Abstract Background: Women with polycystic ovary syndrome (PCOS) often change their metabolic profile over time to decrease levels of androgens while often gaining a propensity for the development of the metabolic syndrome. Recent discoveries indicate that microRNAs (miRNAs) play a role in the development of PCOS and constitute potential biomarkers for PCOS. We aimed to identify miRNAs associated with the development of an impaired metabolic profile in women with PCOS, in a follow-up study, compared with women without PCOS. Methods and materials: Clinical measurements of PCOS status and metabolic disease were obtained twice 6 years apart in a cohort of 46 women with PCOS and nine controls. All participants were evaluated for degree of metabolic disease (hypertension, dyslipidemia, central obesity, and impaired glucose tolerance). MiRNA levels were measured using Taqman^® Array cards of 96 pre-selected miRNAs associated with PCOS and/or metabolic disease. Results: Women with PCOS decreased their levels of androgens during follow-up. Twenty-six of the miRNAs were significantly changed in circulation in women with PCOS during the follow-up, and twenty-four of them had decreased, while levels did not change in the control group. Four miRNAs were significantly different at baseline between healthy controls and women with PCOS; miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p, which were decreased in PCOS. After follow-up, miR-28-3p, miR-139-5p, and miR-376a-3p increased in PCOS women to the levels observed in healthy controls. Of these, miR-139-5p correlated with total testosterone levels (rho = 0.50, p[adj] = 0.013), while miR-376-3p correlated significantly with the waist-hip ratio at follow-up (rho = 0.43, p[adj] = 0.01). Predicted targets of miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p were enriched in pathways associated with Insulin/IGF signaling, interleukin signaling, the GNRH receptor pathways, and other signaling pathways. MiRNAs altered during follow-up in PCOS patients were enriched in pathways related to immune regulation, gonadotropin-releasing hormone signaling, tyrosine kinase signaling, and WNT signaling. Conclusions: These studies indicate that miRNAs associated with PCOS and androgen metabolism overall decrease during a 6-year follow-up, reflecting the phenotypic change in PCOS individuals towards a less hyperandrogenic profile. Keywords: biomarker, metabolic syndrome, microRNA, PCOS, polycystic ovary syndrome, circulation, ovary, non-coding RNA, clinical trial 1. Introduction Polycystic Ovary Syndrome (PCOS), defined by hyperandrogenism, oligo- or anovulation, and polycystic ovary morphology according to the Rotterdam criteria [[40]1], is also characterized by insulin resistance, which is a part of the pathogenesis [[41]2]. The etiology of PCOS is not known in detail but is mainly caused by ovarian and endocrine disturbances, which in younger women primarily manifests as reproductive symptoms including infertility, hyperandrogenism, irregular menstrual cycles, polycystic ovarian morphology, and an increased risk of pregnancy complications if pregnancy occurs [[42]1]. Over time, these symptoms become less prominent, and metabolic disturbances, which are closely related to the pathophysiology of PCOS, become more pronounced. Women with PCOS have an increased risk of developing metabolic syndrome (MetS) and have a four times higher risk of presenting with type 2 diabetes (T2D) [[43]3,[44]4]. MicroRNA (miRNA) are small, non-coding RNA molecules that regulate gene expression by post-transcriptional inhibition of targeted mRNAs [[45]5]. MiRNAs bind to their target mRNA and either inhibit translation or promote their degradation. As present in all body compartments and in circulation, miRNAs are suitable as biomarkers [[46]6]. Furthermore, miRNAs are protected from degradation by Ribonucleases because they are contained in extracellular vesicles, bound in protein complexes or lipoproteins [[47]6]. Studies have shown an altered expression of miRNAs in insulin-sensitive tissues from obese or overweight individuals and patients with T2D, suggesting a potential role of these small RNA molecules in the complications associated with MetS and T2D [[48]7,[49]8]. Another quality of miRNAs as biomarkers is the ability to reflect the changes in clinical disease stages. This has been demonstrated in weight-loss studies and studies of miRNA levels during treatment with insulin sensitizers and bariatric surgery [[50]9,[51]10,[52]11]. While the correlation between metabolic disease and levels of specific miRNAs is well described [[53]12,[54]13], the correlation between miRNAs and reproductive hormones and PCOS is still debated. Several studies have investigated the miRNA profile in women with PCOS [[55]14,[56]15], investigated miRNA biomarkers in serum or plasma [[57]16,[58]17,[59]18,[60]19,[61]20], or searched for miRNAs in follicular fluid [[62]16,[63]21,[64]22] to unravel the biological mechanisms behind hyperandrogenism and anovulation of PCOS. The results of these studies are, however, of great variability, perhaps because of the complexity and heterogeneity of PCOS, and only a few studies have been able to validate previous findings, although some miRNAs have been reported in more than one study. Several studies have reported miR-93 levels altered in both adipose tissue, serum, and granulosa cells in women with PCOS [[65]23,[66]24,[67]25]. Likewise, other research groups have shown that miR-21 is altered in serum, granulosa cells, and follicular fluid [[68]26,[69]27]. Several recent reviews have covered this active field of research [[70]14,[71]15,[72]28]. However, none of these studies addressed the change in miRNA levels over time in PCOS patients, and it is not known how the changes in clinical markers and reproductive hormones of women with PCOS are reflected in the miRNA profile over time; knowledge which is essential in order for miRNA to serve as biomarkers. Thus, with this longitudinal study, we aim to assess selected miRNAs in PCOS women before assisted reproductive therapy and after an average of 6 years of follow-up (FU) to investigate the relationship between the miRNA profile and metabolic changes over time. 2. Materials and Methods 2.1. Study Design and Participants The present study consists of a FU study of 46 women with PCOS and nine controls. Women were diagnosed according to the Rotterdam 2003 consensus criteria [[73]29]. This study was based on the PICOLO cohort of 186 women with PCOS and 38 healthy controls recruited between 2010 and 2013 from three Danish hospitals: Herlev, Hvidovre, and Holbaek Hospital [[74]30]. A flow chart of the participants is shown in [75]Figure 1. Descriptions of the PICOLO cohort can be found in [[76]21,[77]31,[78]32]. The control group and the PCOS group were not matched at baseline (BL) but were similar regarding BMI and age in the FU study. Figure 1. [79]Figure 1 [80]Open in a new tab Flowchart of included women with PCOS and controls at BL and at FU. In the FU study, all participants of the PICOLO study from Holbaek Hospital were invited for FU re-examination at Holbaek Fertility Clinic 6 years after the first visit (Baseline; BL). Exclusion criteria: Oral contraceptives within 8 weeks of examination, endocrine disease, endometriosis, premature ovarian insufficiency, breastfeeding, and pregnancy ([81]Figure 1). Serums from BL and FU were used for miRNA analysis. This study followed the Declaration of Helsinki II, was approved by the Danish Data Protection Agency (REG-31-2016) and the Danish Scientific Ethical committee of region Zealand (Journal no. SJ-525), and was registered at Clinicaltrials.gov ([82]NCT03142633). All subjects gave informed written consent prior to inclusion. 2.2. Anthropometric and Clinical Characteristics at FU Clinical examination at FU included anthropometric measurements (height, weight, blood pressure (BP)), vaginal ultrasound, blood sampling, and an oral glucose tolerance test (OGTT). An OGTT was performed after an overnight fast (>8 h). We collected venous blood samples and measured glucose, serum insulin, and C-peptide at −5, 0, 30, and 120 min after a 75 g glucose load. HOMA-IR was calculated as (fasting insulin (µU/mL) * fasting glucose (mmol/l))/22.5 [[83]33]. Clinical hirsutism was evaluated with Ferriman-Gallwey score (FG-score). Total testosterone (T) was measured using liquid chromatography mass spectrometry (Xevo TQD, Waters, Milford, MA, USA). Sex hormone binding globulin (SHBG), plasma C-peptide, serum insulin, and dehydroepiandrosterone sulfate (DHEAS) were measured on ADVIA Centaur^® XP (Siemens Healthcare, Oxford, UK). Free T was calculated from total T and sex hormone binding globulin (SHBG) described by Vermeulen et al. [[84]34]. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and androstenedione were measured according to standard laboratory procedures at Holbaek Hospital. 2.3. Serum MiRNA Isolation and Analysis MiRNA analyses were conducted using serum from venous blood samples collected in a procoagulant drying tube. Serum and cellular fractions were separated by centrifugation at 2000 rpm for 20 min. Serum was carefully removed, leaving 0.5 mL in order to avoid disturbance of the interface. The serum was stored at −80 °C until analysis. Total RNA was extracted from serum with TriReagent LS (Sigma-Aldrich Denmark, Copenhagen, Denmark), according to manufacturer’s protocol. RNA concentration and purity were determined using NanoDrop ND-1000 (ThermoFisher Scientific, Hvidovre, Denmark). RNA was reverse transcribed using a 96 miRNA custom-designed TaqMan Reverse Transcription Kit (ThermoFisher Scientific, Hvidovre, Denmark) according to the manufacturer’s protocol. The RT-product was pre-amplified with TaqMan^® PreAmp Master Mix and miRNA PreAmp Custom Primer Pools. MiRNA levels were measured by quantitative PCR using human TaqMan low density array (TLDA) cards containing 96 miRNAs (ThermoFisher Scientific, Hvidovre, Denmark) ([85]Table S1). The custom array card was designed with miRNAs from the literature associated with PCOS, metabolic syndrome, insulin resistance or features of the metabolic syndrome, and in some cases circulating miRNAs associated with ovarian cancer along with three miRNAs selected based on an in-house study [[86]32]. Expression data were obtained using the ViiA 7 real-time PCR system and analyzed using QuantStudio software. Amplification plots were manually inspected, and assays with absent or poor amplification or low fluorescent intensity were excluded for further analysis. Cycle threshold (Ct) >32 was considered undetectable following manufacturer’s guidelines and excluded from the analysis. A miRNA had to be present in a minimum of ten subjects to be included in further analysis steps. Thirteen (n = 13) miRNA at FU and nine (n = 9) miRNA at BL did not fulfill these criteria. Data were normalized against the geometric mean of two references, the endogenous small