Combining a nontargeted and targeted metabolomics approach to identify metabolic pathways significantly altered in polycystic ovary syndrome

Abstract

Objective: Polycystic ovary syndrome (PCOS) is a condition of androgen excess and chronic anovulation frequently associated with insulin resistance. We combined a nontargeted and targeted metabolomics approach to identify pathways and metabolites that distinguished PCOS from metabolic syndrome (MetS).

Methods: Twenty obese women with PCOS were compared with 18 obese women without PCOS. Both groups met criteria for MetS but could not have diabetes mellitus or take medications that treat PCOS or affect lipids or insulin sensitivity. Insulin sensitivity was derived from the frequently sampled intravenous glucose tolerance test. A nontargeted metabolomics approach was performed on fasting plasma samples to identify differentially expressed metabolites, which were further evaluated by principal component and pathway enrichment analysis. Quantitative targeted metabolomics was then applied on candidate metabolites. Measured metabolites were tested for associations with PCOS and clinical variables by logistic and linear regression analyses.

Results: This multiethnic, obese sample was matched by age (PCOS, 37±6; MetS, 40±6years) and body mass index (BMI) (PCOS, 34.6±5.1; MetS, 33.7±5.2kg/m²). Principal component analysis of the nontargeted metabolomics data showed distinct group separation of PCOS from MetS controls. From the subset of 385 differentially expressed metabolites, 22% were identified by accurate mass, resulting in 19 canonical pathways significantly altered in PCOS, including amino acid, lipid, steroid, carbohydrate, and vitamin D metabolism. Targeted metabolomics identified many essential amino acids, including branched-chain amino acids (BCAA) that were elevated in PCOS compared with MetS. PCOS was most associated with BCAA (P=.02), essential amino acids (P=.03), the essential amino acid lysine (P=.02), and the lysine metabolite α-aminoadipic acid (P=.02) in models adjusted for surrogate variables representing technical variation in metabolites. No significant differences between groups were observed in concentrations of free fatty acids or vitamin D metabolites. Evaluation of the relationship of metabolites with clinical characteristics showed 1) negative associations of essential and BCAA with insulin sensitivity and sex hormone-binding globulin and 2) positive associations with homeostasis model of insulin resistance and free testosterone; metabolites were not associated with BMI or percent body fat.

Conclusions: PCOS was associated with significant metabolic alterations not attributed exclusively to androgen-related pathways, obesity, or MetS. Concentrations of essential amino acids and BCAA are increased in PCOS, which might result from or contribute to their insulin resistance.

Keywords: Branched-chain amino acids; Insulin sensitivity; Metabolic syndrome; Vitamin D; α-Aminoadipic acid.

Figure 1

Two-dimensional Score Plot of Principal Component Analysis Showing Group Separation Between PCOS and MetS. Each person is represented by 2 replicate data points. PCOS and MetS groups can be seen in clusters separated on 2 components. The first and second components explained 18.51% and 9.67% of variations, respectively. MetS indicates controls with metabolic syndrome (green squares); PCOS, polycystic ovary syndrome (red triangles).

Figure 2

Metabolic Pathways Significantly Different in PCOS vs MetS. The significance of the pathways was evaluated using P values and a false-discovery rate <0.05. CF indicates cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HETE, hydroxyl eicosatetraenoic acid; HPETE, hydroperoxy eicosatetraenoic acid; PPAR, peroxisome proliferator-activated receptor.

Figure 3

Concentrations of Amino Acids Normalized to Account for Technical Variation in Metabolites. A, Essential amino acids as a group were higher in PCOS (P = .03). Among the individual essential amino acids, aside from the observed differences in branched-chain amino acids, only the lysine concentration was higher in PCOS (P = .02). B, Branched-chain amino acid concentrations as a group were higher in PCOS (P = .02). Concentrations of individual branched-chain amino acids were also higher in the PCOS group compared with MetS controls: isoleucine (P = .03), leucine (P = .02), and valine (P = .03). The asterisk indicates P < 05; MetS, metabolic syndrome; PCOS, polycystic ovary syndrome.

Figure 3

Concentrations of Amino Acids Normalized to Account for Technical Variation in Metabolites. A, Essential amino acids as a group were higher in PCOS (P = .03). Among the individual essential amino acids, aside from the observed differences in branched-chain amino acids, only the lysine concentration was higher in PCOS (P = .02). B, Branched-chain amino acid concentrations as a group were higher in PCOS (P = .02). Concentrations of individual branched-chain amino acids were also higher in the PCOS group compared with MetS controls: isoleucine (P = .03), leucine (P = .02), and valine (P = .03). The asterisk indicates P < 05; MetS, metabolic syndrome; PCOS, polycystic ovary syndrome.