Clinica Chimica Acta 429 (2014) 181–188
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Invited critical review
Metabolomics in polycystic ovary syndrome☆ Mora Murri a,b,c, María Insenser a,b,c, Héctor F. Escobar-Morreale a,b,c,⁎ a b c
Diabetes, Obesity and Human Reproduction Research Group, Hospital Universitario Ramón y Cajal, Universidad de Alcalá, E-28034 Madrid, Spain Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), E-28034 Madrid, Spain Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), E-28034 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 27 October 2013 Received in revised form 12 December 2013 Accepted 14 December 2013 Available online 22 December 2013 Keywords: Metabolism Metabolomics Non-targeted Obesity Polycystic ovary syndrome
a b s t r a c t The association of androgen excess with abdominal adiposity, insulin resistance and metabolic derangements characterize many patients with PCOS. However, the mechanisms underlying these associations are not entirely understood, indicating the need for discovery of the origin of these metabolic alterations, and of new metabolic biomarkers for PCOS. This review summarizes the metabolites and metabolic pathways associated with PCOS according to recent metabolomic studies. PCOS-associated metabolites were involved mostly in carbohydrate, fat, and protein metabolism. Obesity, hyperinsulinemia and the intrinsic heterogeneity of PCOS are responsible of the metabolic variation observed in these women. Furthermore, treatment of PCOS seems to modify the levels of some metabolites. Hopefully, advances in the knowledge of metabolism will allow the detection of systemic imbalances in PCOS and will permit the identification of biomarkers that serve to predict the progression of the disease and its future complications. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Metabolomic techniques . . . . . . . . . . . . . . . . . . . Metabolites in PCOS . . . . . . . . . . . . . . . . . . . . . 3.1. Metabolomics profiles in PCOS . . . . . . . . . . . . . 3.2. Metabolites influenced by obesity and hyperinsulinemia . 3.3. Metabolites influenced by PCOS phenotypic heterogeneity 4. Metabolomics and treatment of PCOS . . . . . . . . . . . . . 5. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The polycystic ovary syndrome (PCOS) is the one of the most common endocrine disorders of women of reproductive age [1], and results from complex and poorly understood interactions between environmental and genetic factors [2]. PCOS is defined at present as a mainly hyperandrogenic disorder in which patients present with a constellation of symptoms and signs
☆ GRANTS: Supported by grants FIP PI1100357 and CD11/0030 (Mora Murri is the recipient of a “Sara Borrell” postdoctoral grant) from Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness. ⁎ Corresponding author. Tel./fax: +34 91 336 9029. E-mail address: [email protected] (H.F. Escobar-Morreale). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.018
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that necessarily include clinical and/or biochemical hyperandrogenism together with ovulatory dysfunction and/or polycystic ovarian morphology [3]. The consequences of PCOS are numerous, from cutaneous signs and menstrual and ovulatory dysfunction, to features of metabolic syndrome. A better understanding of this complex disorder and the identification of potential biomarkers of disease may represent a significant advance for the prevention, early and accurate diagnosis, and effective management of PCOS. In the recent years, the development of novel technologies that allow comprehensive metabolic profiling, termed metabolomics, may represent a big step in this direction. Metabolomics is defined as the analysis of multiple small-molecule metabolites, which are the products of metabolism, in biological samples. This term is often confused with metabonomics, which is the study of global, dynamic metabolic response of living systems to
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biological stimuli or genetic manipulation [4]. Metabolomics is a recent addition to the omics family of platforms that also includes, among others, genomics, transcriptomics and proteomics. Metabolomics studies events happening downstream of gene expression and it is considered to be closer to the actual phenotype than either both genomics or proteomics [5]. Metabolites can be intrinsic, resulting from normal cellular physiology, or extrinsic, resulting from the influences of exogenously administered pharmaceuticals [5]. The Human Metabolome Database (HMDB) contained in as many as 41,519 metabolite entries in August 2013, and approximately 2900 of these metabolites are endogenous and may be detectable in the human body. The physiological functions of these endogenous metabolites span over a wide range and include growth, development, and reproduction among other functions [4]. Many patients with PCOS are overweight or obese [6,7] and abdominal visceral adiposity is particularly frequent in these women [8–10]. Moreover, insulin resistance is also common in patients with PCOS independently of obesity [10], and approximately 50–70% of women with PCOS have insulin resistance and compensatory hyperinsulinism [6]. The association of androgen excess, abdominal adiposity, insulin resistance and metabolic derangements in women with PCOS has been explained by the existence of a vicious circle in these women, that may start during early stages of life or even prenatally, whereby androgen excess favoring the abdominal deposition of fat further facilitates androgen secretion by the ovaries and adrenals in women with PCOS [10]. Being included in the definition of the syndrome, increased serum androgen concentrations are the most obvious biomarkers of PCOS. However the mechanisms for the alteration of these levels are not well understood and also, biochemical hyperandrogenism is not always present in women with PCOS. Because PCOS is a complex disorder accompanied in many cases with metabolic derangements, there is clearly a need to discover the exact origin of these metabolic alterations and new metabolic biomarkers for PCOS. The aim of the present review is to summarize the metabolites and metabolic pathways associated with PCOS described in recent metabolomic studies. 2. Metabolomic techniques Metabolomics is an emerging bioanalytical discipline that uses analytical techniques to characterize and quantify in biological samples metabolites – small molecules that are mediators and products of metabolism – with the aim of monitoring metabolism and its fluctuations [4]. Therefore, because metabolites fall downstream of genetic, transcriptomic, proteomic, and environmental variation, metabolomics offers integrated and dynamic measures of phenotypes and medical conditions, providing insights on what is happening in a biological system. Metabolites are present in very broad concentration ranges and exhibit a notable chemical diversity. Therefore, the study of the human metabolome requires a set of instruments, such as the combination of gas (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), or the combination of MS and nuclear magnetic resonance (NMR). The use of MS relies on the fact that every compound has a unique fragmentation pattern. The sample is ionized and the sample ions are separated based on their differing masses and relative abundances. A commonly used MS technique is time-of-flight mass spectrometry (TOF–MS), in which ions are accelerated by a constant electric field of known strength and, therefore, their velocities depend on their mass-to-charge ratio. Hence, the time that the particle takes to reach a detector at a known distance indicates it mass with great accuracy. Before being submitted to TOF–MS identification, molecules need to be separated from the mixture of metabolites that compose the sample under study, either by GC–MS in the case of molecules that can be vaporized without chemical decomposition, or by LC–MS that separates molecules based on their interactions in the mobile and stationary phases.
NMR analyzes the signal that each molecule emits when their nuclei spin orientations are modified after an exposure to a magnetic field and radiofrequencies [11]. This signal is translated into a spectrum that is characteristic for each molecule and can be matched with those of existing databases. Among the most widely used NMR techniques is proton nuclear magnetic resonance (1H NMR), in which the NMR spectrometer is tuned to a specific nucleus, in this case the proton. Both MS and NMR have advantages and disadvantages [12]. MS analysis is more sensitive than NMR and permits the measurement of a broader array of small metabolites through the application of different combinations of chromatographic methods and mass/charge separation techniques. However, MS has the inconvenient of the destruction of the sample and the long sample preparation time required as it usually requires derivatization of metabolites to produce ionic species, whereas NMR is a non-destructive analysis which provides quantitative and reproducible measurements with minimal preparation of the samples, as no separation or ionization steps are necessary. Therefore, studies using a multiplatform approach that combines MS and NMR analysis may provide a more comprehensive understanding of metabolic alterations than studies using only one of these tools [12]. 3. Metabolites in PCOS The application of metabolomics in the field of PCOS is in its early infancy. We have reviewed the few metabolomic studies that compared controls and patients with PCOS. Four studies met this criterion [13–16] and their characteristics are summarized in Table 1. The studies were performed analyzing plasma samples using either 1H NMR [13,15], GC–MS systems [14] or 1H NMR together with GC/TOF–MS [16]. The four studies have high methodological quality according to a modification of the Newcastle–Ottawa Quality Assessment Scale for case–control studies explained in a previous work [17] (Table 1). The altered metabolites found in the studied manuscripts were involved mostly in carbohydrate, fat, and protein metabolism (Figs. 1, 2, Table 2). In the following subsections, we will describe the metabolites that could have a greater interest for PCOS. First, we will describe the metabolites altered in PCOS. Second, we will analyze metabolites that are affected in PCOS by the interaction with obesity, hyperinsulinemia and with the heterogeneity intrinsic to PCOS. Finally, we will describe the single study [18] that addressed the effects of treatment of PCOS on the metabolome, in order to find out which metabolites change after the remission of PCOS symptoms. 3.1. Metabolomics profiles in PCOS Altered carbohydrate metabolism is present in many PCOS patients [1]. The significant increase of lactate and gluconeogenic amino acids, and the reduction of glucose in plasma of non-obese women with PCOS, might be explained by increased glycolysis in muscle and decreased gluconeogenesis in liver [15,16]. Moreover, the increase in lactate levels in insulin resistant non-obese PCOS women [16] suggests insulin stimulated glucose uptake and consumption in the muscle of these PCOS patients, indicating central but not peripheral insulin resistance in these patients. In terms of lipid metabolism, levels of lipoprotein, low-density lipoprotein cholesterol (LDL-c) and very low-density lipoprotein cholesterol (VLDL-c) were increased in PCOS samples compared with controls, accompanied with a decrease of cholesterol and high-density lipoprotein cholesterol (HDL-c) levels [15,16]. These findings are in agreement with the lipid profiles measured by biochemical methods in previous reports [19]. Moreover, levels of free fatty acids (such as palmitic, stearic and linoleic acid) were increased in PCOS samples compared with controls, indicating an increase in lipolysis consistent with the presence of insulin resistance in adipose issue [20,21]. Moreover, linoleic acid may be involved in the inhibition of the maturation and developmental potential of oocytes [22]. Accordingly, the increase
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Table 1 Characteristics of the studies included in this review. Author (year)
Atiomo et al. (2012) Escobar-Morreale et al. (2012) Sun et al. (2012) Zhao et al. (2012)
Biological sample
Plasma Plasma Plasma Plasma
Simple size (n)
Mean age (years)
Mean BMI (kg/m2)
PCOS
Controls
PCOS
Controls
PCOS
Controls
12 36 34 217
10 39 36 48
27.2 26.3 27.2 28.4
28.4 26.5 26.9 29.8
33.2 29.9 24.5 24.5
27.0 29.3 22.6 21.6
in linoleic acid levels in patients with PCOS might be accompanied by a reduction in oocyte maturation and disordered ovulation in PCOS. Additionally, linoleic acid displays potent proinflammatory activities [23], and might also contribute to the chronic low-grade inflammation underlying PCOS [24,25]. However, these interpretations should be considered with caution, as other studies reported conflicting results, with the levels of these lipids being decreased [13,16] or increased [14,16] in PCOS. Another lipid metabolite decreased in patients with PCOS is αtocopherol [14]. α-Tocopherol is a potent antioxidant that serves as a peroxyl radical scavenger that protects polyunsaturated fatty acids in membranes and lipoproteins [26]. The decrease in α-tocopherol may contribute to the association of PCOS with oxidative stress [17]. The aerobic catabolism of glucose, fatty acids, and some amino acids results in acetyl-coenzymeA (CoA), the entry point into the tricarboxylic acid (TCA) cycle. The TCA cycle consists of series of biochemical reactions used by all aerobic organisms to generate energy, through the oxidization of acetyl-CoA into carbon dioxide. Levels of citrate were decreased in PCOS women compared with control women. Additionally, levels of amino acids involved in TCA cycle, such as arginine, citrulline, glutamine, histidine, methionine and proline were also decreased; accompanied by an increase in phenylalanine, threonine, tyrosine, valine, inducing a decrease or increase in elements of the TCA cycle [13,15,16]. This suggests that TCA cycle is impaired in PCOS women, which has also been considered as evidence of the existence of insulin resistance [27]. Hyperandrogenism has been described to alter TCA cycle by decreasing citrate levels [28]. Moreover, TCA cycle has been described to play an important role in oocyte maturation with lower levels of citrate indicating decreased competence [28]. The concentrations of several aromatic and branched-chain amino acids (AAA and BCAA, respectively), such as valine, tyrosine, tryptophan and phenylalanine, were increased in PCOS patients [16]. These amino acids have been linked to insulin resistance and diabetes [29,30]. Moreover, Wurtz et al. [31] reported disorders in AAA and BCAA metabolism preceding hyperglycemia in the general population. Additionally, levels of glycine, which seems to improve the proinflammatory state and
Country
Participant selection
Definition of PCOS
Quality assessment rating
UK Spain China China
Age and BMI matched Age and BMI matched Age and BMI matched Age matched
ESHRE/ASRM NICHD ESHRE/ASRM ESHRE/ASRM
High High High High
upregulate adiponectin gene expression in vitro [32], were decreased in PCOS and the elevated levels of N-acetyl glycoproteins, that serve as inflammatory markers and acute-phase proteins [33], support the existence of low-grade chronic inflammation in PCOS patients [15]. Leucine concentration was decreased in PCOS patients in the study by Sun et al. [15]. Because leucine may rescue insulin signaling via activation of the mTOR pathway, and increasing dietary leucine intake may improve insulin sensitivity [34,35], this decrease in leucine levels could contribute to the insulin resistance of PCOS. Furthermore, Zhao et al. [16] found an increase in leucine levels in PCOS patients, yet this difference was observed only in PCOS patients with insulin resistance. Therefore, they speculated that alterations of plasma levels of leucine in PCOS patients with insulin resistance might be entirely due to the impairment of insulin signaling [16]. 2-Ketoisocaproic acid levels were decreased in patients with PCOS compared with controls [14], suggesting decreased transamination of leucine in the first step of BCAA catabolism instead of the increased transamination previously suggested as a metabolic characteristic of obesity and diabetes [29,36]. Under normal conditions, alanine arising from BCAA nitrogen accounts for 25% of gluconeogenesis from amino acids [37]. These data, together with the decreased alanine levels found by Escobar-Morreale et al. [14] in patients with PCOS, could indicate that BCAAs are being used for protein synthesis in these women, and not for gluconeogenesis as happens in obesity and diabetes [29,36]. However, a decrease in plasma alanine level has been also associated with increased gluconeogenesis in diabetic mice [38]. Levels of dimethylamine were increased in PCOS patients, accompanied by a decrease in choline, phosphatidylcholine and glycerophosphocholine/phosphocholine levels [15]. Symbiotic gut microbiota converts the choline, released by hydrolysis of phosphatidylcholine, into methylamines such as dimethylamine, leading to low circulating levels of plasma phosphatidylcholine, which reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing non-alcoholic fatty liver disease [39]. These results are in accordance with those found in diabetes [40] suggesting a possible relationship of PCOS with these disorders.
Fig. 1. Altered metabolites in PCOS, represented as reported in one or more studies.
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Fig. 2. Interactions of the metabolites described in this review.
3.2. Metabolites influenced by obesity and hyperinsulinemia Escobar-Morreale et al. [14] found substantial metabolic heterogeneity in PCOS that was dependent mostly on the presence or absence of obesity (Table 3). They found that levels of glycerol and long-chain fatty acids were decreased in nonobese women with PCOS compared with nonobese controls, suggesting suppression of lipolysis. This finding requires conserved insulin sensitivity in adipose tissue, and possibly the presence of increased insulin levels might have contributed to reduced lipolysis, although impairment in catecholamine-induced lipolysis in subcutaneous tissue is also characteristic of nonobese women with PCOS [41–43]. Moreover, increased levels of lactate and it precursor, lactic acid, were observed in non-obese women with PCOS when patients were classified for obesity [14,15], indicating increased insulin-stimulated glucose uptake and consumption in the muscle of these non-obese patients [14]. Because such uptake of glucose, similar to the suppression of lypolisis in adipose tissue, requires effective insulin signaling, these findings suggest that insulin resistance is not universal in all the tissues of non-obese patients with PCOS. However, since their insulin concentrations are higher than those of non-hyperandrogenic controls for a similar plasma glucose concentration, a certain degree of central insulin resistance must be present in these women [14]. In conceptual agreement, Harris et al. [44] demonstrated that oocytes from non-obese PCOS women presented increased glucose and pyruvate consumption during overnight in vitro maturation and that treatment with the insulin-lowering drug metformin avoided this increase. This finding indicates that there is no insulin resistance in PCOS oocytes, and that their enhanced glucose consumption may result from exposure to the increased insulin levels resulting from central insulin resistance. On the contrary, glycerol and long-chain fatty acids were increased and plasma lactate concentrations were decreased in obese patients with PCOS, suggesting that in PCOS the development of peripheral insulin resistance requires the coexistence of obesity [14]. Sun et al. [15] and Zhao et al. [16] confirmed that obesity exerted a major influence in the metabolic profile of PCOS (Table 3). Moreover, Zhao et al. [16] also demonstrated an influence of insulin resistance in the
metabolic profile of women with PCOS, even though insulin resistance was entirely dependent on the presence of obesity. In obese women, the increase of long-chain fatty acids and glycerol levels found in those presenting with PCOS, as well as the increase in oleic and palmitoleic acid found in obese women irrespective of PCOS, suggested impaired insulin action at adipose tissue [41,45]. As seen previously, this increase in lipolysis is the opposite result found in the non-obese PCOS patients. The adipose tissue expandability hypothesis states that once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy efficiently and lipids begin to accumulate in other tissues [46]. When this adipose tissue expansion fails insulin can no longer suppress lipolysis. Furthermore, these increased levels of circulating free fatty acid in obese patients with PCOS may contribute further to their central insulin resistance [47], and to the association of PCOS and obesity with the metabolic syndrome and non-alcoholic fatty liver disease [48]. Glycine levels were decreased in obese women [14], which may indicate increased utilization for gluconeogenesis. This finding agrees with the metabolic signature previously associated with obesity that includes increased levels of circulating BCAA [29]. BCAA, which are increased by peripheral insulin resistance as their utilization in tissues such as muscle requires conserved insulin signaling, are used for gluconeogenesis through pyruvate transamination into alanine, thereby contributing to glucose intolerance [29,36]. Furthermore, an increase in BCAA catabolism may explain the increase in phenylalanine levels in the obese women, because large neutral amino acids such as phenylalanine compete with BCAA for transport into mammalian cells [49]. Elevated levels of valine and reduced levels of glycine were also observed in the women with PCOS without insulin resistance as compared with controls [16], and these changes were aggravated when the patients had altered insulin sensitivity. Hence, valine and glycine may be associated with other metabolic disturbances independently of insulin resistance. 3.3. Metabolites influenced by PCOS phenotypic heterogeneity Several metabolites were altered by the heterogeneity inherent to the different PCOS phenotypes (Table 4). Zhao et al. [16] reported that
M. Murri et al. / Clinica Chimica Acta 429 (2014) 181–188 Table 2 Summary of metabolites associated with polycystic ovary syndrome and their changes compared with controls. Carbohydrate related markers Glucose Undentified sugar Lactate
↓ ↑ ↑
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012; Sun et al., 2012
Lipid related markers α-Tocopherol Acetone Cholesterol
↓ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑
Escobar-Morreale et al., 2012 Atiomo et al., 2012 Zhao et al., 2012; Escobar-Morreale et al., 2012 Zhao et al., 2012 Zhao et al., 2012; Sun et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012
↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑
Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Escobar-Morreale et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012
↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↓ ↑ ↓ ↑ ↓ ↑
Atiomo et al., 2012; Sun et al., 2012 Sun et al., 2012 Atiomo et al., 2012 Atiomo et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012; Atiomo et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Escobar-Morreale et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012
TCA related marker Citrate
↓
Sun et al., 2012; Atiomo et al., 2012
Purine related marker Uric acid
↑
Zhao et al., 2012
HDL Phosphatidylcholine Fatty acid Linoleic acid Lipoprotein Palmitic acid Stearic acid Unsaturated fatty acid VLDL/LDL Lipid-CH2CH2CO Protein related markers Creatinine Lysyl-albumin Trimethylamine N-oxide 2-Ketoisocaproic acid Creatine Dimethylamine N-acetylglycoprotein
Amino acids Arginine Choline Citrulline Glutamate Glutamine Glycerophosphocholine/phosphocholine Glycine Histidine Isoleucine Methionine Proline AAA BCAA BCAA/AAA Aspartate Endogenous AAs Gluconeogenic AAs Phenylalanine Serine Threonine Tryptophan Tyrosine Valine Alanine Leucine Lysine Ornithine
185
hormone concentrations, its ratio to follicle-stimulating hormone, AAA levels and BCAA/AAA ratios were much more severe in classic PCOS than in other PCOS phenotypes [16]. Furthermore, the metabolic profile of the ovulatory PCOS phenotype was different compared to that of the classic anovulatory PCOS phenotypes. The levels of endogenous amino acids were decreased in ovulatory PCOS women compared with controls suggesting increased protein synthesis (i.e. in skeletal muscle) in these women. In agreement, Carmina et al. [50] reported increased lean muscle mass in women with PCOS. On the other hand, increased levels of endogenous amino acid including serine, threonine, phenylalanine, tyrosine and ornithine were found in the patients presenting with polycystic ovarian morphology and anovulation, which may indicate increased protein degradation in ovarian dysfunction [16]. Zhao et al. [16] reported that the levels of long-chain fatty acids were reduced in PCOS patients with hyperandrogenism compared with patients without hyperandrogenism. This result disagrees with previous reports describing a positive association between fatty acid levels and androgen excess [19,51,52]. Sun et al. [15] reported that, when taking hyperandrogenism into consideration, the only significant change found was the increased N-acetyl glycoprotein observed in the patients with PCOS and hyperandrogenism compared with the controls. This may represent a link between androgen excess and inflammation in PCOS. 4. Metabolomics and treatment of PCOS Treatment of adolescent patients with PCOS with pioglitazone– flutamide–metformin (Pio/Flu/Met) polytherapy during one month showed changes in serum metabolites [18]. Pio/Flu/Met polytherapy has been described to block androgen receptors, remodel body adiposity, reduce low-grade inflammation, and promote a favorable adipokine profile [18]. Vinaixa et al. [18] reported that 9- and 13-HODE, and azelaic acid, which are downstream metabolic oxidation products of linoleic acid (which is the major polyunsaturated fatty acid in human plasma and LDL [53,54]) and glutaric acid, which can also induce oxidative stress [55], were decreased in serum after 30 days of Pio/Flu/Met treatment. The levels of 1,2-propanediol, acetate, glutamate, lysine and succinate in serum were also significantly decreased after the polytherapy. These results may indicate that Pio/Flu/Met polytherapy reduces the amount of oxidized lipoprotein particles and downstream oxidative metabolites in the serum of PCOS patients [18]. Most of these metabolites participate directly or indirectly, and with lesser or greater importance, in the TCA cycle [56–60], which as described above, is altered in PCOS. Therefore, Pio/Flu/Met treatment seems may influence favorably the metabolism of young patients with PCOS [18]. 5. Limitations Metabolomic studies are not free of limitations. First, the heterogeneity in the criteria used to diagnose PCOS together with the relatively small sample sizes and the different metabolomic techniques used impose important limitations when aiming to integrate the results of the different studies conducted to date. In addition, in not all studies the patients and the controls were matched for BMI [16], did not include insulin resistant controls [16] or did not show data regarding glucose and insulin levels of the women included in the study [15]. Finally, as occurred with other diseases, the findings derived from metabolomic studies still have to demonstrate their usefulness as biomarkers in PCOS and its associated metabolic comorbidities [12]. 6. Conclusions
the classic hyperandrogenic and oligo-ovulatory phenotype of PCOS was related to more adverse biochemical and metabolic changes than other phenotypes when compared with controls. Alterations of luteinizing
The pathogenesis of PCOS remains largely unknown, mostly because our lack of understanding of the molecular mechanisms responsible for
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Table 3 Impact of obesity on the changes observed in metabolomic biomarkers for polycystic ovary syndrome. Obese vs nonobese
Non-obese PCOS vs nonobese control
Obese PCOS vs obese control
↑
Escobar-Morreale et al., 2012
↑ ↑
Escobar-Morreale et al., 2012 Sun et al., 2012
↓
↑
Escobar-Morreale et al., 2012 ↑ ↑ ↑
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↓ ↓
Escobar-Morreale et al., 2012 Escobar-Morreale et al., 2012
↓ ↑ ↓ ↑
Escobar-Morreale et al., 2012 Zhao et al., 2012 Escobar-Morreale et al., 2012 Zhao et al., 2012
↑ ↑ ↑ ↑
Escobar-Morreale et al., 2012 Escobar-Morreale et al., 2012 Sun et al., 2012 Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↑
Zhao et al., 2012
Protein related markers Creatine Dimethylamine N-acetylglycoprotein
↑ ↑ ↑
Sun et al., 2012 Sun et al., 2012 Sun et al., 2012
Amino acids Alanine Arginine Aspartate Choline Glycerophosphocholine/phosphocholine Glycine Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tyrosine Tryptophan Valine Endogenous AAs BCAA AAA BCAA/AAA
↑ ↓ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓
Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012, Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Sun et al., 2012
↓
Sun et al., 2012
TCA related marker Citrate
↓
Sun et al., 2012
Purine related marker Uric acid
↑
Zhao et al., 2012
Carbohydrate related markers Lactic acid Lactate Gluconic acid lactone Gluconeogenic Aas Phosphoglyceryde Undentified sugar Lipid related markers Adipic acid Citramalic acid Glyceric acid Glycerol LDL Linoleic acid Oleic acid Palmitic acid Palmitoleic acid Stearic acid
↑
Escobar-Morreale et al., 2012
↓
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↓
↑
Escobar-Morreale et al., 2012
Escobar-Morreale et al., 2012
this complex disorder. Advances in the field of metabolism will hopefully permit the detection of systemic imbalances in PCOS and the identification of biomarkers that predict the progression of the disorder and its complications. The application of metabolomics to PCOS has rapidly evolved during the past years providing researchers with the opportunity to gain new insights into the molecular mechanisms and metabolic networks involved in its development. Several specific metabolic pathways, including protein, lipid and carbohydrate metabolism and the TCA cycle, appears to be disturbed in PCOS. In addition, treatment with insulin sensitizers and antiandrogens may have a beneficial impact on the metabolic abnormalities of young patients with PCOS. These results represent the basis for the identification of novel PCOS biomarkers, and the development of diagnostic tests and new methodologies for metabolic screening and personalized medicine.
↓
Obese PCOS vs nonobese PCOS
Escobar-Morreale et al., 2012 ↑
Zhao et al., 2012
↓ ↓
Zhao et al., 2012 Zhao et al., 2012
↓
Zhao et al., 2012
Sun et al., 2012
Metabolic heterogeneity is present in patients with PCOS, and it is strongly influenced by hyperinsulinemia, insulin resistance and obesity. The metabolic profile of nonobese patients with PCOS is explained by the hyperinsulinemia resulting from reduced hepatic clearance of insulin and hepatic insulin resistance [61] and the absence of peripheral insulin resistance in the presence of increased circulating insulin concentrations, in conceptual accordance with the previously reported heterogeneity in insulin action between metabolic responses and target tissues [62,63]. In obese patients with PCOS, the metabolic profile indicates the presence of both central and peripheral insulin resistance. As substantial metabolic heterogeneity underlies PCOS, confusion factors such as obesity, hyperinsulinemia, and insulin resistance should be considered when designing diagnostic and therapeutic strategies for the management of this prevalent disorder. The metabolomic study of PCOS is further complicated because the phenotypic heterogeneity of the syndrome. Features of PCOS such as
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Table 4 Impact of the polycystic ovary syndrome phenotype on metabolomic biomarkers. Non-hyperandrogenic PCOS vs controls
Hyperandrogenic PCOS vs controls
Hyperandrogenic PCOS VS Non-hyperandrogenic PCOS
Carbohydrate related markers Glucose
↑
Zhao et al., 2012
Lipid related markers Linoleic acid Palmic acid Stearic acid
↓ ↓ ↓
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Zhao et al., 2012
↓
Zhao et al., 2012
↑ ↓
Zhao et al., 2012 Zhao et al., 2012
↑
Zhao et al., 2012
Protein related markers Creatine Dimethylamine N-acetylglycoprotein Amino acids Alanine Arginine Aspartate Choline Glycerophosphocholine/phosphocholine Leucine Tryptophan Valine TCA related marker Citrate
↑ ↑
↓ ↓ ↓
↓
Sun et al., 2012 Sun et al., 2012
Sun et al., 2012 Sun et al., 2012 Sun et al., 2012
Sun et al., 2012
Purine related marker Uric acid
ovulatory dysfunction, hyperandrogenemia and polycystic ovarian morphology may associate different metabolic alterations. Therefore, the systematic comparison of the metabolic profiles of the different PCOS phenotypes may be helpful in order to understand the metabolic risks and associations of these patients. In conclusion, novel PCOS related metabolites involved in different metabolic pathways have been identified by recent non-targeted metabolomic studies. These findings still need to be replicated by future studies in which well characterized and homogenous PCOS phenotypes are to be compared with carefully matched control women. Only then will these metabolites be considered as biomarkers of the syndrome and its complications, contributing to metabolic screening and personalized medicine in PCOS. Conflict of interest The authors have no conflict of interest to disclose. Acknowledgments Supported by grants FIP PI1100357 and CD11/0030 (Mora Murri is the recipient of a “Sara Borrell” postdoctoral grant) from Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness. References [1] Azziz R, Carmina E, Dewailly D, et al. The androgen excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril 2009;91:456–88. [2] Escobar-Morreale HF, Luque-Ramirez M, San Millan JL. The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome. Endocr Rev 2005;26:251–82. [3] Escobar-Morreale HF. Polycystic ovary syndrome: treatment strategies and management. Expert Opin Pharmacother 2008;9:2995–3008. [4] Nicholson JK, Lindon JC. Systems biology: metabonomics. Nature 2008;455:1054–6. [5] Kovac JR, Pastuszak AW, Lamb DJ. The use of genomics, proteomics, and metabolomics in identifying biomarkers of male infertility. Fertil Steril 2013;99:998–1007. [6] Gambineri A, Pelusi C, Vicennati V, Pagotto U, Pasquali R. Obesity and the polycystic ovary syndrome. Int J Obes Relat Metab Disord 2002;26:883–96.
↑ ↑
Sun et al., 2012 Sun et al., 2012
↓
Sun et al., 2012
↓ ↓
Sun et al., 2012 Sun et al., 2012
↓
Sun et al., 2012
↑
Zhao et al., 2012
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Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
Metabolomics in polycystic ovary syndrome☆ Mora Murri a,b,c, María Insenser a,b,c, Héctor F. Escobar-Morreale a,b,c,⁎ a b c
Diabetes, Obesity and Human Reproduction Research Group, Hospital Universitario Ramón y Cajal, Universidad de Alcalá, E-28034 Madrid, Spain Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), E-28034 Madrid, Spain Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), E-28034 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 27 October 2013 Received in revised form 12 December 2013 Accepted 14 December 2013 Available online 22 December 2013 Keywords: Metabolism Metabolomics Non-targeted Obesity Polycystic ovary syndrome
a b s t r a c t The association of androgen excess with abdominal adiposity, insulin resistance and metabolic derangements characterize many patients with PCOS. However, the mechanisms underlying these associations are not entirely understood, indicating the need for discovery of the origin of these metabolic alterations, and of new metabolic biomarkers for PCOS. This review summarizes the metabolites and metabolic pathways associated with PCOS according to recent metabolomic studies. PCOS-associated metabolites were involved mostly in carbohydrate, fat, and protein metabolism. Obesity, hyperinsulinemia and the intrinsic heterogeneity of PCOS are responsible of the metabolic variation observed in these women. Furthermore, treatment of PCOS seems to modify the levels of some metabolites. Hopefully, advances in the knowledge of metabolism will allow the detection of systemic imbalances in PCOS and will permit the identification of biomarkers that serve to predict the progression of the disease and its future complications. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Metabolomic techniques . . . . . . . . . . . . . . . . . . . Metabolites in PCOS . . . . . . . . . . . . . . . . . . . . . 3.1. Metabolomics profiles in PCOS . . . . . . . . . . . . . 3.2. Metabolites influenced by obesity and hyperinsulinemia . 3.3. Metabolites influenced by PCOS phenotypic heterogeneity 4. Metabolomics and treatment of PCOS . . . . . . . . . . . . . 5. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The polycystic ovary syndrome (PCOS) is the one of the most common endocrine disorders of women of reproductive age [1], and results from complex and poorly understood interactions between environmental and genetic factors [2]. PCOS is defined at present as a mainly hyperandrogenic disorder in which patients present with a constellation of symptoms and signs
☆ GRANTS: Supported by grants FIP PI1100357 and CD11/0030 (Mora Murri is the recipient of a “Sara Borrell” postdoctoral grant) from Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness. ⁎ Corresponding author. Tel./fax: +34 91 336 9029. E-mail address: [email protected] (H.F. Escobar-Morreale). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.018
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that necessarily include clinical and/or biochemical hyperandrogenism together with ovulatory dysfunction and/or polycystic ovarian morphology [3]. The consequences of PCOS are numerous, from cutaneous signs and menstrual and ovulatory dysfunction, to features of metabolic syndrome. A better understanding of this complex disorder and the identification of potential biomarkers of disease may represent a significant advance for the prevention, early and accurate diagnosis, and effective management of PCOS. In the recent years, the development of novel technologies that allow comprehensive metabolic profiling, termed metabolomics, may represent a big step in this direction. Metabolomics is defined as the analysis of multiple small-molecule metabolites, which are the products of metabolism, in biological samples. This term is often confused with metabonomics, which is the study of global, dynamic metabolic response of living systems to
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biological stimuli or genetic manipulation [4]. Metabolomics is a recent addition to the omics family of platforms that also includes, among others, genomics, transcriptomics and proteomics. Metabolomics studies events happening downstream of gene expression and it is considered to be closer to the actual phenotype than either both genomics or proteomics [5]. Metabolites can be intrinsic, resulting from normal cellular physiology, or extrinsic, resulting from the influences of exogenously administered pharmaceuticals [5]. The Human Metabolome Database (HMDB) contained in as many as 41,519 metabolite entries in August 2013, and approximately 2900 of these metabolites are endogenous and may be detectable in the human body. The physiological functions of these endogenous metabolites span over a wide range and include growth, development, and reproduction among other functions [4]. Many patients with PCOS are overweight or obese [6,7] and abdominal visceral adiposity is particularly frequent in these women [8–10]. Moreover, insulin resistance is also common in patients with PCOS independently of obesity [10], and approximately 50–70% of women with PCOS have insulin resistance and compensatory hyperinsulinism [6]. The association of androgen excess, abdominal adiposity, insulin resistance and metabolic derangements in women with PCOS has been explained by the existence of a vicious circle in these women, that may start during early stages of life or even prenatally, whereby androgen excess favoring the abdominal deposition of fat further facilitates androgen secretion by the ovaries and adrenals in women with PCOS [10]. Being included in the definition of the syndrome, increased serum androgen concentrations are the most obvious biomarkers of PCOS. However the mechanisms for the alteration of these levels are not well understood and also, biochemical hyperandrogenism is not always present in women with PCOS. Because PCOS is a complex disorder accompanied in many cases with metabolic derangements, there is clearly a need to discover the exact origin of these metabolic alterations and new metabolic biomarkers for PCOS. The aim of the present review is to summarize the metabolites and metabolic pathways associated with PCOS described in recent metabolomic studies. 2. Metabolomic techniques Metabolomics is an emerging bioanalytical discipline that uses analytical techniques to characterize and quantify in biological samples metabolites – small molecules that are mediators and products of metabolism – with the aim of monitoring metabolism and its fluctuations [4]. Therefore, because metabolites fall downstream of genetic, transcriptomic, proteomic, and environmental variation, metabolomics offers integrated and dynamic measures of phenotypes and medical conditions, providing insights on what is happening in a biological system. Metabolites are present in very broad concentration ranges and exhibit a notable chemical diversity. Therefore, the study of the human metabolome requires a set of instruments, such as the combination of gas (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), or the combination of MS and nuclear magnetic resonance (NMR). The use of MS relies on the fact that every compound has a unique fragmentation pattern. The sample is ionized and the sample ions are separated based on their differing masses and relative abundances. A commonly used MS technique is time-of-flight mass spectrometry (TOF–MS), in which ions are accelerated by a constant electric field of known strength and, therefore, their velocities depend on their mass-to-charge ratio. Hence, the time that the particle takes to reach a detector at a known distance indicates it mass with great accuracy. Before being submitted to TOF–MS identification, molecules need to be separated from the mixture of metabolites that compose the sample under study, either by GC–MS in the case of molecules that can be vaporized without chemical decomposition, or by LC–MS that separates molecules based on their interactions in the mobile and stationary phases.
NMR analyzes the signal that each molecule emits when their nuclei spin orientations are modified after an exposure to a magnetic field and radiofrequencies [11]. This signal is translated into a spectrum that is characteristic for each molecule and can be matched with those of existing databases. Among the most widely used NMR techniques is proton nuclear magnetic resonance (1H NMR), in which the NMR spectrometer is tuned to a specific nucleus, in this case the proton. Both MS and NMR have advantages and disadvantages [12]. MS analysis is more sensitive than NMR and permits the measurement of a broader array of small metabolites through the application of different combinations of chromatographic methods and mass/charge separation techniques. However, MS has the inconvenient of the destruction of the sample and the long sample preparation time required as it usually requires derivatization of metabolites to produce ionic species, whereas NMR is a non-destructive analysis which provides quantitative and reproducible measurements with minimal preparation of the samples, as no separation or ionization steps are necessary. Therefore, studies using a multiplatform approach that combines MS and NMR analysis may provide a more comprehensive understanding of metabolic alterations than studies using only one of these tools [12]. 3. Metabolites in PCOS The application of metabolomics in the field of PCOS is in its early infancy. We have reviewed the few metabolomic studies that compared controls and patients with PCOS. Four studies met this criterion [13–16] and their characteristics are summarized in Table 1. The studies were performed analyzing plasma samples using either 1H NMR [13,15], GC–MS systems [14] or 1H NMR together with GC/TOF–MS [16]. The four studies have high methodological quality according to a modification of the Newcastle–Ottawa Quality Assessment Scale for case–control studies explained in a previous work [17] (Table 1). The altered metabolites found in the studied manuscripts were involved mostly in carbohydrate, fat, and protein metabolism (Figs. 1, 2, Table 2). In the following subsections, we will describe the metabolites that could have a greater interest for PCOS. First, we will describe the metabolites altered in PCOS. Second, we will analyze metabolites that are affected in PCOS by the interaction with obesity, hyperinsulinemia and with the heterogeneity intrinsic to PCOS. Finally, we will describe the single study [18] that addressed the effects of treatment of PCOS on the metabolome, in order to find out which metabolites change after the remission of PCOS symptoms. 3.1. Metabolomics profiles in PCOS Altered carbohydrate metabolism is present in many PCOS patients [1]. The significant increase of lactate and gluconeogenic amino acids, and the reduction of glucose in plasma of non-obese women with PCOS, might be explained by increased glycolysis in muscle and decreased gluconeogenesis in liver [15,16]. Moreover, the increase in lactate levels in insulin resistant non-obese PCOS women [16] suggests insulin stimulated glucose uptake and consumption in the muscle of these PCOS patients, indicating central but not peripheral insulin resistance in these patients. In terms of lipid metabolism, levels of lipoprotein, low-density lipoprotein cholesterol (LDL-c) and very low-density lipoprotein cholesterol (VLDL-c) were increased in PCOS samples compared with controls, accompanied with a decrease of cholesterol and high-density lipoprotein cholesterol (HDL-c) levels [15,16]. These findings are in agreement with the lipid profiles measured by biochemical methods in previous reports [19]. Moreover, levels of free fatty acids (such as palmitic, stearic and linoleic acid) were increased in PCOS samples compared with controls, indicating an increase in lipolysis consistent with the presence of insulin resistance in adipose issue [20,21]. Moreover, linoleic acid may be involved in the inhibition of the maturation and developmental potential of oocytes [22]. Accordingly, the increase
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183
Table 1 Characteristics of the studies included in this review. Author (year)
Atiomo et al. (2012) Escobar-Morreale et al. (2012) Sun et al. (2012) Zhao et al. (2012)
Biological sample
Plasma Plasma Plasma Plasma
Simple size (n)
Mean age (years)
Mean BMI (kg/m2)
PCOS
Controls
PCOS
Controls
PCOS
Controls
12 36 34 217
10 39 36 48
27.2 26.3 27.2 28.4
28.4 26.5 26.9 29.8
33.2 29.9 24.5 24.5
27.0 29.3 22.6 21.6
in linoleic acid levels in patients with PCOS might be accompanied by a reduction in oocyte maturation and disordered ovulation in PCOS. Additionally, linoleic acid displays potent proinflammatory activities [23], and might also contribute to the chronic low-grade inflammation underlying PCOS [24,25]. However, these interpretations should be considered with caution, as other studies reported conflicting results, with the levels of these lipids being decreased [13,16] or increased [14,16] in PCOS. Another lipid metabolite decreased in patients with PCOS is αtocopherol [14]. α-Tocopherol is a potent antioxidant that serves as a peroxyl radical scavenger that protects polyunsaturated fatty acids in membranes and lipoproteins [26]. The decrease in α-tocopherol may contribute to the association of PCOS with oxidative stress [17]. The aerobic catabolism of glucose, fatty acids, and some amino acids results in acetyl-coenzymeA (CoA), the entry point into the tricarboxylic acid (TCA) cycle. The TCA cycle consists of series of biochemical reactions used by all aerobic organisms to generate energy, through the oxidization of acetyl-CoA into carbon dioxide. Levels of citrate were decreased in PCOS women compared with control women. Additionally, levels of amino acids involved in TCA cycle, such as arginine, citrulline, glutamine, histidine, methionine and proline were also decreased; accompanied by an increase in phenylalanine, threonine, tyrosine, valine, inducing a decrease or increase in elements of the TCA cycle [13,15,16]. This suggests that TCA cycle is impaired in PCOS women, which has also been considered as evidence of the existence of insulin resistance [27]. Hyperandrogenism has been described to alter TCA cycle by decreasing citrate levels [28]. Moreover, TCA cycle has been described to play an important role in oocyte maturation with lower levels of citrate indicating decreased competence [28]. The concentrations of several aromatic and branched-chain amino acids (AAA and BCAA, respectively), such as valine, tyrosine, tryptophan and phenylalanine, were increased in PCOS patients [16]. These amino acids have been linked to insulin resistance and diabetes [29,30]. Moreover, Wurtz et al. [31] reported disorders in AAA and BCAA metabolism preceding hyperglycemia in the general population. Additionally, levels of glycine, which seems to improve the proinflammatory state and
Country
Participant selection
Definition of PCOS
Quality assessment rating
UK Spain China China
Age and BMI matched Age and BMI matched Age and BMI matched Age matched
ESHRE/ASRM NICHD ESHRE/ASRM ESHRE/ASRM
High High High High
upregulate adiponectin gene expression in vitro [32], were decreased in PCOS and the elevated levels of N-acetyl glycoproteins, that serve as inflammatory markers and acute-phase proteins [33], support the existence of low-grade chronic inflammation in PCOS patients [15]. Leucine concentration was decreased in PCOS patients in the study by Sun et al. [15]. Because leucine may rescue insulin signaling via activation of the mTOR pathway, and increasing dietary leucine intake may improve insulin sensitivity [34,35], this decrease in leucine levels could contribute to the insulin resistance of PCOS. Furthermore, Zhao et al. [16] found an increase in leucine levels in PCOS patients, yet this difference was observed only in PCOS patients with insulin resistance. Therefore, they speculated that alterations of plasma levels of leucine in PCOS patients with insulin resistance might be entirely due to the impairment of insulin signaling [16]. 2-Ketoisocaproic acid levels were decreased in patients with PCOS compared with controls [14], suggesting decreased transamination of leucine in the first step of BCAA catabolism instead of the increased transamination previously suggested as a metabolic characteristic of obesity and diabetes [29,36]. Under normal conditions, alanine arising from BCAA nitrogen accounts for 25% of gluconeogenesis from amino acids [37]. These data, together with the decreased alanine levels found by Escobar-Morreale et al. [14] in patients with PCOS, could indicate that BCAAs are being used for protein synthesis in these women, and not for gluconeogenesis as happens in obesity and diabetes [29,36]. However, a decrease in plasma alanine level has been also associated with increased gluconeogenesis in diabetic mice [38]. Levels of dimethylamine were increased in PCOS patients, accompanied by a decrease in choline, phosphatidylcholine and glycerophosphocholine/phosphocholine levels [15]. Symbiotic gut microbiota converts the choline, released by hydrolysis of phosphatidylcholine, into methylamines such as dimethylamine, leading to low circulating levels of plasma phosphatidylcholine, which reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing non-alcoholic fatty liver disease [39]. These results are in accordance with those found in diabetes [40] suggesting a possible relationship of PCOS with these disorders.
Fig. 1. Altered metabolites in PCOS, represented as reported in one or more studies.
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Fig. 2. Interactions of the metabolites described in this review.
3.2. Metabolites influenced by obesity and hyperinsulinemia Escobar-Morreale et al. [14] found substantial metabolic heterogeneity in PCOS that was dependent mostly on the presence or absence of obesity (Table 3). They found that levels of glycerol and long-chain fatty acids were decreased in nonobese women with PCOS compared with nonobese controls, suggesting suppression of lipolysis. This finding requires conserved insulin sensitivity in adipose tissue, and possibly the presence of increased insulin levels might have contributed to reduced lipolysis, although impairment in catecholamine-induced lipolysis in subcutaneous tissue is also characteristic of nonobese women with PCOS [41–43]. Moreover, increased levels of lactate and it precursor, lactic acid, were observed in non-obese women with PCOS when patients were classified for obesity [14,15], indicating increased insulin-stimulated glucose uptake and consumption in the muscle of these non-obese patients [14]. Because such uptake of glucose, similar to the suppression of lypolisis in adipose tissue, requires effective insulin signaling, these findings suggest that insulin resistance is not universal in all the tissues of non-obese patients with PCOS. However, since their insulin concentrations are higher than those of non-hyperandrogenic controls for a similar plasma glucose concentration, a certain degree of central insulin resistance must be present in these women [14]. In conceptual agreement, Harris et al. [44] demonstrated that oocytes from non-obese PCOS women presented increased glucose and pyruvate consumption during overnight in vitro maturation and that treatment with the insulin-lowering drug metformin avoided this increase. This finding indicates that there is no insulin resistance in PCOS oocytes, and that their enhanced glucose consumption may result from exposure to the increased insulin levels resulting from central insulin resistance. On the contrary, glycerol and long-chain fatty acids were increased and plasma lactate concentrations were decreased in obese patients with PCOS, suggesting that in PCOS the development of peripheral insulin resistance requires the coexistence of obesity [14]. Sun et al. [15] and Zhao et al. [16] confirmed that obesity exerted a major influence in the metabolic profile of PCOS (Table 3). Moreover, Zhao et al. [16] also demonstrated an influence of insulin resistance in the
metabolic profile of women with PCOS, even though insulin resistance was entirely dependent on the presence of obesity. In obese women, the increase of long-chain fatty acids and glycerol levels found in those presenting with PCOS, as well as the increase in oleic and palmitoleic acid found in obese women irrespective of PCOS, suggested impaired insulin action at adipose tissue [41,45]. As seen previously, this increase in lipolysis is the opposite result found in the non-obese PCOS patients. The adipose tissue expandability hypothesis states that once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy efficiently and lipids begin to accumulate in other tissues [46]. When this adipose tissue expansion fails insulin can no longer suppress lipolysis. Furthermore, these increased levels of circulating free fatty acid in obese patients with PCOS may contribute further to their central insulin resistance [47], and to the association of PCOS and obesity with the metabolic syndrome and non-alcoholic fatty liver disease [48]. Glycine levels were decreased in obese women [14], which may indicate increased utilization for gluconeogenesis. This finding agrees with the metabolic signature previously associated with obesity that includes increased levels of circulating BCAA [29]. BCAA, which are increased by peripheral insulin resistance as their utilization in tissues such as muscle requires conserved insulin signaling, are used for gluconeogenesis through pyruvate transamination into alanine, thereby contributing to glucose intolerance [29,36]. Furthermore, an increase in BCAA catabolism may explain the increase in phenylalanine levels in the obese women, because large neutral amino acids such as phenylalanine compete with BCAA for transport into mammalian cells [49]. Elevated levels of valine and reduced levels of glycine were also observed in the women with PCOS without insulin resistance as compared with controls [16], and these changes were aggravated when the patients had altered insulin sensitivity. Hence, valine and glycine may be associated with other metabolic disturbances independently of insulin resistance. 3.3. Metabolites influenced by PCOS phenotypic heterogeneity Several metabolites were altered by the heterogeneity inherent to the different PCOS phenotypes (Table 4). Zhao et al. [16] reported that
M. Murri et al. / Clinica Chimica Acta 429 (2014) 181–188 Table 2 Summary of metabolites associated with polycystic ovary syndrome and their changes compared with controls. Carbohydrate related markers Glucose Undentified sugar Lactate
↓ ↑ ↑
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012; Sun et al., 2012
Lipid related markers α-Tocopherol Acetone Cholesterol
↓ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑
Escobar-Morreale et al., 2012 Atiomo et al., 2012 Zhao et al., 2012; Escobar-Morreale et al., 2012 Zhao et al., 2012 Zhao et al., 2012; Sun et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012
↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑
Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Escobar-Morreale et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012
↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↓ ↑ ↓ ↑ ↓ ↑
Atiomo et al., 2012; Sun et al., 2012 Sun et al., 2012 Atiomo et al., 2012 Atiomo et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012; Atiomo et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Escobar-Morreale et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012 Atiomo et al., 2012 Zhao et al., 2012
TCA related marker Citrate
↓
Sun et al., 2012; Atiomo et al., 2012
Purine related marker Uric acid
↑
Zhao et al., 2012
HDL Phosphatidylcholine Fatty acid Linoleic acid Lipoprotein Palmitic acid Stearic acid Unsaturated fatty acid VLDL/LDL Lipid-CH2CH2CO Protein related markers Creatinine Lysyl-albumin Trimethylamine N-oxide 2-Ketoisocaproic acid Creatine Dimethylamine N-acetylglycoprotein
Amino acids Arginine Choline Citrulline Glutamate Glutamine Glycerophosphocholine/phosphocholine Glycine Histidine Isoleucine Methionine Proline AAA BCAA BCAA/AAA Aspartate Endogenous AAs Gluconeogenic AAs Phenylalanine Serine Threonine Tryptophan Tyrosine Valine Alanine Leucine Lysine Ornithine
185
hormone concentrations, its ratio to follicle-stimulating hormone, AAA levels and BCAA/AAA ratios were much more severe in classic PCOS than in other PCOS phenotypes [16]. Furthermore, the metabolic profile of the ovulatory PCOS phenotype was different compared to that of the classic anovulatory PCOS phenotypes. The levels of endogenous amino acids were decreased in ovulatory PCOS women compared with controls suggesting increased protein synthesis (i.e. in skeletal muscle) in these women. In agreement, Carmina et al. [50] reported increased lean muscle mass in women with PCOS. On the other hand, increased levels of endogenous amino acid including serine, threonine, phenylalanine, tyrosine and ornithine were found in the patients presenting with polycystic ovarian morphology and anovulation, which may indicate increased protein degradation in ovarian dysfunction [16]. Zhao et al. [16] reported that the levels of long-chain fatty acids were reduced in PCOS patients with hyperandrogenism compared with patients without hyperandrogenism. This result disagrees with previous reports describing a positive association between fatty acid levels and androgen excess [19,51,52]. Sun et al. [15] reported that, when taking hyperandrogenism into consideration, the only significant change found was the increased N-acetyl glycoprotein observed in the patients with PCOS and hyperandrogenism compared with the controls. This may represent a link between androgen excess and inflammation in PCOS. 4. Metabolomics and treatment of PCOS Treatment of adolescent patients with PCOS with pioglitazone– flutamide–metformin (Pio/Flu/Met) polytherapy during one month showed changes in serum metabolites [18]. Pio/Flu/Met polytherapy has been described to block androgen receptors, remodel body adiposity, reduce low-grade inflammation, and promote a favorable adipokine profile [18]. Vinaixa et al. [18] reported that 9- and 13-HODE, and azelaic acid, which are downstream metabolic oxidation products of linoleic acid (which is the major polyunsaturated fatty acid in human plasma and LDL [53,54]) and glutaric acid, which can also induce oxidative stress [55], were decreased in serum after 30 days of Pio/Flu/Met treatment. The levels of 1,2-propanediol, acetate, glutamate, lysine and succinate in serum were also significantly decreased after the polytherapy. These results may indicate that Pio/Flu/Met polytherapy reduces the amount of oxidized lipoprotein particles and downstream oxidative metabolites in the serum of PCOS patients [18]. Most of these metabolites participate directly or indirectly, and with lesser or greater importance, in the TCA cycle [56–60], which as described above, is altered in PCOS. Therefore, Pio/Flu/Met treatment seems may influence favorably the metabolism of young patients with PCOS [18]. 5. Limitations Metabolomic studies are not free of limitations. First, the heterogeneity in the criteria used to diagnose PCOS together with the relatively small sample sizes and the different metabolomic techniques used impose important limitations when aiming to integrate the results of the different studies conducted to date. In addition, in not all studies the patients and the controls were matched for BMI [16], did not include insulin resistant controls [16] or did not show data regarding glucose and insulin levels of the women included in the study [15]. Finally, as occurred with other diseases, the findings derived from metabolomic studies still have to demonstrate their usefulness as biomarkers in PCOS and its associated metabolic comorbidities [12]. 6. Conclusions
the classic hyperandrogenic and oligo-ovulatory phenotype of PCOS was related to more adverse biochemical and metabolic changes than other phenotypes when compared with controls. Alterations of luteinizing
The pathogenesis of PCOS remains largely unknown, mostly because our lack of understanding of the molecular mechanisms responsible for
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Table 3 Impact of obesity on the changes observed in metabolomic biomarkers for polycystic ovary syndrome. Obese vs nonobese
Non-obese PCOS vs nonobese control
Obese PCOS vs obese control
↑
Escobar-Morreale et al., 2012
↑ ↑
Escobar-Morreale et al., 2012 Sun et al., 2012
↓
↑
Escobar-Morreale et al., 2012 ↑ ↑ ↑
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↓ ↓
Escobar-Morreale et al., 2012 Escobar-Morreale et al., 2012
↓ ↑ ↓ ↑
Escobar-Morreale et al., 2012 Zhao et al., 2012 Escobar-Morreale et al., 2012 Zhao et al., 2012
↑ ↑ ↑ ↑
Escobar-Morreale et al., 2012 Escobar-Morreale et al., 2012 Sun et al., 2012 Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↑
Zhao et al., 2012
Protein related markers Creatine Dimethylamine N-acetylglycoprotein
↑ ↑ ↑
Sun et al., 2012 Sun et al., 2012 Sun et al., 2012
Amino acids Alanine Arginine Aspartate Choline Glycerophosphocholine/phosphocholine Glycine Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tyrosine Tryptophan Valine Endogenous AAs BCAA AAA BCAA/AAA
↑ ↓ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓
Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012, Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Sun et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Sun et al., 2012
↓
Sun et al., 2012
TCA related marker Citrate
↓
Sun et al., 2012
Purine related marker Uric acid
↑
Zhao et al., 2012
Carbohydrate related markers Lactic acid Lactate Gluconic acid lactone Gluconeogenic Aas Phosphoglyceryde Undentified sugar Lipid related markers Adipic acid Citramalic acid Glyceric acid Glycerol LDL Linoleic acid Oleic acid Palmitic acid Palmitoleic acid Stearic acid
↑
Escobar-Morreale et al., 2012
↓
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↑
Escobar-Morreale et al., 2012
↓
↑
Escobar-Morreale et al., 2012
Escobar-Morreale et al., 2012
this complex disorder. Advances in the field of metabolism will hopefully permit the detection of systemic imbalances in PCOS and the identification of biomarkers that predict the progression of the disorder and its complications. The application of metabolomics to PCOS has rapidly evolved during the past years providing researchers with the opportunity to gain new insights into the molecular mechanisms and metabolic networks involved in its development. Several specific metabolic pathways, including protein, lipid and carbohydrate metabolism and the TCA cycle, appears to be disturbed in PCOS. In addition, treatment with insulin sensitizers and antiandrogens may have a beneficial impact on the metabolic abnormalities of young patients with PCOS. These results represent the basis for the identification of novel PCOS biomarkers, and the development of diagnostic tests and new methodologies for metabolic screening and personalized medicine.
↓
Obese PCOS vs nonobese PCOS
Escobar-Morreale et al., 2012 ↑
Zhao et al., 2012
↓ ↓
Zhao et al., 2012 Zhao et al., 2012
↓
Zhao et al., 2012
Sun et al., 2012
Metabolic heterogeneity is present in patients with PCOS, and it is strongly influenced by hyperinsulinemia, insulin resistance and obesity. The metabolic profile of nonobese patients with PCOS is explained by the hyperinsulinemia resulting from reduced hepatic clearance of insulin and hepatic insulin resistance [61] and the absence of peripheral insulin resistance in the presence of increased circulating insulin concentrations, in conceptual accordance with the previously reported heterogeneity in insulin action between metabolic responses and target tissues [62,63]. In obese patients with PCOS, the metabolic profile indicates the presence of both central and peripheral insulin resistance. As substantial metabolic heterogeneity underlies PCOS, confusion factors such as obesity, hyperinsulinemia, and insulin resistance should be considered when designing diagnostic and therapeutic strategies for the management of this prevalent disorder. The metabolomic study of PCOS is further complicated because the phenotypic heterogeneity of the syndrome. Features of PCOS such as
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Table 4 Impact of the polycystic ovary syndrome phenotype on metabolomic biomarkers. Non-hyperandrogenic PCOS vs controls
Hyperandrogenic PCOS vs controls
Hyperandrogenic PCOS VS Non-hyperandrogenic PCOS
Carbohydrate related markers Glucose
↑
Zhao et al., 2012
Lipid related markers Linoleic acid Palmic acid Stearic acid
↓ ↓ ↓
Zhao et al., 2012 Zhao et al., 2012 Zhao et al., 2012
↓
Zhao et al., 2012
↓
Zhao et al., 2012
↑ ↓
Zhao et al., 2012 Zhao et al., 2012
↑
Zhao et al., 2012
Protein related markers Creatine Dimethylamine N-acetylglycoprotein Amino acids Alanine Arginine Aspartate Choline Glycerophosphocholine/phosphocholine Leucine Tryptophan Valine TCA related marker Citrate
↑ ↑
↓ ↓ ↓
↓
Sun et al., 2012 Sun et al., 2012
Sun et al., 2012 Sun et al., 2012 Sun et al., 2012
Sun et al., 2012
Purine related marker Uric acid
ovulatory dysfunction, hyperandrogenemia and polycystic ovarian morphology may associate different metabolic alterations. Therefore, the systematic comparison of the metabolic profiles of the different PCOS phenotypes may be helpful in order to understand the metabolic risks and associations of these patients. In conclusion, novel PCOS related metabolites involved in different metabolic pathways have been identified by recent non-targeted metabolomic studies. These findings still need to be replicated by future studies in which well characterized and homogenous PCOS phenotypes are to be compared with carefully matched control women. Only then will these metabolites be considered as biomarkers of the syndrome and its complications, contributing to metabolic screening and personalized medicine in PCOS. Conflict of interest The authors have no conflict of interest to disclose. Acknowledgments Supported by grants FIP PI1100357 and CD11/0030 (Mora Murri is the recipient of a “Sara Borrell” postdoctoral grant) from Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness. References [1] Azziz R, Carmina E, Dewailly D, et al. The androgen excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril 2009;91:456–88. [2] Escobar-Morreale HF, Luque-Ramirez M, San Millan JL. The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome. Endocr Rev 2005;26:251–82. [3] Escobar-Morreale HF. Polycystic ovary syndrome: treatment strategies and management. Expert Opin Pharmacother 2008;9:2995–3008. [4] Nicholson JK, Lindon JC. Systems biology: metabonomics. Nature 2008;455:1054–6. [5] Kovac JR, Pastuszak AW, Lamb DJ. The use of genomics, proteomics, and metabolomics in identifying biomarkers of male infertility. Fertil Steril 2013;99:998–1007. [6] Gambineri A, Pelusi C, Vicennati V, Pagotto U, Pasquali R. Obesity and the polycystic ovary syndrome. Int J Obes Relat Metab Disord 2002;26:883–96.
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