Melatonin-Attenuated Oxidative Stress in High-Risk Pregnant Women Receiving Enoxaparin and Aspirin

Objective In pregnancy, reducing inflammation and oxidative stress is important. Administration of melatonin during pregnancy can improve reproductive performance by improving the placental antioxidant system and inflammatory response. This investigation was carried out to evaluate the beneficial impact of melatonin on the oxidative stress state among high-risk pregnant women receiving enoxaparin and aspirin. Methods In this double-blind, placebo-controlled trial, 40 pregnant women, aged 15–45 years at 6 weeks of pregnancy, were randomly selected and divided into intervention and control groups. The control group received prophylaxis enoxaparin and aspirin once daily between 6 and 16 weeks of pregnancy. The intervention group was taken enoxaparin and aspirin for 9 weeks and melatonin once daily from the sixth week of pregnancy to delivery time. Blood samples were taken to measure some oxidative stress biomarkers including total antioxidant capacity (TAC), malondialdehyde (MDA), total thiol (T-SH), protein carbonyl (PCO), and nitric oxide (NO). The level of high-sensitivity C-reactive protein (hs-CRP) was also determined. Results TAC and T-SH levels increased significantly in the intervention group in comparison with the control group. Melatonin administration compared to the control group led to a significantly decreased level of NO and an insignificant hs-CRP level. Conclusion Melatonin supplementation in high-risk pregnancy had favorable effects on TAC, T-SH, NO, and hs-CRP levels, improved antioxidant activity, and reduced inflammation. More studies are needed in different pregnancy conditions along with the measurement of different biomarkers.


Introduction
Pregnancy is a physiological state associated with increased oxidative stress resulting from increased metabolism and oxygen demand of placental and fetal tissue [1]. Furthermore, oxidative stress is implicated in hypoxia of compromised pregnancies such as preeclampsia (PE), gestational diabetes mellitus, and maternal undernutrition [2,3]. Several studies have indicated that expanded maternal oxidative stress due to increased reactive oxygen species (ROS) production and impaired antioxidant defense mechanisms is related to fetal growth retardation. Tese detrimental impacts of maternal oxidative stress on birth results have been studied by evaluating the maternal oxidative stress status at diferent times throughout pregnancy [4]. At frst, the placenta has a low-oxygen environment, and as it matures and its vessels develop, it changes to an oxygen-rich environment and its abundant mitochondrial mass increases the production of ROS. Nitric oxide (NO) is also produced locally by the placenta contributing to the progress of oxidative stress [5]. While antioxidant activity gradually becomes desirable over oxidation during normal pregnancy, it appears to be an inadequate increase in antioxidants to neutralize the elevated free radicals production [6]. Controlling infammation and oxidative stress is a critical part of a healthy pregnancy. Nevertheless, when infammation is dysregulated, it puts pregnancy at risk owing to some downstream physiological consequences, such as disorders of immune function, fetal development, endocrine activity, and vascular physiology [7]. Tumor necrosis factor-alpha (TNF-α) and hs-CRP are common markers indicating elevated systemic infammation and are associated with disease in pregnancy [8]. Some investigations showed that the use of low molecular weight heparin (enoxaparin) and aspirin to improve pregnancy outcomes has not been so favorable [9,10]. Terefore, the reduction of adverse complications resulting from infammation and oxidative stress in pregnant women requires applying various strategies including antioxidant supplementation and using oxidative stresslowering and anti-infammatory compounds [11].
Melatonin (5-methoxy-N-acetyltryptamine) is an endogenous lipid-soluble hormone, predominantly produced in the pineal gland that is best recognized for its role in providing circadian and seasonal timing cues [12]. Additionally, it has been known as a potent antioxidant. Te mechanism of its performance includes scavenging free radicals, inducing antioxidant enzymes, such as glutathione reductase and glutathione peroxidase, as well as inhibiting the prooxidative enzyme nitric oxide synthase (NOS). Melatonin supplementation for pregnancy has been investigated for a wide range of conditions [13]. Melatonin administration during pregnancy could ameliorate maternal-placental-fetal redox status and reproductive performance by improving the placental antioxidant system and infammatory response [14]. Also, melatonin is important in high-risk pregnancy for its potent antioxidant and anti-infammatory efects, genic receptor expression, and regulation of the circadian rhythm [15]. Considering the increase in oxidative stress and infammation in women with high-risk pregnancy and considering the antioxidant and anti-infammatory properties of melatonin and the fact that the efect of this supplement on high-risk pregnancy has not been investigated, this study aims to investigate the efect of melatonin on the level of oxidative stress and infammatory markers in high-risk pregnant women receiving enoxaparin and aspirin.

Materials and Methods
Tis randomized, double-blind, placebo-controlled trial was conducted in Imam Sajad Hospital, Yasuj, Iran, from 2020 to 2022. Forty persons in the age range of 15-45 among high-risk pregnant women (based on the Venous thromboembolism risk score during pregnancy) [16] receiving prophylaxis enoxaparin and aspirin from the sixth to sixteenth week's gestation were recruited in this study. Exclusion criteria in this study are pregnant women with the following diseases: diabetes mellitus, high blood pressure, metabolic diseases, rheumatological diseases, bone diseases, hemolytic diseases, endocrine diseases, and malabsorption, as well as pregnant women with a history of using corticosteroids, ocular cicatricial pemphigoid (OCP), levothyroxine, progesterone, supplements (such as iron, calcium, and folic acid), smoking, other addictive substances, and alcohol consumption. Finally, a total of 40 pregnant women remained in the study. Participants were randomly divided into intervention and control groups (n � 20 patients per group). Te study was carried out based on the guidelines set out in the Declaration of Helsinki. Te Ethics Committee of the Yasuj University of Medical Sciences approved the study, and written informed consent was acquired from all subjects. Te study is registered in the Iranian Registry of Clinical Trials (IRCTID: IRCT20210608051518N1, https://www.irct.ir/trial/60425).

Study Design.
Te control group received prophylaxis enoxaparin and aspirin once daily between 6 and 16 weeks of pregnancy. Te intervention group took enoxaparin and aspirin between 6 and 16 weeks of pregnancy and melatonin once daily from the sixth week of pregnancy to delivery time. In this study, the assignment of subjects to the two intervention or control groups was unknown to the patients and the investigator, and the coding program was conducted by a third person who was unfamiliar with the study.
Venous blood samples (10 ml) were taken 48-72 hours before the start of administration (baseline) and after delivery, during the study period. Te blood samples were centrifuged at 3500 rpm for 10 minutes, and the serum was aliquoted and stored at −20°C for evaluation of parameters related to oxidative stress and infammation such as MDA, TAC, T-SH, NO metabolite, protein carbonyl, and hs-CRP. Serum hs-CRP concentration was measured using the ELISA kit. Te nitrite level was determined as an index of NO production using the Griess method [17]. Te plasma level of TAC was assayed by the ferric-reducing ability of plasma (FRAP) method suggested by Benzie and Strain [18]. Te protein carbonyl test was performed by a spectrophotometric method [19]. Te plasma MDA level was estimated using the TBA reaction assay [20]. Te total thiol (T-SH) level was measured based on the reaction with DTNB [21].

Statistical Analyses.
Te data were analyzed using SPSS software using descriptive and inferential statistics. Te normality of quantitative data was investigated using the Shapiro-Wilk test. An Independent t-test was used to compare the mean of normal variables between the two groups, and the paired t-test was utilized to compare the scores of variables before and after administration in each group. In cases of non-normality of data distribution, the signifcance of the diference in the average change after administration compared to the baseline state in each group was performed using the Wilcoxon test, and the Mann-Whitney was used to compare the two groups.

Efect of Melatonin on Oxidative Stress and Infammatory
Parameters. As shown in Table 1, TAC and T-SH levels increased signifcantly in the intervention group in comparison with the control group. However, the administration of melatonin compared to the baseline value and the control group led to a considerable decrease in levels of serum NO (p � 0.004). We did not observe a signifcant efect of melatonin on plasma MDA levels. Te PCO content decreased signifcantly in both the intervention and control groups compared with the baseline value. However, the reduction in the control group was more obvious compared to the melatonin supplementation group. Te CRP level reduced dramatically in the intervention group at the end of the study (4.38 ± 0.87) in comparison with the basic feature (12.85 ± 9.67). Nevertheless, this alteration was not considerable compared to control subjects.

Discussion
Gestation is a physiological condition related to elevated metabolic demands and an increased need for tissue oxygen. Tis increased oxygen demand strengthens the rate of ROS generation, particularly in the second half of pregnancy. Te last trimester of gestation is an important time of rising fat catabolism, insulin resistance, and the production of free fatty acids which result in increased hydrogen peroxide production [22]. Te antioxidative defense mechanism sounds changed throughout pregnancy [6,23]. Evaluation of the level of different oxidative stress biomarkers provides guidelines for the detection and treatment of pregnancy complications such as miscarriages and PE [22]. We observed that administration of melatonin among pregnant women receiving prophylaxis enoxaparin and aspirin resulted in a signifcant decrease in NO and hs-CRP, as well as an increase in the serum TAC levels. We did not fnd any signifcant efect of melatonin consumption on MDA and PCO levels.
Our investigation revealed that taking melatonin in highrisk pregnant women led to a signifcant increase in plasma TAC and T-SH levels compared to the control group. Total antioxidant capacity may ofer more remarkable scientifc clues in comparison to the assessment of single biomarkers because of the cumulative efects of the whole antioxidants in plasma. An advanced antioxidant capability in plasma illustrates the improvement of antioxidant status in the body or the presence of a free radical regulatory process [24]. In agreement with our study, Bouroutzika et al. showed that exogenous melatonin increased TAC levels signifcantly in people under heat stress [25]. Moreover, in a study by Mistraletti, melatonin administration resulted in a considerable rise in TAC levels in intensive care unit (ICU) patients compared to the control group [26]. One study proved that melatonin present in beer contributes to the serum total antioxidant, and controlled beer consumption can protect people against oxidative stress [27]. Free radical scavenging activity of melatonin has been shown in several studies [28][29][30]. In this regard, it has revealed better performance in comparison with intracellular scavengers such as glutathione and vitamin E [31,32]. In addition to these direct efects of melatonin, there are also indirect antioxidant efects. Tus, melatonin stimulates GPX activity and inhibits NOS [33]. Total thiol (T-SH) plays an important role in the antioxidant defense system against ROS and other free radicals [34]. In line with our study, T-SH levels increased signifcantly after melatonin administration in hyperglycemia-induced liver damage in rats [35]. Given the increase in TAC and T-SH levels following melatonin consumption, this research confrmed the antioxidant activity of melatonin shown by previous studies.
Following NOS inhibition, melatonin can decrease the production of free radical nitric oxide. NO itself can be toxic; but in addition, it can be broken down into peroxynitrite anions and fnally into the highly toxic OH [33]. Pathophysiology of PE as a multifactorial pregnancy disease could result from abnormal placentation and endothelial dysfunction due to reduced bioavailability of NO. Oxidative stress is suggested to play a critical role in the reduced NO bioavailability in PE pathophysiology through some mechanisms such as inhibiting of eNOS and subsequent defect of NO biosynthesis or via the formation of peroxynitrite by the reaction of NO with the radical anion superoxide [36]. In this study, administration of melatonin from week 6 of pregnancy to delivery resulted in a dramatic decline in the serum level of NO compared with baseline and placebo groups. In a study by Qin et al., results indicated that melatonin considerably inhibited the NOS activity and NO production in LPS-induced acute lung injury in mice [37]. NOS is assumed a prooxidative enzyme, and any factor that decreases its activity would be considered an antioxidant. Melatonin inhibits the activity of NOS, in addition to its NO and peroxynitrite scavenging activity [38].
Exposure of lipids to oxidant compounds leads to stimulation of lipid peroxidation which is a biologically important process, producing various end products such as MDA [39]. We observed that melatonin consumption in the intervention group did not afect serum MDA levels. In contrast with the current study, following melatonin administration in patients with type 2 diabetes mellitus, the serum level of MDA reduced signifcantly [40]. Te same result was obtained in terms of the PCO content. However, in the control group, its level showed a signifcant decrease at the end of the study compared with the basic feature.
Several studies indicate that infammation factors and oxidative stress have negative efects on pregnancy and fetal growth. It can lead to the cause of improper implantation of embryos, premature births, low birth weight, birth defects, and miscarriages [11]. We found that using melatonin reduced serum hs-CRP levels throughout pregnancy. In the present work, Jamilian et al. [41] found that 5 mg melatonin administration twice a day for 12 weeks caused a signifcant reduction in the level of CRP in women with polycystic ovary syndrome. Increased ROS generation can lead to infammation. Melatonin as an antioxidant can play a pivotal role in preventing infammatory processes through its free radical scavenging activity [28]. Among the limitations of this study is the small sample size of patients, which suggests that future studies should be conducted with a larger sample size and in diferent ethnicities.
Evidence-Based Complementary and Alternative Medicine 3

Conclusion
Melatonin supplementation from the sixth week of pregnancy until delivery in high-risk women increased the serum levels of TAC and T-SH, as well as reduced NO and hs-CRP but did not infuence MDA and PCO levels. Tis suggests that melatonin administration can improve antioxidant activity as well as reduce infammation and may provide a useful therapeutic approach for managing pregnancy outcomes associated with ROS production. More investigations with more participants are required to confrm this approach. In addition, further studies should evaluate other biomarkers related to oxidative stress and infammation to investigate the underlying mechanism of the melatonin efect in ameliorating pregnancy complications caused by oxidative stress more efectively.

Data Availability
Te data supporting the fndings of this study are available within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.