Alterations in long noncoding RNAs in women with and without polycystic ovarian syndrome

Long noncoding RNAs (lncRNAs) are RNA transcripts over 200 nucleotides long that are not translated into protein; however, there is increasing evidence of their regulatory functions. To date, there are few studies measuring lncRNA in control women or women with polycystic ovary syndrome (PCOS).


| Study design
This was a cross-sectional study undertaken in 24 medication naïve women with PCOS and biochemical hyperandrogenaemia (age 18-45 years) who presented sequentially to the department of endocrinology and those who fulfilled the criteria of the study were recruited into the local PCOS biobank at United Kingdom (ISRCTN70196169). Twenty-four control women (age 20-44 years) who were age and body mass index (BMI) matched to the PCOS women were included into this study. Subject demographics are shown in Table 1. The diagnosis of PCOS was based on fulfilling all three diagnostic criteria of the Rotterdam consensus, namely clinical and biochemical evidence of hyperandrogenemia (Ferriman-Gallwey score >8; free androgen index >4, respectively), oligomenorrhea or amenorrhoea and polycystic ovaries on transvaginal ultrasound (classical phenotype). 13 Liver ultrasound was performed at the same time to exclude nonalcoholic fatty liver disease. Study participants had no concurrent illness and were not on any medication for the preceding 9 months. None of the women had successful pregnancy or miscarriage at least 5 year prior to study entry. Diabetes was excluded by a 75 g oral glucose tolerance test. Nonclassical 21-hydroxylase deficiency, hyperprolactinaemia, Cushing's disease and androgen-secreting tumours were excluded by appropriate tests. All women gave written informed consent. This study was approved by the Newcastle & North Tyneside Ethics committee, UK.

| Sample collection
Blood samples were taken after an overnight fast during the follicular phase of the menstrual cycle in control women, and serum was stored frozen at −80°C pending analysis. All PCOS women were anovulatory (3 months amenorrhoea; random progesterone <5 nmol/L).
Serum testosterone and androstenedione were measured by isotope dilution liquid chromatography-tandem mass spectrometry (Waters Corporation). Sex hormone binding globulin (SHBG) was determined by an immunometric assay with fluorescence detection on the DPC Immulite 2000 analyser using the manufacturer's recommended protocol. The free androgen index was obtained as the total testosterone ×100/SHBG. Serum insulin was assayed using a competitive chemiluminescent immunoassay performed on the manufacturer's DPC Immulite 2000 analyser (Euro/DPC). The analytical sensitivity of the insulin assay was 2 µU/mL, the coefficient of variation was 6%, and there was no stated cross-reactivity with proinsulin.
Plasma glucose was measured using a Synchron LX20 analyser (Beckman-Coulter), using the manufacturer's recommended protocol. The coefficient of variation for the assay was 1.2% at a mean glucose value of 5.3 mmol/litre during the study period. The insulin Control women (n = 24)

| RNA preparation and analysis following RNA extraction
Approximately 20 ng of total RNA was used to generate strand-

| RNA-seq data analysis
We performed quality check for the read with FastQC. 14 Reads with adaptors and rRNA contamination were removed using BBMap. 15 After filtering, the reads were mapped to human reference genome from Ensembl GRCh38 release 93 16 with STAR 17 using Ensembl 93 gene annotation. 16 For mapped read quantification, we used fea-tureCounts function from Rsubread package 18 in R 19 .
Following quantification, lncRNA was identified and the lncRNAs were then quantified using Wald test from DESeq2.( 20 ) P value < .05 was taken as the cut-off for significance.

| RE SULTS
Baseline characteristics of the 24 PCOS and 24 control women are shown in Table 1. The PCOS and control women did not differ for age or BMI; however, the PCOS women showed greater insulin resistance and hyperandrogenemia (P < .01), androstenedione did not differ.
There were eight significant lncRNAs between the PCOS and control women (

| D ISCUSS I ON
Next-generation sequencing (NGS) has provided a wealth of novel information about genomic organization and regulation of gene expression, with noncoding (nontranslated) RNAs being identified as important regulators of gene expression. 15 Whilst thousands of lncRNAs have now been identified, functional characterization has only been determined in a small subset but has indicated that they are involved in numerous physiological processes, working at the epigenetic, transcriptional and post-transcriptional level. 16 Information to date has shown that their dysregulation is associated with a wide variety of diseases including cancer, 8  women. However, given that the lncRNA did not correlate to either insulin resistance or androgen levels then it may suggest that the differences could be more related to the menstrual cycle, but there are no data on lncRNA throughout the menstrual cycle to answer this.
In conclusion, lncRNA was found to differ between PCOS and control women in the follicular phase of the menstrual cycle, though functional pathway analysis was limited by the unknown functions of many of the lncRNAs. The phase of the menstrual cycle may be important for lncRNA expression and that needs to be taken into account in future studies.

ACK N OWLED G EM ENTS
SLA is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. The authors are further grateful for support provided by the Biostatistics,

Epidemiology and Biomathematics Research Core at Weill Cornell
Medicine-Qatar.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the paper reported.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.