Phytoestrogenic Effects of Various Levels of Soy Isoflavones in Polycystic Ovarian Syndrome CURRENT STATUS: POSTED

Background: Soy isoflavones (SI) has estrogenic effect in tissues by binding to estrogen receptors and might be beneficial for women with Polycystic Ovarian Syndrome (PCOS) by reduction in testosterone, cholesterol , Insulin , weight gain, inflammatory markers and oxidative stress. Methods: The study was plane to examine the effect of various levels of SI on nutrient intake, digestibility, lipid profile, insulin and reproductive hormones of estradiol-valerate (4mg/rat/IM) PCOS induced rat models. Thirty-Six Wistar 45 days old rats weighing 95 ± 5g were divided into 4 groups, each having 9 rats: C (control: without SF), SF10 (SF 0.10g/Kg BW), SF15 (SF 0.15g/Kg BW) and SF20 (SF 0.20g/Kg BW). SF was given through the oral gavage. Food and water were offered ad libitum and intake was recorded daily. Body weight was recorded on weekly basis. During last week of trial, collected feces by total collection method and blood samples were used to calculate nutrient digestibility and biochemical analysis respectively. The Completely Randomized Design and LSD test were used to analyze the data. Results: The significant results were observed in female rats fed SF10, SF15 and SF20 compared to C. Estrogen, progesterone and prolactin were (P<0.05) high in rats fed SF diet that was 4 and 30% increase from C respectively. Insulin, Testosterone, FSH and LH were lowest in rats fed diet SF20. Significant (P<0.05) reduction in cholesterol was observed in rats fed SF15 and SF20 as compared to C. Serum HDL was improved (P<0.05) in all SIF 0.10 , SIF 0.15 and SIF 0.20 as 26.88±1.59 (mg/dl), 32.40±1.42 (mg/dl) and 53.44±1.50 (mg/dl) in comparison to PC. Serum LDL was significantly reduced to 68.89±4.36 (mg/dl) in SIF 0.20 , and to 108.20±4.14 (mg/dl) in SIF 0.15 whereas, insignificant reduction was observed in SIF 0.10 (127.75±4.62 mg/dl) as compared to PC. Highly significant reduction was noted in triglycerides level in SIF 0.20 as 115.00±2.04 (mg/dl), however, significant reduction was observed in both SIF 0.10 and SIF 0.15 as compared to PC after three months of treatment with soy isoflavones. Conclusions: It is concluded that soy isoflavones have potential role on reproductive hormones, insulin levels, weight, lipid profile and nutrient digestibility in PCOS induced rats.


Introduction
In assessing the therapeutic effects of phytoestrogens, a clear dissimilarity must be made between foods having phytoestrogens and dietary supplements, as bioavailability, dosage and physiological effect may vary critically [1]. The majority of phytoestrogens belong to a large group of substituted phenolic compounds known as flavonoids. Isoflavones are polyphenolic compounds that are almost exclusively produced by the members of the Fabaceae family. The major bioactive isoflavones are genistein and daidzein, which are derived mainly from soy and red clover [2]. In particular soybeans, are the richest sources of isoflavones in the human diet.
Isoflavones are identified to exibit weak estrogen hormone-like activity. Because isoflavones are not synthesized by the human endocrine system and can only be ingested or consumed in a diet or as a supplement they are also called as "dietary estrogens" [3]. In human body, estrogen is secreted primarily by the ovaries and in lesser quantities by the adrenal glands. The most active form of estrogen in the body is called estradiol. Estrogen put its effects by binding to estrogen receptors within cells. These estrogen-receptor complexes then combines with DNA to change the expression of estrogen-responsive genes. For simulating the effects of estrogen in tissues, soy isoflavones as phytoestrogens can bind to estrogen receptors [4].
As isoflavones and estradiol are contending for their binding on ERs, the effect of isoflavones also dependent on the level of endogenous estradiol. When there is high levels of endogenous estrogens, isoflavones may hinder full estrogen activity by conquering a part of the ERs. Alternatively, when there is low levels of endogenous estrogens the estrogen activity of isoflavones may become evident [5]. As compared to 17-β-estradiol the affinity of other isoflavones is 100-500 times lower.
Isoflavones act against ERs, but their activity is lesser than that of 17-β-estradiol. At suitably high levels, the effect of isoflavones may approach the effect of endogenous 17-β-estradiol at its physiological level [6]. For this reason, there is emerging trend of using isoflavones as an alternative or complement of HRT in postmenopausal women, particularly when long-term administration is required [7]. Number of studies indicate that, soy being high in isoflavones, can prevent illness and promote good health.
Soy isoflavones have number of benefits for women with PCOS such as reduction in testosterone, Total cholesterol and LDL cholesterol, Insulin, inflammatory markers and oxidative stress. Polycystic ovary syndrome is the most common endocrine disorder among the women of reproductive age [8].
In women with PCOS insulin resistance is common and it causes lipoprotein disturbances that plays a role as predisposing factor for early development of diabetes, hyperlipidemia and cardiovascular disease [9].Two types of interventions: drug treatment and change in lifestyle with exercise and weight loss are mostly used as therapeutic approaches for PCOS. The available common drugs are effective for PCOS, but they have side effects such as amenorrhea, overweight, decreased bone mineral density and depression. Therefore, the use of non-pharmacological treatment methods is considered.
Using soy isoflavones as a non-pharmacological approach for PCOS patients is growing interest in how adding soy to the diet can help address metabolic syndrome and related health conditions. The effects of soy isoflavones, have been studied by several researchers and their biological activity related to estrogen receptor-mediated mechanisms have been observed [10].Soy isoflavones being phytoestrogens are adaptogens. Under increased or decreased levels of estrogen they can be beneficial. When isoflavones attached themselves to estrogen receptors, this hinders the effects of endogenous estrogen. Generally, phytoestrogens have about two percent of the strength of estrogens. Hence, when estrogen levels are high, switching a phytoestrogen for an estrogen means that there will be much less estrogenic activity at a given binding site. Conversely, if estrogen levels are low and estrogen-binding sites are free, phytoestrogens that have almost two percent estrogen to 55% relative humidity under 12-h light: 12-h dark cycle. According to Principles of Laboratory Animal Care (NIH) all animals were kept and treated. The approved experimental procedure by the Animal Ethical Committee was considered. Ad libitum diet and water were offered to the rats.
Isocaloric and isonitrogenous.diets were offered to the rats. The weekly intake of water and feed were monitored at least one week prior to treatments in order to regulate the amount of water drank per experimental animal. The soy isoflavones used for research were purchased from Xi'an Wharton Bio-Tech, Co, Ltd, China.
Different levels of soy isoflavones: Thirty six PCOS induced Wistar female rats were used to observe the effect of different levels of isoflavones. The rats were divided into four groups (9 rats per group) named according to levels viz., PC (Positive control: without isoflavones), SIF 0.10 (soy isoflavone: 0.10 g/kg body weight), SIF 0.15 (soy isoflavones: 0.15 g/kg body weight) and SIF 0.20 (soy isoflavones: 0.20 g/kg body weight). These levels of soy isoflavones were given through oral gavage [12]. All animals included in PC, SIF 0.10, SIF 0.15 and SIF 0.20 were induced PCOS through single intramuscular injection of estradiol-valerate (4 mg/rat/IM).
In this in vivo experimental method, the rats were divided according to Completely Randomized Design. Each group consist of 9 rats and treatment w repeated 3 times to make 12 experimental units, involving 36 rats as a whole.
The trial was of 3 months. The first 2 months were correction period followed by one month collection period. The 30 days before the experimental protocol, 36 rats were given a single intramuscular injection of 4 mg/rat estradiol valerate (Riedeldehaen, Germany) firstly mixed in 0.2 ml oil to induce PCOS [13]. In the last week of study, all rats were bled to get blood samples to carry out biochemical and pathological analysis.
Feed and nutrient digestibility analysis: Digestibility of nutrients were measured by total collection method. All modified diets/feeds and feces were analyzed to calculate ash, moisture, crude protein, crude fat, crude fiber, nitrogen free extract and dry matter content (AOAC, 2000). Nutrient Digestibility (%) = Nutrient intake -Nutrient in feces × 100 Feces sample were collected via designing the cages in such a way that collect samples accurately [14]. Feces were stored at -20 • C [15].

Biochemical Analysis:
In the end of trial 5 cc blood samples of all rats were drawn and prepared for biochemical analysis [13].
The plasma testosterone were estimated by method adopted by [16]. Plasma estrogen levels were checked according to reference method of [17]. The blood glucose were measured using ACCU Check Active® Machine and serum insulin were assessed by using Electrochemiluminescence immunoassay (Roche Diagnostics E170 insulin assay). The lipid profile were determined by the method described by Cholesterol [18], Triglycerides [19]. High density lipoproteins [20], Low density lipoproteins [21].

Statistical analysis:
The data obtained from all experimental sources were subjected to statistical analysis using completely randomized designed (CRD) according to the procedure described by [22]. Tukey's test was used to conclude statistically different groups.

Results
Nutrient Intake: The result showing the effects of different level of soy isoflavones on weight of PCOS induced rats are shown in Table 1. Slightly significant decrease was observed in dry matter intake when compared with PC. Values of crude protein intake were found to be decreased in all rats treated with soy isoflavones as 7.94 ± 0.21 in SIF (0.10) ,8.31 ± 0.23 in SIF (0.15) and as 7.85 ± 0.24 in SIF (0.20) . crude fiber intake was also significantly decreased in all groups with as lows as 0.97 ± 0.16 in SIF (0.15) .Significant reduction in intake of ether extract and ash content of the feed was observed in all rats fed with different levels of soy isoflavones.

Digestibility
The effects of different level of soy isoflavones on digestibility of PCOS induced rats were given in the

Lipid profile:
Effects of different levels of soy isoflavones on lipid profile of PCOS induced rats are given in the Insulin Level: Effects of different levels of soy isoflavones on insulin level of PCOS induced rats are given in the Table 5. Significant reduction was observed in serum insulin level of all rats treated with SIF 0.10 , SIF 0.15 and SIF 0.20 , with the maximum reduction in SIF 0.20 as 5.94 ± 0.37 (mg/dl) as compared to PC.

Discussions
The purpose of this study was to investigate the effects of different levels of soy isoflavones on reproductive hormones, lipid profile, serum insulin, digestibility and weight changes in polycystic ovarian syndrome (PCOS) induced female rats.
In this study significant changes were observed in the levels of reproductive hormones after administration of soy isoflavones on different levels say SIF 0.10 , SIF 0.15 and SIF 0.20 for three months.
Significant increase in progesterone hormone was observed in all levels of soy isoflavones supplementation with maximum increase in SIF 0.20 that is 13.21 ± 0.45 showing 30% increase from PC, whereas, SIF 0.15 and SIF 0.10 showing 13% and 2% change only [23] found similar result with 120 mg/day dosage of soy isoflavones after 8 weeks of supplementation. In PCOS testosterone level become increased because of the high levels of LH or high levels of insulin [24]. This excessive testosterone cause other problematic issues like acne, hirsutism and hair loss and also inhibits normal ovulation. So, reducing testosterone to the normal healthy level is the prime goal of PCOS treatment. Moreover, in FSH/LH ratio, no statistically significant increase in FSH values were noted in SIF 0.10 and SIF 0.15 , but a significant increase of 6.8% was observed in SIF 0.20 . Similar results were found by [10] where no statistically significant change in FSH level was observed after 2 months of supplementation. But increase in FSH concentration in SIF 0.20 was contradictory with all studies, might be this increase was a result of high dosage of soy isoflavone. Whereas, remarkable decrease was observed in LH values with lowest most observed in SIF 0.20 as (13.91 ± 0.67 mIU/ml) [26] suggested similar benefits by using 36 mg/day of soy isoflavones supplementation for 3 months in women having PCOS. LH lowering effects of soy isoflavones was also suggested in other studies [27,28,29,23]. Increase was observed in prolactin values from PC in both SIF 0.10 and SIF 0.20 levels, however, reduction was noted in SIF 0.15 (31.93 ± 1.04 ng/ml) from PC. Similar findings were suggested by [23] after using 120 mg of soy isoflavones per day for 8 weeks. In estrogen concentration significant increase of 46% was observed in SIF 0.20 but, no statistically significant change was noted in SIF 0.10 and SIF 0.15 as compaired to PC. Increase in estrogen levels were noted in a meta-analysis of 47 studies on both pre and post-menopausal women by [29]. Whereas, many studies suggested significant decrease in estrogen level after different level of soy isoflavones administration [30,31,32].
Dyslipidemia is a common problem in women with PCOS [33].  [35,36] showed similar reduction in LDL concentration after soy isoflavones supplementation. Overall, significant decrease was noted in triglycerides concentration, with highly significant in SIF 0.20 as 115.00 ± 2.04 (mg/dl). However, significant reduction was observed in both SIF 0.10 and SIF 0.15 as compared to PC after three months soy isoflavone's supplementation. A significant reduction in triglycerides concentration after 50 mg/day of soy isoflavones intake for 12 weeks in 70 women with PCOS was suggested by [8].
Overall, significant reduction was noted in serum insulin level of all rats treated with SIF 0.10 , SIF 0.15 and SIF 0.20 , with the maximum reduction in SIF 0.20 as 5.94 ± 0.37 (mg/dl) as compared to PC. Similar trial of 70 women with PCOS given 50 mg/day soy isoflavones for 12 weeks was conducted by [8] found significant decrease in circulating serum levels of insulin homeostasis model of assessment estimated insulin resistance and increased quantitative insulin sensitivity check index. But soy isoflavones were found to have more potent effects on fasting insulin level [39,40] .

Conclusion
Our study revealed that soy isoflavones have potential role on reproductive hormones, insulin levels, weight, lipid profile and nutrient digestibility in PCOS induced rats. Significant effects were observed on testosterone, LH, prolactin, estrogen, cholesterol, HDL, LDL, triglyceride and insulin, whereas progesterone and FSH showed statistically significant results only in level SIF 0.20 , however no effects were observed in lower dosage levels, might be due short duration of the study. Although significant results were found in high dosage level of soy isoflavones, but long term administration of this level may cause toxicity. So, long term study with high dosage level of soy isoflavones is required for further investigation.

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