Metformin is widely used for the treatment of polycystic ovary syndrome (PCOS) with variable clinical efficacy. The factors that predict response to metformin and the mechanism of metformin action are not well understood, and identifying the subset of patients who will benefit from treatment remains a challenge. The broad goal of this study is to characterize the factors that predict metformin response and to investigate the mechanisms of metformin action in women with PCOS. Part of the variability in the response to metformin may be explained by genetic heterogeneity of metformin transport proteins. Specific Aim 1 of this proposed study strives to characterize the genetic variants that predict the improved fasting glucose, glucose effectiveness and testosterone levels in response to metformin treatment in women with PCOS. Exon sequencing will be performed to examine functional variants in SLC22A1, SLC22A2, and SLC47A1 in relation to changes in these three outcome measures. Preliminary data from the applicant's ongoing study suggests that metformin does not improve insulin sensitivity in women with PCOS as commonly believed. Rather, metformin improves glucose effectiveness and fasting glucose levels. Furthermore, glucose, insulin, and LH levels did not correlate with testosterone levels and the ovulatory response, highlighting that metformin likely has direct and independent effects on hepatocytes, skeletal muscle and the ovaries. Evidence from the literature suggests that metformin inhibits mitochondrial function, specifically Complex I of the electron transport chain. To date, the effect of metformin on mitochondrial activity has never been studied using 31-phosphorus magnetic resonance spectroscopy (31P- MRS), a well-validated and noninvasive method to examine mitochondrial oxidative phosphorylation in skeletal muscle. Specific Aim 2 of this study proposes to investigate the effect of metformin on mitochondrial activity and its relationship to changes in glucose effectiveness, fasting glucose, and testosterone levels in response to metformin through the use of 31P-MRS. Muscle biopsies will also be obtained for future in vitro cross-validation studies and further examination of Complex I. This study will contribute to the mechanistic understanding of metformin action in women with PCOS and characterize pharmacogenetic factors that predict metformin response.