Levels of circulating insulin cell-free DNA in women with polycystic ovary syndrome - a longitudinal cohort study

Pernille Bækgaard Udesen, Anja Elaine Sørensen, Mugdha V Joglekar, Anandwardhan A Hardikar, Marie Louise Muff Wissing, Anne-Lis Mikkelsen Englund, Louise Torp Dalgaard, Pernille Bækgaard Udesen, Anja Elaine Sørensen, Mugdha V Joglekar, Anandwardhan A Hardikar, Marie Louise Muff Wissing, Anne-Lis Mikkelsen Englund, Louise Torp Dalgaard

Abstract

Background: Women with Polycystic Ovary Syndrome (PCOS) present a heterogeneous reproductive and metabolic profile with an increased lifetime risk of Type 2 Diabetes (T2D). Early biomarkers of these metabolic disturbances in PCOS women have not been identified. The abundance of circulating insulin gene promotor cell-free DNA (INS cfDNA) was shown to be valuable as a predictive biomarker of β-cell death in individuals with Type 1 diabetes (T1D) as well as with gestational diabetes. Since β-cell death is common to the development of T1D as well as in T2D, we aimed to investigate if insulin-coding DNA is more abundant in circulation of PCOS women (vs Controls) and if their levels change after 6 yr. follow-up as a potential measure to predict future T2D.

Methods: A cohort of 40 women diagnosed with PCOS according to Rotterdam 2003 criteria and eight healthy controls were examined at baseline and 6 years follow-up. Clinical measurements for evaluation of glucose homeostasis as well as blood/serum samples were obtained at each visit. Methylated and unmethylated INS cfDNA were quantified using droplet digital PCR. Differences between groups were assessed using Kruskall-Wallis test and Wilcoxon Signed rank test.

Results: At baseline, there was no detectable difference in copy number (copies/μL) of methylated (p = 0.74) or unmethylated INS cfDNA (p = 0.34) between PCOS and Control groups. At follow up, neither methylated (p = 0.50) nor unmethylated INScfDNA levels (p = 0.48) differed significantly between these groups. Likewise, when pooling the groups, there was no difference between baseline and follow up, in terms of copies of methylated or unmethylated INS cfDNA (p = 0.38 and p = 0.52, respectively). There were no significant correlations between counts of unmethylated or methylated cfDNA and the clinical measurements of β-cell function and pre-diabetes.

Conclusion: The circulating level of unmethylated and methylated INScfDNA is similar between PCOS and Controls and cannot be used to predict islet β-cell loss and progression to Type 2 diabetes in a 6-year follow-up.

Trial registration: The Danish Data Protection Agency (REG-31-2016. Approval: 01-12-2015) and by the Danish Scientific Ethical committee of Region Zealand (Journal no. SJ-525. Approval: 13-06-2016), Clinicaltrials.gov, ( NCT03142633 , registered 1. March, 2017, Retrospectively registered).

Keywords: Androgens; Circulating free DNA; Demethylation; Glucose tolerance; Insulin promoter CpG methylation; PCOS; Testosterone.

Conflict of interest statement

Ethics approval and consent to participate

The study was designed and performed according to the Declaration of Helsinki II and approved by the Danish Data Protection Agency (REG-31-2016) and by the Danish Scientific Ethical committee of region Zealand (Journal no. SJ-525). All subjects gave written consent prior to inclusion.

Consent for publication

Not applicable.

Competing interests

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Panel a: Methylated INScfDNA. Panel b: Unmethylated INScfDNA. Panel c: Baseline vs. Follow-up. Number of copies/mL of respectively unmethylated (a) and methylated (b) INScfDNA. PCOS and control group compared at baseline (BL) and follow-up (FU). BL and FU for all samples expressed by copies/uL, are compared in panel c. Results are expressed as medians (95%CI). NS: Not significant
Fig. 2
Fig. 2
Panel a: Total INScfDNA. Panel b: Total INScfDNA. Total amount of INScfDNA (umethylated + methylated copies/uL). PCOS and controls are compared in panel a and BL vs FU in panel b. Results are expressed as medians (95%CI). NS: Not significant
Fig. 3
Fig. 3
Ratio Unmethylated/methylated INS cfDNA. Ratio between unmethylated and methylated INS cfDNA (copies/μL/copies/μL). Results are expressed as medians (95%CI)

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