Metabolomic change due to combined treatment with myo-inositol, D-chiro-inositol and glucomannan in polycystic ovarian syndrome patients: a pilot study

Polycystic ovary syndrome (PCOS) is a heterogeneous syndrome and one of the most common female endocrine disorders, affecting 5–20% of women in reproductive age [1, 2]. Clinical expression is highly variable, but typically includes oligo-ovulation or anovulation, hyperandrogenism and polycystic ovaries [3]. PCOS is associated with an increased risk of type 2 diabetes, cardiovascular events [4] and endometrial cancer [5]. Insulin Resistance (IR) plays a central role in approximately 70–80% of obese women and in 15–30% of lean women with PCOS [3], and represents the pathogenic link between metabolic and reproductive disorders in PCOS [6].

The last decade has seen the introduction of inositols for the treatment of PCOS and a recent review suggests that inositol therapy can reduce insulin resistance, improve ovarian function, and reduce androgen levels in women with PCOS [7]. The two inositol isomers of interest are myo-inositol (MI) and D-chiro-inositol (DCI) which often have a physiological ratio of 40:1 in most healthy tissues [8]. Importantly, the MI:DCI ratio does vary between healthy and disease state [9‐11] thus strengthening the hypothesis that inositol therapy may be beneficial for maintaining metabolic, endocrine, and reproductive health in women with PCOS.

Glucomannan is a water-soluble fiber that is derived from the konjac root [12]. It has been reported as being able to suppress hepatic cholesterol synthesis and increase the elimination of cholesterol-containing bile acids [13]. Some authors have suggested that glucomannan ingestion could also increase the gastric emptying time [14‐16]. This effect may be related to the increased feeling of satiety reported in some studies [17]. The European Food and Safety Authority (EFSA) in 2010 reported a scientific opinion supporting the anti-cholesterol and weight reduction claims for glucomannan-containing drugs [18].

The association inositols-glucomannan may represent a good therapeutic strategy in the treatment of PCOS women with insulin resistance [19].

The use of metabolomics for the study of disease mechanisms is increasing. Metabolites are low-molecular-weight organic and inorganic chemicals that are the substrates, intermediates, and byproducts of enzyme-mediated biochemical reactions in the cell [20]. Several thousand metabolites in human serum have been identified so far, and the application of metabolomics has allowed the development of biomarkers which provides new insight for many diseases [21].

Metabolomic analysis in gynecology has been extended to studies of various malignancies, such as ovarian cancer [22] and endometrial cancer [23, 24]. Moreover, many studies have incorporated a metabolomic approach to better define the pathophysiology of PCOS [25‐31] and to describe how different therapies can modify metabolic profiles [32‐34].

However, the effect of inositols on the metabolomic profile of women with a diagnosis of PCOS has not been described yet. Thus, the aim of this study is to analyze the metabolic profiles in women with PCOS before and after 3 months of therapy with a combination of MI, DCI and glucomannan, and compare these data with a group of healthy control women.

Based on serum metabolomic signatures of PCOS patients and controls a statistical power of 80% resulted from the analysis of at least 15 subjects for each class (See Additional file 1: Figure S1). The demographic and clinical–biochemical characteristics of PCOS patients at baseline (PCOS-T0) and after 3 treatment months (PCOS-T1) and controls (CTRL) are reported in Table 1. All PCOS patients met both the Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group Criteria [4] and the adolescent criteria [35] for the diagnosis of PCOS. All subjects in this study were unmarried, not interested in soon becoming pregnant, and were not currently taking hormonal contraceptives. None of them had previously given birth or had undergone voluntary interruption of pregnancy. None of the controls displayed any biochemical or gynecological abnormalities. Women with PCOS had higher Body Mass Index (BMI) compared with Controls (mean ± standard deviation, 28.4 ± 1.7 vs. 21.6 ± 1.6 [kg/m²], p < 0.05) and BMI decreased significantly (p < 0.05) after 3 months of treatment even if it remained significantly higher compared to controls (26.4 ± 1.7 vs. 21.6 ± 1.6 [kg/m²], p < 0.05). Average blood Glucose, Insulin and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) values were not significantly different, although the mean values for each parameter were greater in PCOS samples. These values did not significantly change after 3 months of treatment. Menstrual cycle regularity improved significantly after treatment. The number of antral follicles and the ovary volume were higher in PCOS patients (6.8 ± 1.2 vs. 15.4 ± 1.7, p < 0.05 and 7.4 ± 0.7 vs. 12.2 ± 2.2 [mL], p < 0.05, respectively) but both decreased after treatment (13.2 ± 2.3 and 10.2 ± 1.9 [mL], p < 0.05, respectively). The same trend was observed for the Ferriman-Galleway and the acne score (see Table 1).

As shown in Fig. 1a, the partial least square-discriminant analysis (PLS-DA) score plots clearly differentiated three groups consisting of controls, PCOS patients before treatment, and PCOS patients after treatment. The 15 highest scoring VIP variables (VIP score > 1.5) identified by PLS-DA are shown in Fig. 1b. The metabolite concentration changes induced by the therapy allowed division of the metabolites into three classes: those with higher concentrations than the controls and which increased further after therapy, those which have higher concentrations than the controls and whose concentration decreased after therapy and, those with lower concentrations compared to controls that did not change after therapy.

As shown in Fig. 2, the concentrations of 3-methyl-1-hydroxybutyl-thiamine diphosphate (FC = 6.94, p < 0.001), valine (FC = 4.87, p = 0.001) and phenylalanine (FC = 10.91, p < 0.001) were higher in PCOS-T0 patients compared to the control and increased even further after the treatments (FC = 1.13, p = 0.001; FC = 1.71, p = 0.002; FC = 1.17, p < 0.001, comparing PCOS-T1 to PCOS-T0, respectively).

Ketoisocapric acid (FC = 3.12, p < 0.001), linoleic acid (FC = 14.76, p < 0.001), lactic acid (FC = 5.31, p < 0.001), palmitic acid (FC = 6.22, p < 0.001) and glucose (FC = 19.92, p < 0.001), were also higher in PCOS patients at enrollment time but they decreased after treatments (FC = 0.93, p < 0.001; FC = 0.90, p < 0.001; FC = 0.93, p < 0.001; FC = 0.93, p < 0.001; FC = 0.89, p = 0.001, respectively).

Glutamine (FC = 0.41, p > 0.05), glyceric acid (FC = 0.85, p > 0.05), creatinine (FC = 0.71, p > 0.05), arginine (FC = 0.50, p > 0.05), citric acid (FC = 0.45, p > 0.05), choline (FC = 0.55, p > 0.05) and tocopherol (FC = 0.59, p > 0.05) were all lower in PCOS patients and had very low concentrations after treatment although these values were not significantly different.

The metabolic pathway analysis of the selected metabolites is summarized in the metabolic systems map shown in Fig. 3. There is a definite interaction of several pathways involving Biopterin metabolism; De novo fatty acid biosynthesis; Di-unsaturated fatty acid beta-oxidation; Glycerophospholipid metabolism; Glycine, serine, alanine and threonine metabolism; Linoleate metabolism; Saturated fatty acids beta-oxidation; TCA cycle; Tyrosine metabolism; Urea cycle and metabolism of arginine, proline, glutamate, aspartate and asparagine; Valine, leucine and isoleucine degradation; Vitamin B3 (nicotinate and nicotinamide) metabolism; Vitamin E metabolism.

This is a prospective case-control study conducted in a single center at University Hospital “San Giovanni di Dio e Ruggi D’Aragona” of Salerno.

The studied population included only young/adolescent women. We have chosen to investigate this specific age group, since younger women may benefit the most from an early treatment in terms of future pregnancy chances and possibly avoiding comorbidities development [36]. Furthermore, the PCOS-inositols treatments do not contraindicate other concomitant therapeutic options [7].

The aim of this study was, firstly, to identify discriminant metabolites in serum of women with a diagnosis of PCOS and, secondly, to define if treatment with inositols can improve metabolomic pathways and metabolic profiles of women with PCOS. Multi-variate statistical analysis identified fifteen metabolites as being particularly important in separating cases from controls: 3-methyl-1-hydroxybutyl-thiamine diphosphate, valine, phenylalanine, ketoisocapric acid, linoleic acid, lactic acid, palmitic acid and glucose were increased in PCOS patients compared to controls, while glutamine, glyceric acid, creatinine, arginine, citric acid, choline and tocopherol were decreased.

According to these results, different metabolic pathways appear to be involved in PCOS pathology. In the serum of PCOS patients at enrollment time in comparison with control group, several metabolites, closely associated with carbohydrate and lipid metabolisms, are significantly dysregulated.

Increased levels of lactic acid have been previously reported in PCOS patients [27, 37] and can be related to an increased muscular gluconeogenesis or can indicate an uptake and consumption of glucose by muscles, suggesting an important role for insulin resistance in these patients [25].

As previously reported [27], linoleic acid and palmitic acid are also increased, likely the result of increased lipolysis that might be related to presence of insulin resistance in fatty tissue [25]. PCOS is associated with increased lipolysis due to presence of IR in adipose tissue [38]. However, IR does not occur in all tissues of all women with PCOS, and different mechanisms may contribute to hyperinsulinemia [39].

Moreover, high abundances of linoleic acid appear to inhibit the maturation and development of oocytes [40]. Due to proinflammatory activity, elevated linoleic acid levels can lead to the chronic inflammation associated with PCOS [41, 42].

Several amino acids and other metabolites associated with the tricarboxylic acid cycle (TCA) are altered in PCOS patients: the levels of arginine, choline, glutamine and citric acid were decreased and those of phenylalanine and valine were increased, compared to the controls. Such alterations to the TCA cycle are also related to insulin resistance [43] and alterations in oocyte maturation [44].

Valine was increased in PCOS patients as previously reported [29] and it might be associated with other metabolic alterations not related with insulin resistance [25]. As with the present study, creatinine has been reported to be reduced in PCOS patients [37].

Nevertheless, our specific cohort showed a low incidence of insulin resistance, according to previous reported data on adolescent population [35, 45], therefore the proposed theories/explanation may not fully apply to the cohort of patients evaluated in this study.

We found a reduction of tocopherol in PCOS patients, consistent with previous work [39]. This metabolite has an anti-oxidant effect and the reduction in concentration might be explained by the increased oxidative stress observed in PCOS.

According to recent guidelines, insulin-sensitizer drugs are the first-line therapy in women with metabolic abnormalities and irregular menstrual cycle with the purpose to improve fertility, whereas a lifestyle change with weight loss and physical activity is the first step in overweight and obese PCOS patients [46]. Inositols belong to a sugar alcohol family comprising nine stereoisomers and two of them, MI and DCI, are involved in the pathophysiology and treatment of PCOS [47]. Inositols work as second messengers of insulin: MI improves oocyte quality mediating glucose uptake and follicle stimulating hormone (FSH) signaling, while the supplementation of DCI, is involved in insulin-mediated androgen synthesis, replenishing stores of the mediator and improving insulin sensitivity [7, 47, 48]. A recent review has shown the efficacy of inositols treatment in the management of PCOS [49], while an Italian study [19] has shown the synergistic action of inositols-glucomannan in the treatment of PCOS women with insulin resistance (IR). So, we can suggest a central but not peripheral role of IR in the present cohort. In another study, after combined treatment with inositols-glucomannan, PCOS patient showed decreased levels of free fatty acids, and patients experienced weight reduction [50]. In addition, the reduction of linoleic acid was also reported after 30 months of polytherapy with pioglitazone-flutamide-metformin and hormonal medication in an adolescent patient group with PCOS [32]. Moreover, a recent study [33] has shown how, in metabolic terms, 3-iodothyronamine administration in a mouse model of PCOS may serve as a promising endogenous supplement in the treatment of dysfunctional lipid metabolism.

It would be reasonable to expect a trend towards normalization of the metabolome upon weight reduction and clinical improvement due to the treatment. Indeed, after 3 months of treatment with a combination of MI, DCI and glucomannan, the clinical symptoms did improve. However, the metabolic profiles of patients after 3 months of treatment were less close to those of controls compared to initial metabolomes. Indeed, metabolites with lower abundances in PCOS-T0 patients either stayed the same or decreased even more after treatment, while those metabolites with higher abundances generally increased after treatment. We offer two potential explanations for these observations: 1) the metabolome is more strongly affected by therapeutically induced rapid weight loss (approximately 400 g/week) in overweight patients compared to PCOS condition; 2) the metabolome of patients with PCOS may adapt to this condition, thus rendering the differences between controls and not-treated patients less prominent. This effect was observed in anorexic women treated with a food therapy [51]. Similarly, it can be argued that the strongly altered metabolome of weight loss patients would normalize if the achieved body weight is maintained. In general terms, we conclude that metabolic processes are seemingly susceptible to changes generated by being overweight and even more so by subsequent weight loss. The modest improvement in menstrual cycle regularity following treatment (Table 1) is statistically consistent with that reported by Pundir’s meta-analysis [52]. This can be related to the lower incidence of IR in our cohort; indeed a large amount of evidence indicates that lowering insulin levels with insulin-sensitizing drugs in IR-PCOS women can reduce circulating androgen levels, increase SHBG levels, and restore ovulatory menstrual cycles [53]. Moreover, although only 27% of the post-treatment subjects reported menstrual cycle characteristics consistent with a “regular” definition (i.e., a cycle lasting between 25 and 36 days and menstrual blood loss between 20 and 80 mL), all the patients reported some kind of improvement in menstrual cycle regularity.

Our study has a few limitations. The first is the lack of a metabolomic comparison among PCOS patients undergoing inositols-only treatment. As a result, we cannot address which changes were due to the combined treatment and which ones were due to inositols. A further limitation is the small number of patients enrolled, although acceptable statistical power [54] is still achieved. Our PCOS patients did not show higher free and total testosterone levels compared with reference values of the Italian population. However, many factors are considered for diagnosis of hyperandrogenism. The Androgen Excess Society criteria for diagnosis of PCOS requires clinical or biochemical evidence of hyperandrogenism [55].

Clinical diagnosis of hyperandrogenism is identified by hirsutism (as scored by the Ferriman-Gallwey exam), acne, and alopecia [56]. Biochemical diagnosis of hyperandrogenism includes measuring serum total testosterone, free testosterone, free androgen index, androstenedione, and dehydroepiandrosterone sulfate [57]. Amirie et al. [58], conducted a meta-analysis of 6593 PCOS patients and confirmed that the Ferriman-Gallwey clinical diagnostic criteria significantly correlates with biochemical diagnostic criteria. Concentrations of androstenedione and dehydroepiandrosterone sulfate correlated with the Ferriman-Gallwey diagnosis of PCOS; while, as with our study, a significant correlation was not found with testosterone and free testosterone [58]. Indeed, the most commonly quantified biochemical parameters of hyperandrogenism are free testosterone and free androgen index; however, the increased levels of androstenedione or other androgen precursors are, for many patients, the only indicators of hyperandrogenism [55, 58, 59]. Thus, the fact that free testosterone and total testosterone was not higher in our PCOS cases does not indicate the patient did not have PCOS. Therefore, all of our PCOS cases met the clinical criteria for hyperandrogenism, which is considered the cornerstone of PCOS [55]. Finally, our PCOS cases were also well defined by ultrasonography and Acne scores.

Despite the limitations, the present pilot study allowed us to follow the patients over a 3-month treatment regimen. Indeed, our pilot study is an ideal model because it allows metabolomic analysis of pre- and post-treatment serum taken from the same patient. This in turn allows comparative analysis of metabolite profiles from subjects for which the genetic, hormonal and environmental backgrounds have remained constant. In this way, changes in metabolome occurring during the treatment can be traced independently from the physiological inter-variability among people.

Large functional and prospective studies, including clinical trials, are needed to verify our preliminary results and to separate the role of inositols and of the combined treatment with inositol and glucomannan. Such studies will continue to shed further insights into these metabolite profiles and determine whether some of them are biomarkers or actual mediators of PCOS. A better understanding of the role of these biomarkers in obesity and obesity-related PCOS pathogenesis might finally lead to novel targets for therapeutic/preventive intervention. Furthermore, this kind of study can contribute to understanding the role of weight reduction in PCOS patients.