Polycystic Ovary Syndrome in Type 1 Diabetes

Differences in insulin sensitivity between study subgroups

Study of sensitivity to insulin action by hyperinsulinaemic euglycaemic clamp. Clamping will be conducted in the follicular phase fo women's study participants. In this protocol free fatty acids will be also determined in stored samples.

Fat mass percentage

Body composition studies: Fat mass percentage with respect to total body weight. Methodology: Bioelectrical impedance analysis by Monitor VitalScan Medeia® System device (United States, CA).

Phase angle 50 KHz

Methodology: Bioelectrical impedance analysis by Monitor VitalScan Medeia® System device (United States, CA).

Peritoneum-vertebral column fat thickness

Ultrasound assessment of fat compartments, determined using Toshiba Nemio ZG SSA-580ª ultrasound equipment (Toshiba Medical Systems, S.A., Alcobendas, Madrid) following the protocol previously validated and reported by our research group (PMID: 23386652).

Identify genomic variants in genes/proteins related to insulin signalling and action, adiposity, inflammation, steroidogenesis, and PCOS itself

This approach will permit a detailed characterization of all the genetic variants present in the coding portion of the human genome, covering not only genes already identified to be associated with PCOS, but also other genes related to the pathophysiology of PCOS in women with T1D. Bioinformatic analysis of WES will be focused on genes that have been linked to PCOS and functional hyperandrogenism in populations of European ancestry in previous candidate gene studies and genome wide association studies, leaving open the possibility of analyzing other genes of interest depending on the results obtained in other studies of the project and the data published by other groups. Genomic DNA will be isolated from peripheral bood with the FlexiGene DNA Qiagen kit and submitted to an external biotech company (Macrogen Spain) for conducting these studies. Genomic DNA will be isolated from peripheral blood with the FlexiGene DNA Qiagen kit and submitted to an external biotech company.

Description of differential DNA methylation patterns in deep subcutaneous adipose and skeletal muscle tissues (Epigenomics)

Oxford Nanopore sequencing enables direct detection of methylated cytosines from isolated genomic DNA without the need for bisulphite conversion. The Adaptive Sampling RRMS panel (Reduced Representation Methylation Sequencing) protocol enables to target the regions of human genome highly enriched for CpG sites, including a total number of 7-8M CpGs, and multiplexing up to 4 samples on a single PromethION flow cell.

Description of differential gene expression profiles in adipose and muscle tissues (Transcriptomics)

RNA from adipose and muscle will be isolated using the Fatty Tissue and Total RNA Purification kits (Norgen Biotek). RNA quantity and quality shall be evaluated with the Qubit fluorimeter and Agilent Bioanalyzer. RNAseq libraries will be prepared following the protocol included in the Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit. Sequencing will be performed using an Illumina NovaSeqXPlus (1x100bp) with \>40M reads/sample. Targeted expression of selected genes (chosen according RNAseq results) will be assessed by real-time qPCR in a LightCycler480 with products from AnyGenes company.

Description of differential proteomic profiles in adipose and muscle tissue (Proteomics)

Comparative proteome analysis with isobaric tandem mass tag (TMT). Samples will be processed according to the protocol described in Carmona L. et al. (PMID: 37171157) on S-Trap columns. The resulting peptides will be labelled with TMT18plex labels according to the manufacturer's instructions (Thermo Scientific). The labelled peptides shall be separated on an Ultimate 3000 nanochromatograph on a C18 reverse phase column.

Insulin requirements/day adjusted for weight

To evaluate the interaction between T1D and PCOS on parameters of metabolic control and morbidities associated with T1D itself.

Time in range

Interstitial blood glucose monitoring: time in range \[between 70 and 180 mg/dl (%)\]

Ambulatory blood pressure

Blood pressure determined according to 24-hour ambulatory blood pressure monitoring

Subclinical atherosclerosis: Differences in carotid intimomedial thickness between study subgroups

Imaging will be conducted using a high-resolution 7-15 MHz phased-array transducer (EPIQ 5, Philips Healthcare, Bothell, WA, USA) by the same trained operator in all study participants. This operator will be blinded to the women's features and their group allocation. Under controlled light and temperature conditions, studies will be performed by positioning the women in a supine position with a 35-degree incline of the head and torso, and a 45 degrees right-turn or left-turn, respectively, of the head. The left and right common carotid arteries will be explored in B-mode in longitudinal and transversal planes, to rule out the presence of plaque that might interfere with cIMT measurements. The posterior carotid wall at 1 cm of the common carotid bulb will be imaged and cIMT estimated by visual assessment (average of five manual measurements) of the distance between the lumen/intima and intima/adventitia interphases in longitudinal frames acquired during arterial diastole.