The Effectiveness of Astaxanthin Supplementation on the Clinical Symptoms and Cardio-metabolic Profile in Women with Polycystic Ovary Syndrome

Introduction Polycystic Ovary Syndrome (PCOS) is a significant endocrine disorder affecting women of reproductive age, associated with common symptoms, including anovulation, infertility, metabolic dysfunction, cardiovascular issues, dyslipidemia, as well as clinical manifestations related to hyperandrogenism. his condition not only leads to reproductive issues but is also linked to psychological disorders, such as depression. The exact etiology of PCOS remains incompletely understood; however, it is suggested that, alongside genetic factors, insulin resistance, an abnormal rise in circulating testosterone levels, and obesity-related inflammation may contribute to ovarian dysfunction.

lifestyle intervention is the current recommendation that could enhance anthropometric indices, cardio-metabolic markers, and reproductive characteristics of women with PCOS. Recent evidence indicated that the administration of lipid-soluble antioxidants may influence lipid profiles and lipid peroxidation levels in individuals with PCOS. Furthermore, previous studies have reported the positive effects of supplementation with antioxidants on other complications associated with PCOS, including inflammatory markers and insulin resistance.

Astaxanthin (ASX) is a carotenoid fat-soluble pigment with potential antioxidant properties naturally found in certain seafood and a specific type of algae. As a consequence of the unique molecular structure of this component, the beneficial effects of ASX on various health aspects involving lipid profile, blood pressure, and depression have been proposed by recent studies . Several experimental investigations also indicated the anti-oxidative and anti-inflammatory impact of ASX supplementation in various PCOS models; however, there are limited studies on the impact of ASX on the cardio-metabolic profile, inflammatory biomarkers, and clinical manifestation of women with PCOS. This paper will describe the design, methodology, and potential clinical implications of the impact of ASX supplementation in women with PCOS.

Thus, this trial aims to investigate the effect of 12 weeks of 10 mg/day astaxanthin administration compared to a control group on insulin sensitivity, lipid profile, circulating Malondialdehyde (MDA) levels, severity of hirsutism, and depression in women with PCOS.

Material and methods Study setting and recruitment This is a single-center double-blind, randomized controlled clinical trial with two parallel groups: one intervention group and one control group. Eligible women with PCOS who are referred to the collaborating gynecologist clinic in Isfahan, Iran, in 2024-2025, will be recruited in this study. After outlining the study's objectives, written informed consent will be obtained from all participants. All participants will be assigned a project-specific code that will be used throughout all data analyses.

Randomization and allocation Data on general information and socio-economic status will be obtained through face-to-face interviews. After recording information at baseline, participants will be randomly divided into two intervention and placebo groups. Using the Permuted Block Randomization method and a table of random numbers, patients will be randomly allocated to receive ASX supplements or placebo after stratifying for age, and metformin intake.

Subjects in the intervention group will receive one capsule containing 10 mg ASX per day made by the "Zyest Technology Tarawat Zendig" institute in Iran. The dosage of ASX is determined based on the safe amount of ASX consumption recorded in the relevant articles. The placebo group will take one capsule containing corn starch in a wrapped and covered form and the appearance of ASX supplementation (blinding). The pharmaceutical company coded both participants in the ASX and placebo groups (codes A and B). The investigators and participants will remain unaware of the allocation, and the code will be kept concealed until the study concludes.

To ensure participant adherence, all women are requested to return the supplement packs at each visit (every two weeks), and the remaining prescribed supplements will be counted within that timeframe. Additionally, the compliance of study participants will be monitored daily through social networks. Participants will be instructed not to alter their common diet and physical activities during the study.

Outcome measures Evaluation of general, demographic, and anthropometric characteristics Participant's general information including age, history of illness, desired disease phenotype (diagnosis by a doctor with the phenotype section in the demographic information questionnaire and smoking will be collected through a general information questionnaire by interview. The questionnaire will be carefully filled with questions from the participant and the patient's contact number will also be recorded. Data collection, including laboratory data, blood pressure, and anthropometric measurements will be assessed at baseline and 12 weeks (post-intervention). For anthropometric evaluations, all measurements will be performed according to the method provided by the World Health Organization. Standing height will be measured using a wall-mounted height meter (Seca 206, Germany) with an accuracy of 0.1 cm without shoes, heels attached to the wall knees straight, looking forward and shoulders in a normal position. A weight measurement of people with minimal clothes and without shoes will be done using a digital scale with an accuracy of 100 grams. BMI will also be calculated by dividing weight (in kilograms) by the square of height (in meters).

Dietary intake assessment The individual's dietary intake will be evaluated using a 24-hour dietary recall in the four scenarios (at the baseline, 4-week, 8-week, and the end of the study) by a trained nutritionist. the reported amounts of each food and drink consumed will be converted to grams per day using the household guidebook. Diet information analysis will be done using Nutritionist IV software (First Databank, Hearst Corp, San Bruno, CA, USA).

Physical Activity Assessment Questionnaire The physical activity evaluation of the participants will be recorded by the short form of the International Physical Activity Questionnaire (IPAQ) 4 times (at the baseline, 4-week, 8-week, and the end of the study). To obtain the overall physical activity score based on MET-minutes/week, the score obtained from 3 activity categories including walking, moderate physical activity, and vigorous physical activity will be recorded.

Blood pressure measurement To measure blood pressure, patients were asked to rest for 10 minutes, then the measurement was done using a mercury sphygmomanometer (Riester, Germany). The blood pressure of each person will be measured twice with a time interval of 10 minutes, in a sitting position, from the right arm and at the level of the heart. The average of these two measurements was considered as the patient's blood pressure. Before taking blood pressure, patients will be asked about smoking or drinking coffee in the previous 2 hours.

Evaluation of the severity of hirsutism To assess the severity of hirsutism, the amount of total body hair in nine sits (upper lip, chin, chest, upper and lower abdomen, thigh, upper and lower back, and upper arm) will be evaluated according to the modified Ferriman-Gallwey (mFG) scoring system. In this system, each body sit will be visually scored on a scale of 0 to 4, and the maximum score of each participant could be 16. The final score of 0 to 3 will be measured as non-hirsutism, 4 to 7 will be considered mild hirsutism, and 8 Up to 11 and 12 to 16 will reflect moderate hirsutism and severe hirsutism, respectively. Subjects will be asked to not use any removing hair methods during the study.

Evaluation of biochemical indicators On days zero (before the start of the intervention) and day 80 (the end of the intervention), 10 cc of blood is taken from the patient in the morning and collected in a plastic tube. It is left with 3,600 revolutions for 3-4 minutes and poured into microtubes containing 0.5 ml. The samples are stored in a freezer at -70°C. To reduce the measurement error, all samplings are done at 8-10 in the morning and in the fasting state. Blood samples taken from patients at the beginning and end of the study are centrifuged for 10 minutes at 300 rpm to separate their serum. Serum values of lipid profile (triglyceride (TG) level, total cholesterol, Low-density lipoprotein cholesterol (LDL-c), and High-density lipoprotein cholesterol (HDL-c) serum) are measured by enzymatic colorimetric method. To measure serum cholesterol, cholesteryl ester enzymes are then added to the solution, and cholesterol oxidase is finally converted into cholesterol-4-en-3-one H₂O₂ and the cholesterol concentration of the sample is obtained using the quinoneimine colorimetric marker. Lipase enzyme is used to measure TG, which converts TG into glycerol and fatty acid. Glycerol turns into glycerol phosphate and finally dihydroxyacetone phosphate, H₂O₂. using the quinoneimine colorimetric indicator, the TG concentration of the sample is obtained. To measure LDL, first, all lipoproteins except Very Low-density lipoprotein cholesterol (VLDL), HDL-c, and chylomicrons are removed, and with the addition of cholesterol esterase, cholesterol oxidase enzymes, H₂O₂ cholestenone is finally made, and LDL-c concentration is obtained by measuring the color resulting from the reaction. To evaluate serum HDL-c, firstly, lipoproteins containing apolipoprotein B such as LDL-c, VLDL, and chylomicron are separated with phosphotungstic acid, magnesium ion, and using quinonimine colorimetric marker and enzyme method, HDL-c concentration of the sample is obtained. Serum glucose is converted to glucuronic acid, H₂O₂ by the activity of glucose oxidase, and the glucose concentration is calculated using the quinonimine indicator. Fasting insulin is measured by the enzyme immunoassay method. Monoclonal antibodies are added to the serum to connect to two epitopes of insulin molecules. After adding buffers, insulin concentration is obtained by measuring light absorption. The HOMA-IR index is used to calculate insulin resistance. To calculate the HOMA-IR index, fasting insulin in the U/mlμ scale and fasting glucose in the /l nmol scale are used: HOMA-IR = fasting insulin (μU/L) x fasting glucose (nmol/L)/22.5. Serum Malondialdehyde (MDA) values are measured by the TBARS method (Arsam Far Biot Company kit) and with a spectrophotometer or fluorimeter.

Masking In this study, the individual conducting the blood test, the one prescribing the supplements, and the person analyzing the data are unaware of the division of participants into two groups: drug and placebo. Since the astaxanthin supplement and placebo are identical in color and shape, patients are also unaware of this division.

Blinding All baseline assessments will be conducted before randomization, and certain assessments will be performed with blinding for group allocation.

Sample size People with polycystic ovary syndrome will be selected by convenience sampling method. The number of samples will be calculated by considering the first type error α=0.05 and the second type error β=0.2 (80% power) and using the following formula.

=((Z\_(1-α/2)+Z\_(1-β) )\^2×〖(S〗\_1\^2+S\_2\^2))/d\^2 Based on the primary outcome considered on inflammatory factors and by placing the numbers obtained from a study conducted on the effect of astaxanthin supplementation on Malondialdehyde (MDA) \[18\], the value of d significant difference observed in the level (p \< 0.05) and equal to 0.884 mmol/liter will be equal to 17 people in each group, which will be 44 people, including the drop of 25% of people during the study.

Statistical analysis In this study, the quantitative variables will be reported as mean (standard deviation), and qualitative variables will be reported as number (p