Recruitment of Subjects
A total of 178 participants were recruited for this case-control study. Among these 178 participants, 73 were healthy controls and 105 were drug naïve PCOS women. PCOS cases were recruited from the patients attending the outpatient clinic of the Department of Obstetrics and Gynaecology, Govt. Medical College, Srinagar, Jammu and Kashmir, and Department of Endocrinology, Sher-e-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India for PCOS related complications from October 2017 to March 2020. PCOS patients were recruited based on the Rotterdam criteria16. For exclusion criteria, participants who presented with PCOS mimicking disorders like hyperprolactinemia, Cushing’s syndrome thyroid dysfunction, androgen secreting tumours and congenital adrenal hyperplasia were excluded from the study. Controls recruited for the study were age matched healthy women without any biochemical or clinical signs of hyperandrogenism, had regular cycles, with no history of autoimmune or endocrine disorders. All of the participants were ethnic Kashmiris living in Kashmir province and did not receive any hormonal therapy for at least past six months
Ethics statement
The study was ethically approved by the ethical committee Government Medical College Srinagar under ethical approval no. 94/ETH/GMC/ICMR. The participants were recruited only after written informed consent was obtained from them. All methods and protocols were performed in accordance with the relevant guidelines and regulations.
Anthropometric and clinical evaluation
The study participants recruited underwent general anthropometric measurements that include height, weight, waist, hip, waist-hip ratio (WHR), body mass index (BMI) and extent of hirsutism (FG score). A detailed history of clinical symptoms like menstrual cycles, hirsutism, acne, alopecia, and acanthosis nigricans was also taken from all participants. Weight was determined using a weighing balance while wearing light clothing and height was determined in a standing position without shoes. In order to measure waist circumference, the minimum measurement between the iliac crest and lateral costal border was established while the measurement for hip circumference was the maximum measurement over the buttocks. The waist-hip ratio was determined by dividing the waist circumference by the hip circumference. BMI was calculated as weight(kg)/Height(m²). The Ferriman-Gallwey scoring system was applied to measure the hirsutism score. Transabdominal ultrasonography was performed on all PCOS patients to determine the ovarian volume and/or number of peripheral ovarian cysts.
Hormonal and Biochemical assessment
The peripheral venous blood samples were collected from the participants on the second or third day of their menstrual cycle after an overnight fast in clot activator vials for hormonal and biochemical assessment. The hormones include testosterone, luteinizing hormone, follicle stimulating hormone. thyroid stimulating hormone and Prolactin were estimated on Beckman coulter UniCelDxl 800 (Access Immunoassay system) by Radioimmunoassay (RIA) using RIA kits (Immunotech S.R.O, Prague, Czech Republic). Fasting insulin, androstenedione, dehydroepiandrosterone sulphate, and Sex Hormone-Binding Globulin were measured by enzyme-linked immunosorbent assays using Calbiotech, CA, USA, and DGR Instruments GmbH Marburg ELISA kits using SkanIt RE 4.0 software on Thermo Scientific Multiskan FC ELISA reader.
The biochemical parameters done included oral glucose tolerance test, Lipid profile, Liver function and kidney function tests. All the biochemical parameters were determined by using ERBA bioassay diagnostic kits ERBA Chemtouch 7, Semi-Automatic Biochemistry Analyzer, Wiesbaden, Germany.
Insulin resistance was determined by calculating HOMA IR
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MzMzPQdR337t3r+JxBZaAMWxFk1UQbRCbGqB3FF198EQDws5/9DABw/fp1fO9734tVllqthuXlZd9LDkNDQ76JMfh2Y6+Q3ZkzZ87EDuQ+OTkZujse/FtaWur4PNkFiFJw//73vwH4X6oANpWASjEDm4pwaWkJuVzOnXiCLyrIyxphO6UyAYY59gPAd77zHd/1d999F4B65yJsd3hhYaFt1yQMmRCCZQpriyjjXSaNoNGxsLAAwG9wSVupXsLyIotFVX+K0eN9bqd+kp1OANi/fz+efPJJXL9+HU899RTq9XrHXQ2R27hf/+mGZDKJfD6P2dlZty3L5TLq9TquX7/uSxsVDWEnyrYdHj58CF3Xsby83NXXz8J2HScnJ5U7sh9//DGAdgOqUqkAaB+XwOapVrVaRSKRwMmTJ5Ufs/nwww8BhO+UihGpknvZmf3mN7/pux62w9utPIYhO55helBOtbz5iwGv+k2cdupG/oFw47XXetKLlGFjYyMyXSc6bQ5pmub7d6+iIXQiGA1hbm4O9Xod+/fv99kDEtVETluBR7pYxqwXsVs6zSnj4+NotVo9qcvjyEAZtmKIlsvlyLdcxdgzDCNygIyOjsIwDJim6SqQuLswN27cQDqdVhqG8rWoqJBi2yGZTOL3v/89AODkyZN9+ZyfHJN/8YtfDE0jOwhBJGqEagIENo2my5cvu18xO3HihO++TCZhR78yAar6Xgzy4NFk1BGnlMlLrVZDvV6PdVzpJXhMLm0RNDyjjHdpV+/bxlKeoHEiBqvqKFbFE0884ft3o9GAaZrKndyofhLDIZvNwnEcPHjwAEtLS7h8+XJXO5GyOOo1r7zyCgzDwMsvv4yhoSFcuHABi4uLOH78uJsmLBqC7LhIH/WCRqPRNol3c+JTq9Xw7rvv4u7du0ilUjh37lxsvSB9FVyAhiG7kEFkUfDMM88o74+OjmJjYwPZbBazs7Nti3JZQIWND5F71c6j6CPvPVkcRhmpceUxjA8++ACA2tALO9XqZBx2aqdu5F/GsKoNdkJPBpHF7VaJcjtsNBpt+ne7bgiqaC3BeUzlhvDss88in8+3/Ul/ZLNZ5PN5AI8W7UH5FFshbpjRfoTZ3CsMlGE7OjqKXC6HVquF119/XZnGtm1cuHABmqZ1PO4EgLNnzwLYjD/bKVac0Gg0sLKyEuqbdfz4cXfw7pQfzOjoqCsoR44c2XXjtl6vdzyuU/kmNRoNd2chGAZGwuIIhw4dwtNPP92mHGUykUWIZVm+sFVizKnCzEg7eQ3yOK4NXmzbdv1W4xoDKhqNhntcH9z9kjqojHcxLKT+Uh5N09omUemDrfqlix+i9yQjTj+JgRHcUQEQ6rrgZTvtGoexsTGcPXsWGxsbcBwHGxsbPqMWULshAI9CF168eLFnMauvXr0auqju1BaWZeHSpUv46U9/iuHhYfdE58SJE7HK98c//hFA/MWPSuaLxaKbl9dQKpfLbbuOskEhO79C0DCrVCo+o07kXmWI1ev1tl3JTq4NQaLkUYXs7oYZerJgCOav2kHtpp26kX8pgyou8E7rye3E9ZW5+Je//GVomqtXr+Lb3/62r3zbcUOwbTtyHpUxEXRDADb7YnJysu1PZPfFF190w38dOnQIhmG0LVhkvMY5NV5YWIj9LpAX27b33Ceft8KeNGzv378PAG1O8oIcaQX/H9j8hnMul8Ps7KwvvqZt2yiXy+4q+J133vEpYNu2cf/+fdy/f9+X5/Hjx6HrOnRd9x2PSnpgU5HIbyzLwqlTp6DreuRLKqJgZmdnO8bstG3bVVbB8lmW5R7nBONkTk5OIp1Oo9Vq4ciRIx13sntFnJ0Q4NGRotStVqvh3LlzvuOtWq2GiYkJd2fh2rVrbh1qtRrW1tbc+MiC7JjVajU3tmzwCFKoVCo+o1cmFe/YkF1BTdNgWVZb3Etd17G2tgbLsmBZFl566SVX8cY1BsSvU8aStIX4KUb56QbrEKz/Sy+9hHw+7x5Neetw7NgxaJqGt99+261To9FAJpPxjUtR2jdu3IBt225cxPn5eZRKJd8kGqefhoeHYRiG79RCYssmEomOu7biq67a9ReZ39jY8PWTHO15XZWkLtKOUt56vY6zZ89ibGwMY2NjyGQymJ6e9hloYW4ImUwGqVQK9XodY2NjqFQqqFQq7mJnYWHBnbRs23aNpf/85z/KumQyGWUsZKm7tIUKy7KQTqdx9uxZd0JPJpNuYPlOMVzF1xQA/vnPf4am8yKnCFK+YrGIRqPhc8MqFosoFov48MMPUS6X3cnUtm1XjwXrpWka1tbWXPm4ePFi6O7b9PS028Zh+kgMwieeeMKNVStsRx4FeTej1Wop21h2ZoO7iqZpQtM0V8ZqtVpX7dSN/HuRsaaiF3oyiOhIlSEVNecBj2Rsfn4eU1NTvoWw+OqbpulzlYlyQ5C42IB6kS/PVJ2Q3blzB7quuzow7KMM3SCbbt7Y6xMTE8hms74FbiKRwMjIiNuGMt+99tprW/p4xauvvorDhw93tEn2PLsVfiEOYWG+vGE0VPdV1ahWq27MTUmTSqWcQqHQFgpGla83z1KppAzRofpN8FpUyA7vX1h4lKi8VM8J5inxGzuVqZeEhTUKS6tpmqNpmpPL5Zxms+mGS/LGZzRN0/1GvNRD13Xld70lNqq0azDcmcSc9T7fccLD4TjOoz4Le573U8zeWKrdhFTytoU3LqUqbIy3DtJuUeWJqoM3xqv8RhUOR5VO9Y3zuP3kjQkq8qkKKxSGfBPdS5gMqmRFQi2p5F7aXSVfmqY5f/vb3yLDTnnjxwJw4+Z626KbsIaq/kin0x3DKqnkXpVvGN7+RkjYJxUSbkrXdTcUlYSOkjZwnEdxvr16WjWuJK2k88qt937w+d58gyGxvHF1w54XVx5VZe2k31OplDJcmjf+tbR3N+3Ujfx754dg2Lpe68kgEi8+LPxd1JwsFAqFNtmWeT5IKpUKDQMXVw5VIbS8dVhcXGwLJxhFVGhDCcsXlCMvuVzOHRO6rjvZbHZbYb5EbrvRw3uRPWXYksFAJvRB+/50EO/HN4KkUqlYE6DjbLaXSmmHfSSBbCLxN3sdr1HiQKuQ4O47vTjshIyNQZexfkB53D0KhcKWPli0V5DFajcbGGTn2ZOuCOTxRo6pBu37014sy8LJkyeV/o2VSgX1eh2vvfZax+eUy2XMzs4qffbefPNNAP5QQOQRhw4dQqlUQi6X6+lzT506FTp2k8kkEolErKPoneTNN99EoVAYaBnrB5TH3eX06dN4/vnnH9uj74sXL+K3v/3troctI9F8qt8FIIPF9PQ0TNN0Iz8MOvfv34dlWUgmk7BtG6urq64vVDeBuN9//30cPHgQw8PDsCwLt27dwpUrV1AqlbbkK/VJQdq4XC73LPC5aZo4f/48Pv74Yzz33HNuJIh//etfuH37Ns6fP9/XiaxcLmPfvn2xPpRBtgblcfeYmZnB1NQUGo3GY7VQm5qawk9+8pPHqsyfGPq9ZUwGAzmm8/qFDTryfXcE/Lu28rnkoD/nXjjufpyoVqtdt3sY4ifs9S/Vdd3J5XJ9/0zl4uLiJ0a++gXlsT+USqXHpo0LhQLdgPYwQ47T4fu0hBBCCCGEPAbQx5YQQgghhAwENGwJIYQQQshAQMOWEEIIIYQMBDRsCSGEEELIQEDDlhBCCCGEDAQ0bAkhhBBCyEBAw5YQQgghhAwENGwJIYQQQshAQMOWEEIIIYQMBDRsCSGEEELIQEDDlhBCCCGEDAQ0bAkhhBBCyEBAw5YQQgghhAwENGwJIYQQQshAQMOWEEIIIYQMBDRsCSGEEELIQEDDlhBCCCGEDAQ0bAkhhBBCyEBAw5YQQgghhAwE/w8a6KZWN1OJ+wAAAABJRU5ErkJggg==)
Isolation Peripheral blood mononuclear cell
Total RNA was isolated from peripheral blood mononuclear cells (PBMCs). For PBMCs separation 2ml of whole blood sample in Na2-EDTA vials was obtained from all the subjects. The whole blood sample was diluted by the addition of an equal volume of phosphate buffer saline (PBS). The density gradient centrifugation was applied for the isolation of PBMCs. Ficoll-Paque plus (GE Healthcare Bio-Sciences Sweden) having a density of 1.077 g/ml was used as a density gradient medium. PBMCs have density slightly lesser than Ficoll-Paque plus (<1.077 g/ml) thus allowing PBMCs to form a buffy coat at the interface of plasma and Ficoll. After density gradient centrifugation PBMCs were isolated and washed twice with PBS to remove any contamination of Ficoll-Paque and plasma. The PBMCs thus obtained were stored at -80°C for further processing.
RNA isolation and cDNA Synthesis
Total RNA was isolated from PBMCs using TRIzole reagent (Life Technologies, Carlsbad California)17. Traces of genomic DNA in extracted RNA samples could potentially interfere with expression analysis studies. So, all of the RNA samples were subjected to DNase treatment before cDNA synthesis. Sigma Aldrich DNase treatment kit (DNase I) was used as per the manufacturer’s protocol in order to eliminate any traces of genomic DNA. All of the RNA samples were analyzed qualitatively and quantitatively using NanoDrop (Thermo Scientific). RNA samples having absorbance A260/280 ratio between 1.9–2.0 were subsequently used for cDNA synthesis. Thermo Scientific RevertAid First Strand cDNA Synthesis Kit was used to reverse transcribe 1.5 g of total RNA into cDNA according to the manufacturer’s protocol in an applied biosystems thermal cycler.
Quantitative real-time polymerase chain reaction
The relative expression levels of lncRNAs PRNCR1 and PCGEM1 were measured by real-time quantitative polymerase chain reaction (qPCR). SYBR Green I assay was employed for qPCR quantification of lncRNAs. 10ml pre-formulated real-time master mix containing buffer, dNTPs, DNA polymerase and SYBR Green I dye was used (KAPA SYBR® FAST), 0.3ml of forward and 0.3ml of reverse primers and cDNA less than 100ng (1ml) in a 20ml reaction following manufacturer’s protocol. The relative quantification was performed in Roche LightCycler® 480 Instrument II on 96 well plate having the following reaction protocol pre-incubation at 95°C for 5mins, a 40 cycle amplification at 95°C for 20 sec, 58°C/56°C for 15 sec (for PRNCR1 and PCGEM1 respectively) and 72°C for 15 sec. Amplification was followed by melting curve analysis with the following conditions 95°C for 5sec, 60°C for 60sec and 95°C continuous. The product size was confirmed by running the amplified products on 2% agarose gel. The relative expression of PRNCR1 and PCGEM1 was estimated by the Livak method and Beta-Actin was used as a reference gene18. Each reaction of qPCR was performed in triplicates.
The following primers were used to quantify the lncRNA PRNCR1 levels: 5´-CTCTGTGGAAGCATTGTGGA-3´ (forward) and 5´-TATCAGCCCTTGGAATCTGG -3´ (reverse), For lncRNA PCGEM1 following primers were used 5´-GGTGCCTTTGCCAATGTTAT-3´ (forward) and 5´-AGCATGCTCTCTGCAAAGGT-3´ (reverse) and β-Actin RNA was quantified as a control to normalize differences in total RNA levels using the following primers 5´-ATCGGAACGGTGAAGGTGACA-3´ (forward) 5´-TGGCAAGGGACTTCCTGTAAC-3´ (reverse)
Statistical Analysis
The quantitative baseline variables were expressed as number percentage and mean ± SD. All parametric variables including hormonal, anthropometric and biochemical were compared by unpaired students t-test between PCOS and controls. The anthropometric, hormonal and biochemical parametric variables between PCOS and controls. The association of expression of lncRNAs PRNCR1 and PCGEM1 with PCOS was evaluated by using the Chi-square test. The relation between various metabolic and hormonal parameters with the expression of lncRNA PRNCR1 and PCGEM1 was determined by Spearman or Pearson rank correlation coefficient. The distribution of expression of lncRNA PRNCR1 and PCGEM1 were divided into binary groups among both PCOS and healthy controls. The limits for binary groups were derived from the control group. For PRNCR1 limits derived for binary group cut offs were <1.46 for the low expression, >1.46 for the high expression and for PCGEM1 binary groups cut offs were <1.54 for the low expression, >1.54 for the high expression. Sigma Plot 10 was used to determine the Receiver operator characteristic (ROC) and area under curve (AUC). The statistical computing tool vassarstats (http://vassarstats.net/) was used for statistical analysis. Data at a P-value of <0.05 was considered statistically significant.