sci | Vol. 7, No. 2, March-April 2021: 108 – 112 Introduction Uncontrolled pregnancy is one of the most important chal- lenges of the century.1,2 Oral contraceptive pills (OCP) contin- ued to be one of the most common methods of contraception used by the majority of women. Across all ages, women in their 20s are the most likely to use OCP.3 The OCP is the commonest contraceptive method used among Saudi women.4 Combined pills and progestin-only pills are the two types of OCP.5 The usage of OCP has been shown to cause oxidative stress by enhanced depletion of anti- oxidant and increased lipid peroxidation.6 Oxidative stress is defined as an imbalance between anti- oxidants and pro-oxidants in the cells, which are manifested by high levels of free radicals.7 Free radicals are deleterious to the human body because they can retrieve electrons from var- ious molecules provoking the formation of oxidized forms so that severe oxidative stress can even trigger cell apoptosis and necrosis.8 Antioxidants systems, both non-enzymatic and enzy- matic, are produced in the body to block too much free rad- icals production.9 Non-enzymatic antioxidants are known as synthetic antioxidants or dietary supplements, including vita- min C, vitamin E, β-carotene, and so on.10 Mounting evidence suggests that oxidative stress could play a pivotal role in the pathogenesis of several diseases including inflammatory, mus- cular, cardiovascular, and neurodegenerative diseases.11 Nitric oxide (NO) is a key organizer of the endothelia functional in the cardiovascular system. Change of the redox equilibrium in the vascular system intervenes with NO prod- uct and modifies vascular homeostasis bioavailability, pro- moting the development of metabolic and cardiovascular diseases.12 Estrogen generally has diverse vascular actions via increasing the bioavailability of NO on activation of NO syn- thase, and NO is inversely associated with oxidative stress.13 Lobysheva et al.14 observed a correlation between the use of combined oral contraceptives pills (COCP) and increased oxidative stress and the decreased NO in women who used OCP. Hassan et al.15 reported that NO is significantly reduced in COCP users than nonusers. Vitamins also immediately scavenged ROS and upregu- lated the activity of antioxidant enzymes. Vitamin E is con- sidered one of the most vital antioxidants; vitamin E prevent peroxyl radicals generated by peroxidation of polyunsaturated fatty as a result of ROS, therefore, protecting cells against oxi- dation of membrane phospholipids.16 Hassan et al.15 concluded that serum vitamin E levels are significantly lower among the OCP users’ groups. Vitamins B6 and B12 play crucial inter-re- lated roles in DNA synthesis throughout the lifecycle, espe- cially during childhood, adolescence, and the reproductive years for women.17 Low vitamin B6 status has been associated with an increased risk of cardiovascular disease.18 The use of OCP causes many biochemical changes in; clotting factors, thrombosis, platelet changes, atherosclerosis, and inflamma- tory profiles.19-21 Jamil et al.22 concluded that hemoglobin levels are normal in the OCP users. Baker et al. (2016) reported that the WBC count elevates in the woman using OCP due to that the OCP may cause some internal infection in the women. CRP is generated via the liver, and CRP levels rise within the presence of any inflammatory in the body.23 Serum levels of CRP are often used to detect inflammatory signs to esti- mate cardiovascular sickness and stroke.24 Ferreira et al. (2017) found a significant increase in CRP levels, especially among overweight women who use COCP more than 3 mg/dL (a boundary value related to the evolution of cardiovascular dis- eases). This study aimed to evaluate the effect of OCP on oxi- dative stress in Saudi women. Materials and Methods The study was carried out on 55 Saudi women were divided into two groups: women taking an OCP (N=30) and women who had never taken the OCP (N=25). The ethical approval was obtained through the local ethics committee at the King Effect of oral contraceptive pills on oxidative stress in Saudi women Nadia N. Osman1,2, Dalal Mhammed Al-mutairi1,3* 1 Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. 2 Food Irradiation Research Department, National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt. 3 Biochemistry Department, Faculty of Science, Tabuk University, Tabuk, Saudi Arabia. *Correspondence to: Dalal Mhammed Al-mutairi (E-mail: dalal_9911@hotmail.com) (Submitted: 05 January 2021 – Revised version received: 29 January 2021 – Accepted: 21 February 2021 – Published online: 26 April 2021) Abstract Objective The aim of this study was to evaluate the effect of oral contraceptive pills (OCPs) on oxidative stress in Saudi women. Methods A total of 55 Saudi women were divided into two groups: users of OCP (N=30) for at least 1 year and non-users (NOCP, N=25). The demographic data were obtained through face-to-face interviews performed by the researcher. Blood specimen from both groups were drawn after 8 h of fasting to estimate serum total antioxidant (TAOC), nitric oxide (NO), C-reactive protein (CRP), vitamins (E, B6, B12), and some hematological parameters: hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC). Results The results showed a significant decreased in serum TAOC, NO, vitamins E, and B6 accompanied with high significant increase in CRP level while no significant changes B12, HB, RBC, and WBC were observed in the OCP users as compared to the control group. Conclusions The results of this study indicated that the use of OCP resulted in low levels of the total antioxidant, nitric oxide, vitamins E, and B6 with a significant increase in CRP. Women who used OCP may be more susceptible to oxidative stress by enhanced depletion of antioxidants. 109 Original Effect of oral contraceptive pills on oxidative stress in Saudi womenNadia N. Osman, Dalal Mhammed Al-mutairi J Contemp Med Sci | Vol. 7, No. 2, March-April 2021: 108 – 112 Abdul-Aziz University Hospital. All relevant information was recorded on predesign questionnaires. A blood specimen (6  ml) was collected by venipuncture after 8–12 h of fasting were collected to estimate various parameters: total antioxi- dants, NO, vitamins E, B6, and B12 by colorimetric method using ELISA Kit. Hb, RBC, and WBC were estimated using a hematology analyzer. The CRP level was also determined by a commercial Kit from SIMENS. Statistical Package for Social Science (SPSS) computer software was used for data analysis. Means ± standard devi- ation (SD) was calculated. Independent t-test and Spearman’s correlation were used to analyze the differences between bath OCP users and non-user control. The results were considered statistically significant at p ≤ 0.05. Results The current results showed that total antioxidant and nitric oxide were significantly decreased (p=0.000) among women using oral contraceptive when compared to control group (Table 1). As shown in Table 2, there was a highly significant (p=0.000) reduction in the vitamins E and B6 in OCP users compared to the control group. However, there was no signifi- cant difference (p=0.32) in vitamin B12 between the cases and control. Data demonstrated in Table 3 showed that OCP induced a non-significant changes in Hb, RBC, and WBC (p=0.06, 0.52, 0.90 respectively), associated with a highly significant increase (p=0.000) in CRP, compared to NOC. From Table 4, it could be observed that there was a sig- nificant changes association between duration of OCP use and TAOC, NO, CRP, vitamins E, and B6. Moreover, Hb was significantly higher started after 5 years, while, vitamin B12 and WBC were significantly different started 3–4 years. No significant differences in the RBC were noticed along with the tested duration (1–7 years) (Table 4). Discussion Oral contraceptive steroids are one of the factors that enhance oxidative stress and lead to the formation of free radicals. Oxidative stress constitutes a disturbance caused by an imbal- ance between the generation of free radicals and the antiox- idant system, which causes damage to biomolecules. This, in turn, may lead to the occurrence of many chronic degenerative diseases.25 The free radical-mediated peroxidation of membrane lip- ids increases permeability and membrane fluidness with loss of its integrity that leads to cell damage.26 Antioxidants work cooperatively in biological systems, and it is important to be able to correlate antioxidant measurements with antioxidant defenses and disease prevention. It is therefore recommended to study “total antioxidant capacity”, rather than monitoring individual antioxidant levels, which may be less affected by dietary habits.27 In this study, the total antioxidant level was significantly decreased among women using OCP. This result was in agreement with Palan et al. (2010) and Adejumo et al.28 The behavior of molecules related to oxidative stress can differ according to types and doses of estrogen, progestogen, or the particular compounds of estrogen and progestogen.29 Estrogens display an antioxidant activity by inhibiting the expression and function of the NADP+/NADPH oxidase,30 by increasing the expression and level of activation of the endo- thelial isoform of the nitric oxide synthase (eNOS)31 and by stimulating the expression and activity of the manganese SOD (MnSOD) and of the extracellular SOD (ecSOD).32 These anti- oxidant activities of estrogens are counteracted by progestins via the activation of the NADPH oxidase and the inhibition of the expression and activity of the MnSOD and of the ecSOD.33 Therefore it implies that the counteractive effect of progestin would result in a decrease in the serum total antioxidant status especially, in women taking either COCP.28 In the current study, there is a significant decrease in serum NO in the OCP users as compared to the control group. Table 1. The effect of OCP on Total antioxidant and NO. Variables Group N Mean p-value Sig TAOC NOC 25 21.82 0.000 H. Sig OCP 30 4.89 NO NOC 25 0.000 H. Sig OCP 30 22.05 NOC non-oral contraceptives, OCP oral contraceptives pills, TAOC total antioxidant, NO nitric oxide. Values are as Mean±SD, p<0.05 significant as compared to the non-users. Table 2. The effect of OCP on vitamins. Variables Group N Mean p-value Sig Vitamin E NOC 25 0.000 H. Sig OCP 30 24.379.87 Vitamin B6 NOC 25 627.65123.38 0.000 H. Sig OCP 30 274.1870.24 Vitamin B12 NOC 25 267.4867.05 0.32 N. Sig OCs 30 248.46 NOC non-oral contraceptives, OCP oral contraceptives pills. Values are as Mean±SD, p<0.05 significant as compared to the non-users. Table 3. The effects of OCP on Some Hematological Parameters and CRP. Variables Group N Mean p-value Sig Hb NOC 25 0.06 N. Sig OCP 30 12.16 1.77 RBC NOC 25 4.32 0.58 0.52 N. Sig OCP 30 4.23 0.42 WBC NOC 25 6.99 1.73 0.90 N. Sig OCP 30 7.08 3.59 CRP NOC 25 0.16 0.000 H. Sig OCP 30 NOC non-oral contraceptives, OCP oral contraceptives pills, HB haemoglobin, RBC red blood cells, WBC white blood cells, CRP C-reactive protein. Values are as Mean±SD, p<0.05 significant as compared to the non-users. 110 Original Effect of oral contraceptive pills on oxidative stress in Saudi women Nadia N. Osman, Dalal Mhammed Al-mutairi J Contemp Med Sci | Vol. 7, No. 2, March-April 2021: 108 – 112 A similar result was reported by Lobysheva et al.14 Hassan et al.15 observed that taking COCP resulted in a significant decrease in NO level. Estrogen is generally has diverse vascular actions via increasing the bioavailability of NO on activation of NO synthase, and NO is inversely associated with oxidative stress.13 Decreased bioavailability of NO may result in abnor- mal reactions between the vessel wall and platelets and is thus involved in the initiation and progression of atherosclerosis.34 The present study reported a significant decrease in vitamins E, B6 and no significant change in B12 in the OCP users as compared to the control group. A similar result was reported by Palan et al. (2010), Wilson et al. (2011) and Singh et al. (2018). The study by Hassan et al.,15 observed a decrease in the serum vitamin E, especially in women taking either COCP. In contrast to our results, Mcarthur et al. (2013) and Berenson and Rahman,35 have reported that the young women using OCP had significantly lower serum vitamin B12 concentrations. Vitamin E level was significantly decreased among women using OCP. The mechanism of such an effect is unknown, but it was thought that, at its high level, certain estrogens, in par- ticular diethylstilbestrol, may produce reactive oxygen species that may attack a number of biological substances, including lipids and DNA, and could natural antioxidants may be con- sumed to trap such compounds.36 The OCP may have some relatively specific effect on tryptophan’s metabolism, which is independent of vitamin B6 levels. This effect may be pri- marily on the activity of tryptophan oxygenase since studies in rats have shown directly as well as adrenal-mediated effects of estrogens on the activity of this enzyme.37 Previous stud- ies38,39 indicated that steroids or steroid conjugates could affect the activity of the PLP-dependent enzymes of the kynurenine pathway, i.e., kynureninase and kynurenine aminotransferase. Thus, it seems most likely that the altered tryptophan metabo- lism of oral contraceptive users is the summation of hormonal effects on tryptophan oxygenase and kynureninase rather than the production of a general vitamin B6 deficiency.40.41 Vitamin B12 level was no significant difference between OCP users and nonusers because that absorption is not affected and that redistribution of B12 throughout the body could be responsible.42 In this study, level Hb and WBC were showed a non-sig- nificant decrease in non-oral contraceptive users. Regarding hematological parameters, different results were observed by several researchers. Toryila et al.43 have reported a reduction in Hb, RBC, WBC among women using COCP compared to the controls. Jamil et al.22 showed Hb levels are normal in the OCP. Al-Zayadi44 and Yeasmin et al.45 have reported increases in Hb level comparing with control groups. Toryila et al.46 observed an increase in WBC count in COC-treated female Wistar rats. The difference may be as a result of the use of dif- ferent COC with different concentrations of estrogen and pro- gesterone and the duration of use. Mooij et al. (1992) reported no significant difference on hematological parameters due to OCP use in women, but serum iron status was significantly increased for the users of OCP. In this study a no significant change in RBC among women using OCP. A similar result was reported by Al-Zayadi.44 Due to the confounding of the effects of dose and the type of hor- mone, and the formulation of the drug, it was not easy to esti- mate the effect of OCP on RBC. Even though the combination of estrogen and progesterone would be considered as advance formal of the OCP, the use of it still strongly associated with vascular disease.47 Table 4. Effect of duration of OCP use on biochemical parameters. Control N = 25 Duration of OCs 1-2 years n = 7 3-4 years n = 10 5-7 years n = 13 TAOC 21.828 ± 5.674 5.357 ± 1.651P= 0.000* 4.860 ± 1.706 P= 0.000* 4.669 ± 2.200 P= 0.000* NO 171.316± 34.8 45.771 ± 21.229P= 0.000* 41.220 ± 25.322 P=0.000* 40.892 ±21.422 P= 0.000* CRP 0.160 ± 0.071 1.614 ± 0.467P= 0.000* 1.440 ± 0.384 P=0.000* 1.608 ± 0.528 P=0.000* Vit E 96.524±25.026 23.843 ±11.652P= 0.000* 22.460 ± 8.921 P=0.000* 26.139 ± 10.081 P= 0.000* Vit B6 627.652±123.389 284.343 ± 69.892P= 0.000* 289.060 ± 92.414 P= 0.000* 257.262 ± 50.212 P= 0.000* Vit B12 267.480 ± 67.053 278.00 ± 50.471P= 0.523 224.300 ± 70.539 P=0 .049* 251.154 ± 85.517 P= 0.508 HB 11.204 ± 2.054 11.450 ± 2.441P= 0.600 12.019 ± 1.125 P= 0.547 12.657 ± 1.760 P= 0.047* RBC 4.321 ± 0.588 4.134 ± 0.360P= 0.218 4.234 ± 0.260 P=0.371 4.289 ± 0.555 P= 0.712 WBC 7.156 ± 1.604 6.370 ± 1.382P= 0.210 5.729 ± 0.811 P=0.008* 6.869 ± 1.673 P= 0.470 The values are the mean ± S.D. of parameters measured. Significantly different from control value at p<0.05*, 0.01**, 0.001*** 111 Original Effect of oral contraceptive pills on oxidative stress in Saudi womenNadia N. Osman, Dalal Mhammed Al-mutairi J Contemp Med Sci | Vol. 7, No. 2, March-April 2021: 108 – 112 The results indicated a significant increase in CRP in the OCP users as compared to the control group; this finding is in agreement with Divani et al.48, Ferreira et al., (2017), Guedes et al.49, and Oliveira et al.50 It was indicating that OCP leads to an increased subclinical inflammatory process. Divani et al.51 established that progestin increases interleukin 6 (IL-6, an inflammatory-mediated stimulation) of CRP in combination with conjugated equine estrogen and showed that IL-6 change was negatively linked to CRP change when subjects were treated only with equine estrogen. The involvement of proges- tin has been suggested to induce IL-6-mediated CRP stimula- tion, whereas other mechanisms are likely to be responsible for the development of CRP in women receiving only conjugated equine estrogen.52 This study evaluates the effects of duration of OCP use on oxidative stress, which is represented by a significant decrease in TAOC, NO, vitamins E, and B6 levels. In contrast, CRP has shown a significant increase. Egoro et al. (2018) demonstrated that elevated levels of plasma CRP in long-term users of OCs containing lower doses of estrogen and progestin composition for ≥2 years as compared to (control group). However, it was observed a significant change in Hb, WBC, and B12 after 3 years of using OCP. Similarly, Toryila et al.43 reported that long- term use of COCP might lead to more complications than short- time use. However, the long-term users of the OCP on RBC has shown no significant difference (p≥ 0.05) com- pared to the mean value of non-users of OCs (control group). Conclusion The results of this study indicated that the use of OCP resulted in low levels of total antioxidant, NO, vitamins E, and B6 with a significant increase in CRP. Moreover, there is a statistically significant relationship between OCP use and increased oxi- dative stress. Conflict of Interest None References 1. Minahan, C, O’Neill, H, Sikkema, N., Joyce, S, Larsen, B, Sabapathy, S. Oral contraceptives augment the exercise pressor reflex during isometric handgrip exercise. Physiol Rep, 2018;6:e13629. 2. Meier, TB, Drevets, WC, Teague, TK, Wurfel, BE, Mueller, SC, Bodurka, J, Dantzer, R, Savitz, J. Kynurenic acid is reduced in females and oral contraceptive users: Implications for depression. Brain Behav Immun, 2018;67:59-64. 3. Al-Mass, AA, Al-Shahrani, BS, Al-Mweisheer, AN, Tulbah, SA, Syed, S, Anwer, R, Haque, S. 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This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i2.956