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.
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.
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) . Plasma TAC concentrations were measured using the method of ferric reduction antioxidant power developed by Benzie and Strain . Plasma GSH using the method of Tipple et al. , and MDA concentrations by the thiobarbituric acid reactive substances spectrophotometric test were determined with inter- and intra-assay CVs below 5% . 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 . 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.
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 . 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 .
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.
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 , 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.
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).