Introduction

Polycystic ovary syndrome (PCOS) is a very common and heterogeneous endocrine disorder with unknown etiology, affects approximately 5–10 % of premenopausal woman [1]. Clinically, PCOS patient often presents insulin resistance and confers a substantial risk for developing type 2 diabetes mellitus (T2DM) and coronary artery diseases [2, 3]. Moreover, by molecular level, previous studies have showed that various genes variants, such as INS, INSR, IRS1/2, ENPP1, PPARγ, CAPN10, related to insulin resistance in PCOS [4].

However, these studies remained largely on the nuclear genes that involved in the clinical manifestation of PCOS, little is known the impact of mtDNA mutations with PCOS. Since mitochondria use oxidative phosphorylation (OXPHOS) to convert dietary calories into usable energy, releasing reactive oxygen species (ROS) as a toxic by-product [5]. We hypothesized that mtDNA variants may lead mitochondrial dysfunction, thus, influence the clinical expression of PCOS.

Most recently, Victor et al. [6] reported that defect in mitochondrial Complex I was observed in PCOS patients; moreover, a reduction of ROS production was also found in PCOS patients with insulin resistance. In addition, Skov et al. [7] demonstrated that pioglitazone therapy restored the insulin resistance in woman with PCOS, by coordinated up-regulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. Those studies shed new light on the pathogenesis of mitochondria dysfunction involved in PCOS.

With the purpose of understanding the molecular basis of PCOS, we performed a mutational screening of mtDNA sequence variants in PCOS patients from Hangzhou area in Zhejiang Province. We first evaluated the clinical characterization of PCOS patients and controls, moreover, we employed polymerase chain reaction (PCR) amplification to detect mtDNA variants and discussed the potential role that mtDNA played in PCOS patients.

Materials and methods

Clinical presentation

Since 2005, a total of 57 women with the presence of PCOS clinical features and 38 healthy women with age, body mass index (BMI) matched, were recruited in Hangzhou First People’s Hospital in Zhejiang Province, China. The selection for those unrelated women with PCOS in this study is in accordance with the Rotterdam criteria [8]: (i) a through personal and family medical history, including the symptoms, completing a physical examination; especially for the woman who presents the clinical signs of hyperandrogenism; (ii) polycystic ovaries symptom; (iii) the ratio between luteinizing hormone (LH) levels and follicle stimulating hormone (FSH) levels: LH/FSH >2.0, or the testosterone level >2.64 nmol/L. Patients with hyperprolactinemia, thyroid and adrenal diseases, 21-hydroxylase deficiency, and androgen-secreting tumors were excluded.

Mutational analysis of mitochondrial genome

Genomic DNA was extracted from the peripheral blood samples of the patients and controls using the phenol-chloroform method as described elsewhere [9]. The entire mitochondrial genome was PCR amplified in 28 overlapping fragment spanning the light strand and the heavy strand [10]. Each fragment was purified and subsequently analyzed by the direct sequencing in an ABI 3700 automatic DNA sequencer using Big Dye Terminator Cycle sequencing reaction Kit. The resultant sequence data were compared with the updated Revised Cambridge Sequence (GenBank Accessible No. NC_012920) [10].

Statistical analysis

Clinical and molecular data were presented as mean ± SD or percentage. Statistical analysis was performed using the SPSS statistical package (version 11.5) utilizing Pearson Chi-square test or Fisher’s exact test, a P value <0.05 was considered as statistically significant.

Results

Clinical characterization of PCOS patients and controls

The patients’ characteristics and biochemical profile were presented in Table 1. The group was adequately and closely matched for age and BMI. As expected, the testosterone score was significantly higher (P < 0.001) in PCOS than in the control subjects. No difference in glucose, insulin or triglyceride was observed.

Table 1 Clincial and biochemical features of PCOS and control subjects
Full size table

Genotyping analysis of mitochondrial genome

In order to understand the mtDNA contribution to the pathogenesis of PCOS, we performed a systematic mutational screening of mtDNA variants in PCOS and controls. As listed in Table 2, a distinct set of mtDNA variants have been identified in PCOS patients and controls. These sequence variants including 16 bases changing in D-Loop gene, 7 variants in 12S rRNA gene, 3 variants in 16S rRNA, other sequence variants occurred in mitochondrial polypeptide-coding genes suggested that mitochondrial dysfunction might be involved in the pathogenesis of PCOS patients. Moreover, we also identified six tRNA polymorphisms in PCOS patients but were absent in control group, suggested that variants in tRNA genes may be associated with PCOS. We further performed the statistical analysis of the mtDNA polymorphisms in PCOS and control groups; a Chi Square was carried out to determine the P value, interestingly, the 9 bp deletion occurred at np. 8281–8291, showed the statistically significant (P < 0.05).

Table 2 Mitochondrial DNA variants in PCOS patients and controls
Full size table

Discussion

In this study, we have performed clinical, genetic and molecular characterization of 57 patients and 38 controls to analysis mtDNA mutations in clinical manifestation of PCOS. In fact, PCOS is one of the most common endocrinopathy affecting the woman in the reproductive age [11], woman with PCOS always presents with a wide spectrum of clinical phenotypes, including hyperandrogenism, hirsutism, insulin resistance, obesity, infertility and increase the risk of developing T2DM and cardiovascular disease [12, 13]. Statistical significance was observed in testosterone level with PCOS and the control did emphasis the hormone deficiency in PCOS patients. Furthermore, considering that the ovary is a relative highly energy consumption organism, and acquired more mitochondria to generate ATP, had droved us to analysis mtDNA variants in PCOS patients.

As shown in Table 2, 57 patients with PCOS and 38 health controls harbored distinct sets of sequence variants. There are 16 variants in the non-coding region D-Loop, 7 variations in 12S rRNA gene and 3 in 16S rRNA gene, while the other variants are mainly focused on OXPHOS complex. Most of the single nucleotide polymorphisms are well-known mutational hotspots and none of these variants could be defined as “novel” [14]. Among these variants, the ND1 G3316A, changed the alanine into threonine, T3394C variant caused tyrosine to histidine, have been found to be associated with T2DM [15] and Leber’s hereditary optic neuropathy (LHON) [16]. Moreover, mutations involved the amino acid substitution such as C5178A (L237M), A5301G (I287V) and G5460A (A331T) in ND2 gene, G7853A (V90I) in CO2 gene, C8414T (L17F) in A8 gene, G8584A (A20T), C8684T (T53I) and A8860G (T112A) in A6 gene, A10398G (T114A) in ND3 gene, A12026G (I503 V) in ND4 gene, G12406A (V24I), T12811C (Y159H), G13145A (A26T), G13708A (A458T), G13759A (A475T) and G13928C (S54T) in ND5 gene, A15326G (T194A) in Cytb gene. It is interesting to note that the common 9 bp deletion which located between tRNALys and CO2 gene, had a statistically significance (P < 0.05), our previous study also indicated that this deletion has a potential role in clinical manifestation of PCOS patients [17].

All these protein-coding variants were further analyzed by phylogenetic and sequences from other organisms including mouse [18], bovine [19], and Xenopus laevis [20], except ND1 T3394C, ND5 T12811C, showed no evolutionary conservation. It is interesting to note that the well-known ND5 T12811C mutation, the A6 G8584A and C8684T mutations which have been considered as a risk factor in increasing and decreasing the clinical expression of LHON [21, 22], respectively, presented only in PCOS patients but absent in health controls [23], implicating a potential pathogenic role in PCOS development.

Because most of the mitochondrial proteins are nuclear encoded and mitochondrial tRNAs acted as key effectors in translation and linked to metabolic activity, variants in tRNAs may be another contributing factor that involved in the pathogenesis of PCOS. In this study, we found six tRNA variants in PCOS patients but were absent in control subjects, these variants (Fig. 1) localized at highly conserved nucleotides of corresponding tRNAs and may cause potential structural and functional alternations. Of these, T4395C variant located at the sixth base of mitochondrial tRNAGln accept arm, while the mutant 4395C created a new G-C (6C-64G) base-pairing, by literature searching, this variant has been found to be associated with maternally transmitted essential hypertension [24]. Furthermore, A7543G variant occurred at anti-coden stem of tRNAAsp [25] and was first reported in an 11-year-old girl with myoclonic seizures, developmental delay, and severe behavioral problems [26], previous investigation also showed that in yeast Saccharomyces cerevisiae, the mutant A7543G altered the stem and ultimately affected the tRNA stability level [27]. Therefore, it is anticipated that abolished base-pairing 29A-41T of tRNAAsp by A7543G variant likely altered tRNAAsp metabolism. Similarly, G5821A variant disrupted a highly conserved base-pair of tRNACys [28]. Moreover, tRNALys A8343G, tRNAArg T10454C and tRNAGlu A14693G variants occurred at the highly conserved nucleotide of T loop of corresponding tRNAs, A to G transition at position 8343 (conserved nucleotide at 54) has been reported as a risk factor underlying the respiratory chain deficiency [29] and a possible PD risk factor (http://www.mitomap.org/MITOMAP), while tRNAArg T10454C, tRNAGlu A14693G variants have been implied as potential modified factors in increasing the penetrance and expressivity of deafness-associated 12S rRNA A1555G mutation [30, 31]. Consequently, variants in these tRNAs caused a reduction in tRNA stability level and affected the tertiary structure of the mitochondrial tRNAs [32].

Fig. 1
figure 1

Location of six tRNA variants in PCOS patients but absent in control subjects

Full size image

In conclusion, our study found a relative high frequency of mitochondrial OXPHOS genes mutations and tRNA variants in PCOS patients, and mtDNA mutations may play an important role in PCOS patients. The limitation of this study is the small sample size and further efforts should be taken to determine the function of these mitochondrial variants.