The Effects of Thylakoid-Rich Spinach Extract Combined with the Calorie-Restricted Diet on Cardiometabolic Risk Factors in Obese Women with Polycystic Ovary Syndrome: A Randomized Controlled Clinical Trial

Background: The polycystic ovary syndrome (PCOS) was associated with a higher risk of cardiovascular diseases, related to metabolic dysfunction, due to its peculiar hormonal pattern, dyslipidemia, and inammatory state. This study was aimed to investigate the effects of thylakoid-rich spinach extract supplementation combined with a hypocaloric diet on cardiometabolic risk factors in obese women with PCOS. Methods: In a double-blind, placebo-controlled design, forty-eight participants (age 20–45 years) consumed in random order either a thylakoid-rich spinach extract powder (5g) or a matched placebo (5g starch), given daily combined with the hypocaloric diet for 12 weeks. The samples were collected at baseline and after the end of the supplementation. The primary outcome was the measure of anthropometric changes, glycemic indices, and lipid prole. The secondary outcomes included inammatory status and blood pressure. Results: Thylakoid-rich spinach extract supplementation combined with the restricted-calorie diet was associated with signicant reductions in abdominal obesity indicators, insulin resistance, lipid accumulation product (LAP), and diastolic blood pressure (DBP) (P< 0.05). Signicant decreases were also seen in serum triglycerides (TG), insulin, high-sensitivity C-reactive protein (hs-CRP), and total testosterone compared with the placebo group (P< 0.05). However, no signicant differences in systolic blood pressure (SBP) and in serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and fasting blood glucose were evident between groups at the end of study ( P>0.05). Conclusions: Thylakoid-rich spinach extract supplementation combined with the calorie restriction for 12 weeks may improve the cardiometabolic risk factors in obese women with PCOS. Fresh baby spinach leaves (Spinacia oleracea) used to prepare of thylakoid membranes according to the previously 23, 24]. The required spinach collected from Tabriz, East 2018; some plant samples were the Herbarium The scientic name of the collected specimen is Spinacia oleracea L. belonging to the Oleracea family with the herbarium number TBZ-fph-1898. The thylakoid supplement used in this investigation was prepared based on the method described by Emerk et al. [24], at an experimental scale in the Synthesis Laboratory of Drug Applied Research Center, Tabriz University of Medical Sciences. Fresh spinach leaves after removing the stems and veins, were washed and drenched. 1000 g spinach leaves were homogenized with 1250 ml water in a blender and ltered through four layers of Monodur polyester mesh (20 µm). This obtained ltrate was diluted 10 times with distilled water, and its pH adjusted to 4.7 with Hydrochloric acid (HCl). PH 4.7 is the isoelectric point of the thylakoids, and maximum precipitation occurs at this pH. The thylakoids occulated, and a green precipitate with a clear, a bit yellowish supernatant was obtained after 4 h standing in the cold (-4 o C). The supernatant was removed, and the green precipitate was collected from the ltrate thylakoids at pH 4.7 and washed in water by repeated centrifugation; the precipitation was repeated at the same pH. The washed thylakoids were collected, and after adjusting to the desired pH (pH 7.0), the nal sediments freeze-dried to obtain a green thylakoid powder. Large scale production, of this freeze-dried thylakoid powder, was conducted by the fatty acid; NF-κB, nuclear factor kappa B; NO, nitric oxide; OCP, Oral contraceptive pill; PCOS, polycystic ovary syndrome; PUFA, polyunsaturated fatty acid; REE, Resting energy expenditure; ROS, reactive oxygen species; SBP, systolic blood pressure; SCFAs, short-chain fatty acids; SD, standard deviation; SFA, saturated fatty acid; SHBG, sex hormone-binding globulin; TC, total cholesterol; TG, triglycerides; TyG, triglyceride glucose index; VAI, visceral adiposity index; WC, Waist circumference; WHR, waist-to-hip ratio; WHtR, Waist to height ratio.

women with PCOS. The PCOS was diagnosed by a gynecologist, based on the Rotterdam criteria, which includes suffering from two of the following symptoms: (i) amenorrhea or oligomenorrhea with eight or fewer menstruations in the previous 12 months, (ii) biochemical and/or clinical signs of hyperandrogenism, and (iii) polycystic ovaries on ultrasound examination (> 12 follicles, 2 to 9 mm in diameter and/or increased ovarian volume > 10 mL) [19]. The study population was recruited from the gynecology and infertility clinics of Alzahra Hospital in Tabriz, Iran, or Sheykholrayis Polyclinic in Tabriz, Iran. Medical records of patients in these clinical settings were carefully reviewed. Of those, forty-eight obese women (body mass index (BMI): 30-40 kg/m 2 ) with PCOS and aged 20-45 years, were recruited. Exclusion criteria were as follows: any evidence of thyroid disease, adrenocortical dysfunction, or hyperprolactinemia (Prolactin > 30 mg/mL), being menopause, or pregnant, or lactating; smoking or being exposed to cigarette smoke; having co-morbidity with other gynecologic or endocrine disease, or hepatic, renal, or cardiovascular disease, diabetes and/or impaired glucose tolerance; taking any nutritional or herbal supplements during two months prior to the study, using ovulation induction agents or drugs affecting metabolic or insulin statuses such as statins, thiazolidinediones, corticosteroids, insulin, antiobesity and anti-diabetic drugs (including Metformin). Comprehensive interviews on the general characteristics of the participants, containing age, family history of PCOS, disease and medication history or previously utilized treatments, and their lifestyle, were conducted at the beginning of the trial. If the patients had adopted a diet and/or a speci c physical activity program, or any changes in medications, or experienced any detrimental events during the study, they were withdrawn from this clinical trial. This clinical trial was registered in the Iranian Registry of Clinical Trials (http://www.irct.ir; registration no. IRCT20140907019082N9).

Ethics approval and consent to participate
The eligible patients were given a detailed explanation of the study procedures. Written informed consent was obtained from those willing to participate in the trial. The study protocol was conducted according to the Declaration of Helsinki guideline and approved by the ethics committee of Research Vice-Chancellor of Tabriz University of Medical Sciences (Tabriz, Iran; Ethics code: IR.TBZMED.REC.1397.447).

Sample size
The sample size was calculated based on the previous study [20]. By considering the changes in FBS, with a con dence level of 95%, alpha = 0.05 and power of 90%, a total of 21 participants were calculated for each group, which was increased to 24 to cover a probable dropout rate of 15% (total sample size: 48 obese women with PCOS). For this calculation, Power Analysis and Sample Size Software (PASS; NCSS, LLC, US) version 15 was used.

Study protocol
The study participants were randomly allocated into one of the two experimental groups (in a 1:1 ratio) by a research assistant not otherwise involved in the study, using of the Random allocation software (RAS) and randomized block procedure of size two (age (< 33 vs. ≥33) and BMI (< 35 kg/m 2 vs. ≥35 kg/m 2 )). The researchers and participants were blinded regarding the randomization, allocation, and subjects' groups until the end of the study and the completion of nal analyses. To ensure the blinding in the evaluation process, the person (completely irrelevant to the study) who prepared the supplement packages assigned a three-digit code to each of the treatments. The eligible subjects were assigned to receive 5 g/day of thylakoid-rich spinach extract or matching placebo as 5 g/day raw starch (one sachet before lunch) for 12 weeks. The sachets were completely identical in all other aspects (shape, weight, color). The primary outcomes were changes in anthropometric and metabolic status (FBS, insulin, insulin resistance, and lipid pro le). The secondary outcomes were blood pressure, triglyceride glucose (TyG) index, lipid accumulation product (LAP), visceral adiposity index (VAI), and hs-CRP. At rst, demographic and clinical questionnaires, a 3-days food record, and international physical activity questionnaire-short form (IPAQ-SF) were completed for all participants. Next, anthropometric assessments and blood pressure measurements were done. All mentioned assessments were performed at baseline and at the end of the study. For biochemical evaluations, blood samples were taken from each patient, after 10-12 h of overnight fasting, at baseline and end of the study. The participants were asked to keep their regular medication (i.e., OCP) and usual levels of physical activity throughout the study period. They were also advised to inform the researchers for any changes in their medical therapy program and also any adverse effects of the supplements.

Intervention protocol
Participants randomly recruited to a 12-week intervention arm consisting of 5 g/day of thylakoid-rich spinach extract powder + low-calorie diet (n = 24) or to a control arm of 5 g/day powdered raw starch as placebo + low-calorie diet (n = 24). The choice of 12-weeks intervention duration and a dose of 5 g/day thylakoid, was based on observed bene cial effects of thylakoid supplementation on obesity status and related metabolic pro les in consumer subjects [21]. All of the participants received a calorie-restricted diet planned by an expert dietitian. For planning this diet, total energy expenditure was calculated based on resting energy expenditure (REE), which was calculated based on the Mi in equation [22], physical activity level, and thermic effect of food (10% of total energy expenditure). After calculating the daily required energy for each participant, by a 500 kcal de cit from it, individualized diets were designed. The assigned diet contained 30% fat, 55% carbohydrate, and 15% protein. The participants were requested to follow healthy eating recommendations, including changing cooking methods to healthier ways and limiting fast foods, saturated fats, high-fat foods, sugar, sweets, and sugar-sweetened beverages. Food Exchange utilization was thoroughly explained to the participants, and replacing the foods they did not have access to, by the foods of equal calorie from the corresponding food groups was instructed. Adherence to the recommended diet was evaluated using a 3-day food diary (2 weekdays and 1 weekend day) at baseline and end of the study. Daily intakes of macro-and micro-nutrients were analyzed by Nutritionist IV software (First Databank Inc., Hearst Corp., San Bruno, CA, USA).

Preparation of spinach thylakoids and placebo
Fresh baby spinach leaves (Spinacia oleracea) were used to prepare of thylakoid membranes according to the previously registered protocols [16,23,24]. The required spinach was collected from Tabriz, East Azerbaijan Province, Iran in spring, 2018; some plant samples were delivered to the Herbarium Center of the Faculty of Pharmacy, Tabriz University of Medical Sciences. The scienti c name of the collected specimen is Spinacia oleracea L. belonging to the Oleracea family with the herbarium number TBZ-fph-1898. The thylakoid supplement used in this investigation was prepared based on the method described by Emerk et al. [24], at an experimental scale in the Synthesis Laboratory of Drug Applied Research Center, Tabriz University of Medical Sciences. Fresh spinach leaves after removing the stems and veins, were washed and drenched. 1000 g spinach leaves were homogenized with 1250 ml water in a blender and ltered through four layers of Monodur polyester mesh (20 µm). This obtained ltrate was diluted 10 times with distilled water, and its pH adjusted to 4.7 with Hydrochloric acid (HCl). PH 4.7 is the isoelectric point of the thylakoids, and maximum precipitation occurs at this pH. The thylakoids occulated, and a green precipitate with a clear, a bit yellowish supernatant was obtained after 4 h standing in the cold (-4 o C). The supernatant was removed, and the green precipitate was collected from the ltrate thylakoids at pH 4.7 and washed in water by repeated centrifugation; the precipitation was repeated at the same pH. The washed thylakoids were collected, and after adjusting to the desired pH (pH 7.0), the nal sediments freeze-dried to obtain a green thylakoid powder. Large scale production, of this freeze-dried thylakoid powder, was conducted by the Iran Darook Pharmaceutical Co., Tehran, Iran. Placebo consisted of corn starch, which was colored in edible green color, and like thylakoid powder, avored with kiwifruit essence. Therefore, green powders with kiwifruit avor were made of thylakoid or placebo, which appearances (shape, size, and color) and also, avor were similar in placebo and thylakoid. Next, they were packed in completely identical sachets; and each sachet contained 5 g of thylakoid or 5 g of starch. The contents of the sachets were dissolved in a glass of water and drunk before lunch. Both participants and researchers were unaware of group assignment. Packages were coded; and distributed monthly by a third person, who was irrelevant to the study. For reminding the consumption of the supplements, a chart was designed for the participants to complete and return in each visit. Participants received a brief daily cell phone reminder and weekly a phone call to take the supplement and to minimize withdrawal and ensure their adherence to the study protocol. The participants were requested to return the remaining sachets at each visit; counting these sachets enabled us to evaluate compliance. Consuming ≥ 80% of the supplements was considered compliance.

Assessment of anthropometric variables and blood pressure
The body weight, height, waist, and hip circumferences of the participants were measured at baseline and at the end of the study. Body weight (to the nearest 0.1 kg) and height (to the nearest 0.1 cm) were measured using a Seca digital scale (Hamburg, Germany), in an overnight fasting state with minimal clothing and no shoes on. BMI was calculated as weight (kg) divided by height squared (m 2 ). The hip circumference (HC) was measured at the level of the maximum extension of the buttocks, and waist circumference (WC) was measured at the midpoint between the lower ribs and the iliac crest. Waist to height ratio (WHtR) was calculated as WC (cm) divided by height (cm). Waist to hip ratio (WHR) was calculated as WC (cm) divided by HC (cm). Blood pressure was measured in a sitting position three times after resting for 10 min, by a digital sphygmomanometer (Omron M3, Kyoto, Japan), and the mean of three measurements was reported as the nal systolic and diastolic blood pressure.

Blood sampling and measurements of biochemical parameters
The venous blood sample (10 ml) was drawn from each participant at baseline and end of the study after overnight fasting, between 8:00 a.m. and 9:00 a.m. during the early follicular phase (d 2-5) of a spontaneous or P-induced menstrual cycle. Blood samples immediately centrifuged at 3500 rpm for 10 minutes to the separation of serum samples from whole blood. Serum samples were used to measure lipid pro le, FBS, insulin, testosterone, sex hormone-binding globulin (SHBG), and hs-CRP. The biochemical parameters, including a lipid pro le, glycemic parameters, and hs-CRP were immediately measured after sample collection, and the remaining serum was frozen immediately at − 80 °C, until the end of the study. Serum FBS, triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) concentrations were measured through enzymatic methods by an auto-analyzer (Hitachi-917, Tokyo, Japan) using the colorimetric technique, by commercial kits (Pars-Azmoon Co., Tehran, Iran). The Friedewald equation was used to calculate low-density lipoprotein cholesterol (LDL-C) [25]. Serum insulin level was measured by chemiluminescence (IMMULITE 2000, SIEMENS); and the homeostatic model of assessment for insulin resistance (HOMA-IR) was calculated, on the basis of the suggested formula [26]. Serum hs-CRP concentration was determined using an immunoturbidimetric assay (Pars Azmoon Co., Tehran, Iran). Testosterone and SHBG concentrations were determined using ELISA kits (Bioassay Technology Laboratory, Shanghai Korean Biotech, Shanghai City, China) according to the manufacturer's instructions with inter-and intra-assay coe cient variances (CVs) lower than 7%. The free androgen index (FAI) was calculated based on suggested formulas [27]. TyG index, LAP, and VAI were calculated based on suggested formulas as follows [28]:

Statistical analysis
All statistical analyses were performed using SPSS version 23 (SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was performed to determine the normality of data distribution. Distribution of data was expressed as mean (SD) for normally distributed and median (percentiles 25 and 75) for not normally distributed quantitative data and frequency (percent) for qualitative data. To compare the two groups at the baseline, independent sample t-tests or Mann-Whitney or chi-squared tests were used. Assessments of differences within the group were made by paired-samples t-tests or nonparametric Wilcoxon signed-rank test, and sign test. A comparison of the two groups at the end of the study was completed by the analysis of covariance (ANCOVA) after adjusting for the baseline parameters and covariates. Post hoc paired comparisons were made by using a Sidak test. Results with P values of < 0.05 were considered statistically signi cant.

Baseline characteristics of participants
In this study, from 179 eligible participants, initially, 48 participants were recruited, whom 44 participants completed the trial (intervention group, n = 21; control group, n = 23; Fig. 1). The reasons for the loss to follow up are explained in the study ow diagram (Fig. 1). No subject reported any adverse event throughout the study. In this study, adherence and compliance rate were high, which were assessed monthly by counting the number of used sachets. The average consumption was greater than 90% in both groups. The mean age and weight of the participants were 31.95 ± 2.59 years and 88.65 ± 6.85 kg, respectively, 68.2% of the participants were married, and half of the subjects (50%) had an academic education level. No signi cant differences were seen between the two groups regarding the initial characteristics at baseline (P > 0.05; Table 1). Additionally, as shown in Table 1, no signi cant differences were found between the two groups in terms of components of metabolic syndrome. There were no signi cant differences between the two groups regarding physical activity levels, at baseline, and end of the study (P > 0.05).

Dietary intakes
Dietary intakes of participants were indicated in Table 2. No signi cant between-group differences were observed in average daily intake of energy, macronutrients, and dietary ber at baseline (P > 0.05), except for SFA and MUFA, which were higher in the thylakoid group than the placebo group (P < 0.05; Table 2). As expected, after 12 weeks of the intervention, energy intake signi cantly decreased in both groups compared to baseline, due to receiving a calorie-restricted diet by both groups. The dietary intake of carbohydrate, total fat, SFA, PUFA, and MUFA signi cantly decreased during the intervention period in both thylakoid and placebo groups (P < 0.05). But, the intakes of protein, ber, and cholesterol were not signi cantly changed during the intervention period in both groups (P > 0.05). However, at the end of the study, between-group analysis adjusted for baseline values and confounders showed no signi cant differences in dietary intakes between the two groups (P > 0.05; Table 2). Cardiometabolic Parameters Table 3 presents the cardiometabolic parameters of subjects, including abdominal obesity indicators, glycemic indices, lipid pro le, blood pressure, total testosterone, and hs-CRP at baseline and after the intervention. There were no statistically signi cant differences in these cardiometabolic variables between the two groups at baseline (P > 0.05). As expected from the experimental design, all subjects in the thylakoid group and placebo group lost body weight (-6.97 ± .52 kg vs. -3.19 ± 0.72 kg) at the 12th week, due to the daily energy de cit. Comparison between two groups adjusted for the baseline values and potential confounders revealed that there was a signi cant reduction in BMI in the thylakoid group compared to the placebo group after the 12-week intervention (Table 1) testosterone, TG, and hs-CRP decreased signi cantly in both thylakoid and placebo groups compared to baseline (all P < 0.05). Results of the post-intervention analysis of covariance showed that these reductions remained signi cant after controlling for the potential confounders, including changes in physical activity, total energy intake, and weight (P < 0.001). Accordingly, the differences between the two groups were signi cant in terms of these parameters (signi cant reductions in the thylakoid group than the placebo group), except for FAI, which was no signi cant difference between the two groups (P = 0.12).
At the end of the study, the levels of TC (P = 0.03), VAI (P = 0.01), SBP (P = 0.05), DBP (P = 0.01), SHBG (P = 0.03), and TyG index (P < 0.001) decreased signi cantly in the thylakoid group compared to baseline. But, these parameters were not signi cantly changed in the placebo group compared to baseline (all P > 0.05). As shown in Table 3, results of an analysis of covariance after adjusting for the baseline values and potential confounding factors (changes in weight, physical activity, and total energy intake) revealed no signi cant between-group differences by the end of the study for these parameters, except for levels of TyG index and DBP, which decreased signi cantly in thylakoid group compared to the placebo group (P < 0.05). Overall, we found that after adjustment for baseline values, age, BMI, and confounders (changes in weight, physical activity levels, and total energy intake throughout the study), daily supplementation with thylakoid had no signi cant effects on SBP, FAI, VAI, and serum levels of FBS, HDL, LDL,TC, and SHBG.

Discussion
The present clinical trial investigated the effects of 12 weeks intake of thylakoid-rich spinach extract on the cardiometabolic risk factors in obese women with PCOS under a calorie-restricted diet for the rst time. The results of this study showed that thylakoid-rich spinach extract supplementation with the daily dose of 5 g for 12 weeks, concurrent with a low-calorie diet could signi cantly decrease the body weight, BMI, abdominal obesity indicators (WC, WHR, WHtR), diastolic blood pressure, LAP, HOMA-IR, TyG index, serum levels of insulin, TG, testosterone, and hs-CRP. We observed signi cant decreases in VAI, FAI, TC, and SBP, as well as a signi cant increase in SHBG level in the intervention group compared to the baseline. But, these changes were not signi cant compared to the control group after adjusting for potential confounders. Serum concentrations of FBS, LDL, and HDL did not change signi cantly in both groups compared to the baseline and did not differ signi cantly between the two groups (Table 3). During this study, due to receiving a low-calorie diet by all participants, intakes of energy and macronutrients changed signi cantly compared to baseline. But, these changes in the intervention group were not signi cant compared to the placebo group (Table 2). To our knowledge, clinical trials evaluating the effects of thylakoid-rich spinach extract supplementation in women with PCOS do not exist. After adjusting for confounders, we found that both general and central or abdominal obesity-related indicators signi cantly decreased in the thylakoid consumers compared to the placebo recipients. BMI, as an assessment tool for general obesity, WC, as a screening instrument for determining abdominal obesity, WHR, as an instrument for assessing central obesity and visceral fat, and WHtR, as an anthropometric indicator for assessing central adiposity, were evaluated [29,30]. Our ndings regarding the reductions in body weight, BMI, WC, WHR, and WHtR are in agreement with the ndings of a recent systematic review of the anti-obesity effects of the thylakoid-rich spinach extract [15], and other studies [16][17][18][31][32][33][34] that demonstrated weight loss, body fat and body mass reductions following including thylakoid-rich spinach extract into the diet. Several proposed mechanisms involved in these effects are as follows: inhibition of the pancreatic lipase/co-lipase activity, consequently, retarding the digestion and absorption of dietary fat; hence, post-prandial high-fat content in the small intestinal lumen curbs hunger and stimulates satiety signals including, the gut-derived satiety hormone cholecystokinin (CCK)and glucagon-like peptide-1(GLP-1) as well as the adipose-derived satiety hormone leptin. Moreover, a signi cant reduction in serum levels of ghrelin, a stomach-derived hunger hormone, was demonstrated. Resultant satiety promotion and suppressed appetite to eat contribute to reduced food intake and subsequent weight loss in subjects [16,17,21,31,32,[34][35][36]. In our study, there were no signi cant differences between the intervention group and the placebo group regarding the intake of energy and dietary nutrients. Our ndings were in agreement with the previous studies [17,32] in which indicated no signi cant changes in total calorie and nutrient intakes of thylakoid consumers in trials, and food intake at the next meal following thylakoid intake was not different between the placebo and the thylakoid groups. Thus, the exact mechanism of thylakoid affecting anthropometric indices should be explored. The present study indicated that consumption of 5 g thylakoid-rich spinach extract for 12 weeks signi cantly decreased insulin and HOMA-IR, but did not affect FBS in obese women with PCOS. This was consistent with the results of the study by Köhnke et al.; they reported a 37% reduction in the insulin levels in the thylakoid group compared to the control group, without signi cant changes in blood glucose levels between the two groups [31]. Signi cant decreases in insulin levels in thylakoid consumers compared to controls, without considerable changes in glucose level probably, suggest an elevated insulin-stimulated glucose uptake in cells due to increased insulin sensitivity as a result of thylakoid supplementation in our study.
Considering the weight loss and central obesity reduction, as well as insulin sensitivity improvement and insulin resistance attenuation following thylakoid supplementation without changes in glucose concentration in our study, which has also been demonstrated in some studies [17,32], thus, it presents good implications of spinach thylakoid for obesity treatment. On the contrary, regarding glucose levels, multiple animal studies reported the hypoglycemic action of spinach-derived thylakoids [23,[37][38][39]. Park et al. found that spinach extract inhibited intestinal α-glucosidase in vitro, and the digestion of disaccharides (− 19.6%), hence, by lowering the uptake of dietary glucose, glycemia might be improved [40]. In another study, intake of 0.5 g/kg body weight of spinach thylakoid in a single dose, led to blood glucose suppressed after an oral glucose tolerance test, without changes in insulin levels in pigs [39]. The discrepancy between the ndings of studies might result from the differences in type and dosages of spinach thylakoids, duration of intervention, design of the study, and acute or chronic administration of the spinach thylakoids. There are some probable mechanisms for the bene cial effects of thylakoid-rich spinach extract on glucose metabolism as follows: reducing intestinal uptake of glucose by localization of thylakoids as large complex structures onto the mucosa and also, by binding to the starch and/or amylase, increasing CCK and GLP-1 secretions, and postponing gastric emptying. Additionally, the antiobesity and bene cial effects of thylakoid-rich spinach extract on anthropometric indices might play a crucial role in the improvement of glycemic parameters [23,38,41]. The ndings of the current study demonstrated that after the 12-week intervention, total testosterone levels decreased signi cantly in both groups compared to baseline. But, these changes were signi cantly different between the two groups, and spinach-derived thylakoid supplementation contributed to a signi cant reduction in total testosterone levels, even after adjusting for all potential confounders. In this regard, no other study is available investigating the effect of spinach thylakoids supplementation on testosterone levels and hyperandrogenism status. However, our ndings are in accord with reports elsewhere on weight loss in PCOS [13,42]. It has been demonstrated that high testosterone in women with PCOS is often associated with increased adiposity and insulin resistance. Furthermore, insulin directly stimulates androgen synthesis by the theca cell. Therefore, the improvement of insulin sensitivity and the resultant decrease in circulating insulin could decrease testosterone levels, as previously reported, that improvement in insulin resistance through weight loss or the use of insulin-sensitizing drugs leads to a decrease in hyperandrogenemia [43,44]. For the rst time, we observed that thylakoid-rich spinach extract with a hypocaloric diet decreased DBP and serum levels of hs-CRP, without effect on the SBP in obese women with PCOS after 12 weeks. There are limited studies with contradictory ndings on the effects of spinach extract contained thylakoids on the in ammatory status and blood pressure. Rebello et al. reported that 5 g of spinach extract rich of thylakoid had no signi cant effect on the hs-CRP levels in overweight or obese males and females. In this study, each participant was tested with one-time thylakoid-rich spinach extract ingestion on 2 days, and the hs-CRP level evaluated two hours after lunch [32].Moreover, Stenblom et al. reported that the concentrations of TNF-α decreased signi cantly from 0 to 240 min after the thylakoid supplementation in healthy overweight women. Blood samples were evaluated over a 4-h period [35]. It seems that the differences in duration of intervention or design of the study, individual differences, and baseline values may result in different ndings. PCOS is associated with increased hs-CRP concentration. It is demonstrated that hs-CRP be as an indicator to identify the risk of cardiovascular events, and a high level of hs-CRP, as a marker of chronic in ammation, was associated with the risk of hypertension development [45,46]. Previous studies revealed the documented potential roles of spinach extract in the restoration of these conditions by suppressing reactive oxygen species(ROS), lowering oxidative damage, and inhibiting NF-κB activation and consequent pro-in ammatory markers [15,47,48].
Lipid components of thylakoid mainly, galactosyldiacylglycerols, have shown anti-in ammatory activities in several studies [49,50]. Considering, hyperandrogenism may be the initiator of chronic low-grade in ammation, and increased abdominal adiposity contributes to the in ammatory load in PCOS, the reducing effect of thylakoid-rich spinach extract on the hs-CRP level can be explained by the decrease in abdominal obesity and insulin resistance, which causes a decrease in hyperandrogenism by decrease in LH-mediated androgen secretion and reduced peripheral aromatization [51]. The most common pigment of thylakoid, chlorophyll, is rich in magnesium. According to previous studies, magnesium-rich foods can help to lower hypertension; so, thylakoid-rich spinach extract because of its high magnesium content, as the central atom of the chlorophyll molecule, might exert antihypertensive effects through providing a large amount of this mineral [52].Additionally, it is reported that spinach extract, as the richest source of nitrate, shows cardioprotective effects through inducing an increase in postprandial plasma nitrate and nitrite concentrations and lowering blood pressure. It is hypothesized that these effects are mediated by alterations in oxidative stress. In this regard, the increase in NO and its bioavailability, lead to the inhibition of adhesion molecules expression and ROS production suppression, with consequent action on vascular in ammatory condition [53]. Furthermore, it may exert antihypertensive effects by modulating body weight and insulin resistance in this trial. As we know, no reports are available indicating the effects of spinach thylakoid supplementation on blood pressure and in ammatory status in women with PCOS.
Regarding lipid pro le, we found that supplementation with thylakoid-rich spinach extract ( [17,54]. A number of animal studies revealed the potential of the spinach extract containing thylakoids to lower blood triglycerides [37,55,56]. Due to the lack of studies regarding the effects of spinach-derived thylakoids on lipid metabolism, the mechanism by which thylakoid-rich spinach extract might affect lipid pro le are still unknown. The existing evidence shows that the prebiotic effect of spinach thylakoids, and hence, modulation of gut microbiota may be involved in the lipid-lowering properties of the thylakoid-rich spinach extract [38,57]. In this regard, it is demonstrated that spinach thylakoid consumption induces fatty acid oxidation and inhibits de novo synthesis of lipids through increasing the generation of shortchain fatty acids (SCFAs). Additionally, both population and diversion of gut microbiota were affected by the intake of spinach thylakoids. The bacterium Lactobacillus reuteri was increased in the distal ileum in the receiving thylakoids group. It is reported that Lactobacillus reuteri can reduce serum cholesterol levels [38,58,59]. However, further studies are needed to investigate the prebiotic effects of thylakoid-rich spinach extract and its effect on the gut microbiota, particularly those with cardio-metabolic bene t properties.
This study, as the rst clinical trial, had several strengths including, monitoring the patients' diets by designing calorie-restricted dietary plans with a focus on amount and type of the macronutrients, and individual preferences, tracking physical activities and dietary intakes before, through, and after the intervention. Other principal strengths of this study were: a low drop-out rate and a high compliance rate of the participants to the treatment in groups, frequent visits and phone calls, the double-blindness of this study, and the adjusting of measured biochemical parameters for the known confounding factors. Limitations of our study include relatively small sample size, the absence of a follow-up evaluation of ovarian masses by sonographic assessment, and not testing for a dose-response relationship between thylakoid intake and appeared changes in the measured parameters. In this trial, the appetite and desire to eat in a short duration were not assessed. We did not assess the prebiotic effects of spinach extract and also, the gut microbiota of PCOS patients.

Conclusions
In conclusion, the present trial, for the rst time, revealed that supplementation with 5 g/day thylakoid-rich spinach extract in combination with calorie-restrictive diet for 12 weeks in obese PCOS women could signi cantly improve abdominal obesity parameters and insulin resistance, which are the key components of the therapeutic plan for obese women with PCOS. Along with these changes, DBP and serum levels of TG, testosterone, and hs-CRP decreased signi cantly compared to the calorie-restrictive diet alone. These data suggest that the thylakoid-rich spinach extract supplementation in combination with a calorie-restrictive diet might be applied as an adjunct intervention in conjunction with other pharmacological procedures aimed at achieving improvement in cardiometabolic disturbances in obese women with PCOS. However, data in this regard were limited and further investigations organized as placebo-controlled trials with larger sample size and longer duration are proposed.   This is a list of supplementary les associated with this preprint. Click to download.