Introduction

Preeclampsia (PE) and related hypertensive disorders of pregnancy complicate 2–5 percentages of all pregnancies in developed countries and are major contributors to maternal and neonatal morbidities and mortalities [1, 2]. PE and eclampsia rank second or third in all worldwide ranking of maternal death causes. In Iran, PE is reported to be involved in 12% of pretermed neonate deliveries and 18.2% of maternal deaths. Also, the prevalence of the disease is not equal among different ethnic groups [3,4,5]. PE is a major disease of human pregnancy, marked by hypertension and proteinuria, appearing during the second or third trimester of gestation [6, 7]. There are risk factors and clinical factors that significantly increase PE risk, including the extremes of maternal age, chronic hypertension, family predisposition for PE and eclampsia, polycystic ovarian syndrome, nulliparity, new male partner, multiple gestations, obesity, sleep disordered breathing, preexisting diabetes, and various infections such as periodontal disease, urinary tract infections, renal disease, and helicobacter pylori. Also, in terms of obstetric history, vaginal bleeding for at least 5 days during pregnancy increases PE risk [8, 9]. Despite several studies on PE, its exact etiology remains poorly understood. However, it is known that placental changes occur early in pregnancy, associated with an imbalance between the antioxidant defense system, and the generation of reactive oxygen species (ROS) characterizing oxidative stress. Also, there is a generalized inflammatory process, as well as the presence of progressive vascular endothelial damage, which culminates in placental dysfunction [10, 11]. However, the placenta is naturally protected by some antioxidant defenses. These antioxidant enzymes’ defense systems help to prevent the placenta from undue harm. This is the basis of the involvement of micronutrients and other antioxidants in the etiopathogenesis of PE [12]. In addition, hypertension, dyslipidemia, insulin resistance, endothelial dysfunction, dysfunction hemoglobin, platelets, hepatic transaminases, protein/creatinine ratio, and uric acid, and vascular impairment have all been observed in PE [13]. Also, PE has negative effects on the psycho-emotional state (depression and anxiety), sleep patterns, and psychopathological symptoms [14, 15].

Se is an essential micronutrient, which plays an important role in preserving many natural body functions. The biological function of Se is related to its incorporation through selenocysteine (SeCys) into the structure of proteins important for metabolism [16,17,18]. Se is present in both inorganic and organic forms. The organic form is the Se contained in amino acids termed as selenomethionine (SeMet) and SeCys, while the inorganic forms (e.g., selenate, selenite, selenide, and elemental Se). The inorganic form of Se is a major source of dietary Se in the human population. Animals that are raised on Se-containing soil are a rich source of selenoproteins (SePs) [16, 19]. SePs play various functions in the human body, including cell signaling and redox reactions, selenium transport (selenoprotein P), and activation and proliferation of immune cells (selenoprotein K) [17, 20]. It constitutes an integral part of selenoproteins and some antioxidant enzymes such as thioredoxin reductase (TRxR), glutathione peroxidase (GPx), and iodothyronine deiodinase (DIO), which protect cells from the damaging effects of free radicals produced during oxidation [16]. Also, Se is a component of other enzymes, particularly DIO which catalyzes the deionization of thyroxine (T4) to triiodothyronine (T3). Deiodinases play a key role in the regulation of thyroid hormones. They are responsible for the control of proper growth, development, and cell metabolism [16]. Additionally, Se plays a key role in the immune system regulation. The element stimulates the immune system to enhance the production of antibodies (IgM and IgG) and causes enhanced activity of macrophages and T cells [21]. Also, the protective effects of Se in the etiology of cancer result from its effect on cell membranes protecting against oxidative stress, and the stabilizing effect on DNA and increase of cellular immune response. It has been indicated that Se inhibits tumor cell proliferation via the effect exerted on Bcl-2 apoptosis suppressor gene and the expression of p53 tumor suppressor gene [18, 22]. Protein hydrophobic interactions induced by a variety of nutritional and environmental factors can provide rational explanation for a number of pathologic conditions. The deficiency of Se leads to elevated pathological changes, inflammation, and oxidative stress [23, 24]. Several studies have assessed the relationship between the level of body Se and the incidence, severity, and mortality of different disease such as cancer, COVID-19, Alzheimer, respiratory diseases, hepatic diseases, polycystic ovary syndrome (PCOS), cardiovascular disorders, neurologic, and other degenerative diseases [25,26,27,28,29].

Previous studies have reported controversial results between Se levels and PE, recurrent abortions, and intrauterine growth restriction (IUGR) [30, 31]. Se is a component of some vital enzymes that play roles in the antioxidant activities, so if serum values of these enzymes are low, it can result in numerous pathways that can lead to PE [32]. Also, with increased understanding of the pathogenesis of PE, clinical prediction models of the disease have gradually diversified. Doppler has more recently shown some promise [33]. First and second trimester uterine artery Doppler assessments have high predictive value for clinical outcome (prediction of PE and IUGR) [34]. Uterine artery PI has been used as a marker of PE and fetal growth restriction (FGR), in the presence of which PI increases due to the elevation in uterine artery impedance [35]. Combination of the clinical, biochemical, and biophysical markers is likely to increase the predictive power of screening examinations [36]. Currently, the implementation of serum biomarker screening quad test (AFP, alpha-fetoprotein; BHCG, beta-human gonadotropin; uE3, unconjugated estriol; and IHA, inhibin-A) for fetal aneuploidy has commenced worldwide, and many studies have shown that additional information derived from such screenings, other than aneuploidy risk estimation, can also be used to identify the risk of adverse obstetric outcomes, such as fetal growth restriction, PE, and preterm birth [37, 38].

The hypothesis of this clinical trial is that clinical outcomes, metabolic profiles, PI of uterine artery, and mental health might improve among high-risk mothers in terms of PE screening with quadruple tests. A literature review revealed limited information about the effects of Se on clinical profiles, mental health, PI and metabolic biomarkers in high-risk mothers screening with quadruple tests. Therefore, in this clinical trial, we aimed to evaluate the effects of Se in pregnant women at the risk of PE screened with quad test.

Methods

Trial Design and Participants

This research was a prospective randomized double-blind placebo-controlled clinical trial. The current study was done among 60 high-risk pregnant women in terms of PE that were screened by quad test, aged 18–40 years who referred to Shahid Beheshti Hospital, Kashan, Iran, between May 2020 and December 2020. Participants who consumed Se supplements during the past 12 weeks and pregnant women with diabetes mellitus, thyroid disorders, hypertension, chronic proteinuria, abnormal scan anomaly, multiplication, AIDS, hepatitis B or hepatitis C, intolerance to yeasts containing selenium supplements, and bone and chromosomal abnormalities were excluded.

Ethics Statements

This clinical trial was done according to the guidelines laid down in the Declaration of Helsinki. This study was approved by the ethics committee of Kashan University of Medical Sciences (IR.KAUMS.MEDNT.REC.1399.008) and registered on the Iranian Registry of Clinical Trials website (http://www.irct.ir: IRCT20200608047701N1). All subjects provided informed written consent before recruitment.

Clinical Trial Procedures

Randomization was done using computer-generated random numbers by a trained staff at the obstetrics and gynecology clinic. Randomization and allocation were concealed to the researchers and participants until the final analyses were completed. Another person at the clinic, who was not involved in the trial and not aware of random sequences, assigned the participants to the numbered bottles of supplement. The treatment group (n = 30) received 200 µg/day Se as Se amino acid chelate (Alfa Vitamins, Florida, USA) from 16 to 18 weeks of gestation for 12 weeks with meals. Placebo group (n = 30) received capsules in size, shape, color, packaging, taste, and smell as Se. Participants were asked not to take other Se supplements during the 12-week intervention, however maintaining their regular diet and physical activity throughout the intervention period. Adherence to supplements and placebos was determined by counting the tablet containers. To increase compliance, all participants received brief daily cell phone reminders to take the Se and Placebo.

Assessment of Anthropometric Measures

Weight and height of participants were determined in an overnight fasting status using a standard scale (Seca, Hamburg, Germany) at the baseline and after the 3-month intervention. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared.

Assessment of Outcomes

The primary outcome measurement was high-sensitivity C-reactive protein (hs-CRP), and the secondary outcome measurements were clinical outcomes, other metabolic profiles, and uterine artery PI.

Biochemical Measures

At first and after the end of the clinical trial, 10 mL of blood samples and a urine sample were taken from each subject at the Kashan Reference Laboratory in an early morning after an overnight fast (Kashan, Iran). Blood was collected in two separate tubes: (A) one without EDTA to separate the serum, in order to determine serum Se, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), uric acid, prolactin, and hs-CRP concentrations and (B) another one containing EDTA to examine plasma nitric oxide (NO), total antioxidant capacity (TAC), total glutathione (GSH), and malondialdehyde (MDA). Also, protein/creatinine ratio was measured in urine samples. Blood samples were immediately centrifuged (Hettich D-78532, Tuttlingen, Germany) at 3500 rpm for 10 min to separate serum and plasma. Then, the samples were stored at − 80 °C before analysis at the KUMS reference laboratory. Hemoglobin, hepatic transaminases, protein/creatinine ratio, and uric acid were measured on the day of blood and urine sampling with enzymatic kits (Pars Azmun, Tehran, Iran) with intra-assay coefficient of variations (CV s) below 5%. Platelet counts in venous whole blood samples were determined with Sysmex KX-21 Haematology analyzer (Sysmex Corporation, Japan). Also, the samples were analyzed to determine the serum Se levels, using atomic absorption spectrometry method (A Perkin-Elmer) [39]. Plasma TAC concentrations were measured using the method of ferric reduction antioxidant power developed by Benzie and Strain [40]. Plasma GSH using the method of Tipple et al. [41], and MDA concentrations by the thiobarbituric acid reactive substances spectrophotometric test were determined with inter- and intra-assay CVs below 5% [42]. A commercial ELISA kit (LDN, Nordhorn, Germany) with inter- and intra-assay CVs below 7% determined serum hs-CRP levels. The plasma NO levels were determined using Griess method [43]. Measurements of metabolic biomarkers were performed in a blinded fashion, in duplicate (pre- and post-intervention) after the intervention, in the same analytical run, and in random order to reduce systematic error and inter-assay variability.

Clinical Measures

Mean validated automated devices (3BTO-A2, Micro life, Taipei, Taiwan) measured arterial pressure. Women were in the sitting position with their arms supported at the level of the heart, and a small (22 cm), normal (22–32 cm), or large (33–42 cm) adult cuff was used, depending on the mid-arm circumference. After resting for 5 min, two recordings of blood pressure were made in both arms simultaneously. We calculated the final MAP as the average of all four measurements [44]. In the present study, Beck’s Depression (BDI), Anxiety (BAI), and the Pittsburgh Sleep Quality Index (PSQI) inventories were used to assess level of depression, anxiety, and sleep quality, respectively [45,46,47]. BDI is a 21-question and BAI is a 20-question inventory, where each question scored between 0 and 4, and higher scores indicate the higher levels of depression and anxiety, respectively. The Persian versions of both inventories were validated in the previous studies [48, 49]. The PSQI includes a scoring key for calculating a patient’s seven sub-scores, each of which can range from 0 to 3. The sub-scores are tallied, yielding a “global” score that can range from 0 to 21. A global score of 5 or more indicates poor sleep quality; the higher the score, the worse the quality [47].

Radiology Measures

Trans-abdominal color Doppler ultrasound was performed before and after the intervention to evaluate the left and right uterine artery PI, and the average value was recorded. This examination was performed using trans-abdominal ultrasound with color-flow mapping. When three similar consecutive wave forms were provided, the PI was measured on each side.

Sample Size

We did not detect a similar trial about the effects of Se supplementation on uterine artery PI, clinical signs, and metabolic profiles in high-risk mothers in terms of PE screening with quad marker for determining the sample size based on primary outcome. Hence, the sample size was calculated based on the effects of Se supplementation on hs-CRP status in pregnant women at risk for intrauterine growth restriction (IUGR). Type one (α) and type two (β) errors were defined as 0.05 and 0.20 (power = 80%), respectively. Based on a previous study [50], we used a standard deviation (SD) of 1.8 and 1 for Se and placebo groups, and a difference in mean (d) of 1.2, considering hs-CRP as the key variable. We needed 24 patients in each group. Assuming a dropout of 6 persons per group, the sample size was considered to be 30 patients in each group.

Statistical Analyses

The Kolmogorov–Smirnov test was used to determine the normality of data. To detect the differences in anthropometric parameters among two groups, we used the independent-samples t-test. Multiple linear regression models were used to assess treatment effects on study outcomes after adjusting for baseline concentration of variables. The effect sizes were demonstrated as the mean differences with 95% confidence intervals. P values < 0.05 were considered statistically significant. Statistical analyses were done using the Statistical Package for Social Science version 18 (SPSS Inc., Chicago, Illinois, USA).

Results

Six subjects in the supplements group and four subjects in the placebo group dropped out for personal reasons (Fig. 1). Finally, 50 patients (selenium (n = 24) and placebo (n = 26)) completed the trial. No difference in mean age and anthropometric parameters was seen between the two groups (Table 1). Overall, the compliance rate was high, such that more than 90% of capsules were consumed throughout the study in both groups. No side effects were reported following the intake of Se in patients with PE throughout the study.

Fig. 1
figure 1

Summary of patient flow diagram

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Table 1 General characteristics of study participants
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Se supplementation resulted in a significant improvement in depression scores (β − 5.63; 95% CI, − 6.97, − 4.28; P < 0.001), anxiety scores (β − 1.99; 95% CI, − 2.56, − 1.42; P < 0.001), and sleep quality scores (β − 1.97; 95% CI, − 2.47, − 1.46; P < 0.001) compared with the placebo (Table 2). Compared with the placebo, Se supplementation resulted in a significant elevation in TAC (β 82.88 mmol/L; 95% CI, 3.03, 162.73; P = 0.04), and GSH (β 71.35 µmol/L; 95% CI, 5.76, 136.94; P = 0.03), and a significant reduction in hs-CRP levels (β − 1.52 mg/L; 95% CI, − 2.91, − 0.14; P = 0.03). Additionally, Se supplementation significantly increased serum Se levels (β 22.25 mg/dL; 95% CI, 18.3, 26.1; P < 0.001), and a significant decrease in UAPI (β -0.09; 95% CI, − 0.14, − 0.04; P = 0.04) compared with the placebo. Se supplementation did not affect other metabolic profiles.

Table 2 Clinical parameters, metabolic profile, uterine artery PI, and mental health scale at baseline and after 12 weeks of the intervention in high-risk pregnant women in terms of PE screening with quad marker
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Discussion

In the current study, we investigated the effects of supplementation of Se on clinical symptoms, metabolic status, and uterine artery PI in high-risk pregnant women in terms of PE screening with quad marker. We found that taking Se supplements for 12 weeks by high-risk pregnant women in terms of PE screening with quad marker improved the depression, anxiety, sleep quality scores, UAPI, Se, hs-CRP, TAC, and GSH levels, but did not affect blood pressure, hemoglobin, platelets, AST, ALT, LDH, creatinine, uric acid, protein/creatinine ratio, MDA, and NO levels. Based on these findings, supplementation of Se may be an appropriate adjunct therapy for high-risk pregnant women in terms of PE screening with quad marker. To our knowledge, this evidence is the first study to evaluate the effects of Se on clinical outcomes, metabolic biomarkers, and uterine artery PI in high-risk pregnant women in terms of PE screening with quad marker. Previous evidence has demonstrated that a small increase in Se intake in pregnant women with inadequate Se status could protect them against PE [51]. In the current study, no side effects were seen following supplementation with Se in high-risk pregnant women in terms of PE screening with quad marker throughout the study. It must be considered that mean dietary plus supplemental Se administration in our study patients was lower than upper limits (400 μg). Se is an element with a very narrow quantitative range of levels between deficiency, physiological responses, and toxic dose [16]. Excess of Se in the diet causes chronic food poisoning signs (e.g., nausea, vomiting, and diarrhea). Acute exposure to high amounts of Se leads to a general weakness and neurological disorders [52]. Also, chronic toxicity by an excess of Se leads to selenosis signs, which is manifested by changes and fragility of fingernails, hair loss, infertility, skin rash, gastrointestinal disorders, unpleasant odor in exhaled air, and nervous system disorders [16, 53]. However, data on the toxic effects of Se supplementation on health situation are conflicting. In the study conducted by Burk et al. [54], it indicated that administration of moderate 200 μg/day to large dose 600 μg/day Se was safe in volunteers ages ≥ 18 years for 16 weeks. On the other hand, gastrointestinal signs, hair loss, and memory disorder were reported as the adverse effects of Se intake [55]. However, further evidences are required about potential teratogenicity/toxicity of long-term increased Se intake in high-risk pregnant women in terms of PE screening. Although the beneficial effects of Se (100 μg/day) from the first trimester of their pregnancy until delivery for a period of approximately 6 months were reported [56], we believe that further studies are required to confirm our results.

Effects on Clinical Outcomes and Uterine Artery PI

Previous studies have shown that Se administration has the potential to decrease the risk of PE in pregnant women with low Se levels. Se is known as an essential trace mineral that has many important functions at the concentration of the cell and organism in human and animal health, and so it is relevant to various pathophysiological conditions [57]. We found that supplementation of Se after 12 weeks to high-risk pregnant women in terms of PE screening with quad marker improved the Se levels, and UAPI, but it did not affect the total clinical outcomes, compared with the placebo. Data documenting the effects of supplementation of Se on clinical outcomes and uterine artery PI in high-risk pregnant women in terms of PE screening with quad marker are limited. In a study by Gomez et al. [58], successive changes in uterine artery blood flow patterns between the first and second trimesters of pregnancy and their association with risk of hypertensive disorders and fetal IUGR were investigated. The results showed that compared to normal pregnancies, complication pregnancies have a higher prevalence of bilateral notch and uterine artery PI in each of the first and second trimesters. Pregnant women with sustained abnormal PI are at greater risk for adverse perinatal outcomes. In addition, in a study conducted by Mesdaghinia et al. [50], it was observed that supplementation with Se for 10 weeks led to a higher percentage of women in the Se group had PI of < 1.45 than in the placebo group.

Effects on Metabolic Biomarkers

There are placental changes in PE, linked to an imbalance between the production of ROS and the antioxidant defense system, characterizing the placental oxidative stress that leads to an increase in the production of pro-inflammatory cytokines [11]. Our study indicated that taking Se supplements for 12 weeks in high-risk pregnant women in terms of PE screening with quad marker resulted in a significant rise in plasma GSH and TAC, and a significant reduction in serum hs-CRP levels compared with the placebo, but unchanged plasma MDA and NO levels. As there is evidence that Se supplementation may have antioxidant and anti-inflammatory effects [59, 60]. In a study done by Kamali et al. [61], it was seen that the intake of 200 μg/day Se supplements for 4 weeks to coronary artery bypass grafting (CABG) surgery had beneficial effects on hs-CRP, GSH, and MDA levels, but did not affect other metabolic profiles. Also, total antioxidant status (TAS), and GSH levels had significant increase following supplementation with Se after 45 days in patients with refractory epilepsy, and epilepsy [62]. In addition, results of a meta-analysis in randomized controlled trials indicated a reduction in serum CRP and increased the glutathione peroxidase level in patients with coronary heart disease supplemented with Se [63]. However, in a study conducted by Savory et al. [64], Se supplementation at a dosage of 200 μg daily for 3 weeks among overweight adults did not alter GSH, and TAS levels. Se appeared to provide a protective effect against oxidative stress, and Ca (2 +) entry through modulation of neutrophil transient receptor potential vanilloid 1 (TRPV1) calcium channels in patients with PCOS [65]. Also, Se intake may decrease inflammation and oxidative stress through increasing selenoprotein biosynthesis, inhibiting production of ROS/RNS, and the inhibition of nuclear factor kappa light chain-enhancer of activated B cells (NF-kappa B) by modulating selenoprotein gene expressions [66,67,68].

Effects on Mental Health Parameters

Se deficiency occurred significantly more among the PE pregnant women compared to the normotensive controls [32], and low Se status might experience relatively depressed moods, and support the idea that Se plays a special role in the brain [69]. PE is a pregnancy-associated condition with complex disease mechanisms, and a risk factor for various long-term health outcomes for the infant and mother [70]. PE women most often experience complications of mood disorders in comparison with females with uncomplicated pregnancy [71]. Mental health disorder (depression, anxiety, and sleep) are highly prevalent in high-risk pregnancy such as PE [72, 73]. In our study, a 12-week Se supplementation by high-risk pregnant women in terms of PE screening with quad marker reduced depression, anxiety, and sleep quality scores compared with the placebo. In a study by Mokhber et al. [74], it showed that supplementation with 100 μg/day selenium during pregnancy might be an effective approach for the prevention of postpartum depression level in Iranian women. In addition, Se therapy (200 μg/day) to HIV/drug users led to a significant improvement in decrease anxiety but did not affect depression or distress [75]. Also, current data supports the beneficial effect of Se on central nervous system, depression, and sleep disorders [76]. However, Hawkes and Hornbostel [69] reported that selenium supplementation did not promote improvements in mood in persons eating a typical US diet. While the neuroprotective effect of Se has yet to be definitively characterized, the protective role of Se in psychological disorders could be related to its important action in antioxidant defense [77]. Additionally, Se has been shown to influence health status through its important role in immune function and neurotransmitter turnover rate [78, 79]. Also, Se intake may affect mental health parameters through modulating thyroid function, which in turn play a role in the reduction of depression score [80].

Limitations

The current study had few limitations. Firstly, sample size in our study was small. Future studies with longer duration and higher participant are needed to confirm the validity of our finding. In addition, we did not evaluate dietary intakes of study patients; however, we requested patients not to change their regular dietary intakes and physical activity. Furthermore, additional future studies are needed to examine the effect of Se supplementation on gene expression involved in metabolic profiles, and inflammation factors, as well as on Se-dependent antioxidant enzymes including thioredoxin reductase and GPx isoforms. Unfortunately, because of limited funding, we did not examine the effects of Se supplementation on these measures. In addition, the beneficial effects of Se supplementation on lipid profiles and glucose homeostasis would be of interest, and it is suggested for a future study.

Conclusion

Overall, our study demonstrated that Se supplementation for 12 weeks in high-risk pregnant women in terms of PE screening with quad marker resulted in a significant improvement of depression, anxiety, sleep quality scores, UAPI, Se, GSH, TAC, and hs-CRP levels but did not affect other clinical symptoms and metabolic profiles. This suggests that Se supplementation for 12 weeks at dosage 200 µg/day might confer advantageous therapeutic effects for high-risk pregnant women in terms of PE screening with quad marker. Further evidence is required in other diseases and for longer periods to determine the safety of selenium supplement.