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Additional file 1: Figure S1: Expression of lncRNA TUG1 transcript variants in male and female human skeletal muscle. Figure S2: Optimisation of Tug1 knockdown in C2C12 myocytes and uncropped western blot images. Figure S3: Mitochondrial morphology in myotubes with Tug1 knockdown, supplement to Fig. 3. Figure S4: Tug1 knockdown in cells used for mitochondrial respiration experiments, related to Fig. 4. Figure S5: Supporting data for transcriptomic responses to EPS and Tug1 KD, related to Figs. 6 and 7. Individual data are available at DOI: 10.6084/m9.figshare.20175770 [24].

STUDY QUESTION Do ovarian hormone changes influence the levels of cell-free or circulating microRNA (cf-miRNA) across the menstrual cycle? SUMMARY ANSWER This exploratory study suggests that fluctuations in hormonal levels throughout the menstrual cycle may alter cf-miRNAs levels. WHAT IS KNOWN ALREADY cf-miRNA levels vary with numerous pathological and physiological conditions in both males and females and are regulated by exogenous and endogenous factors, including hormones. STUDY DESIGN, SIZE, DURATION A prospective, monocentric study was conducted between March and November 2021. Since this was a pilot study, the sample size was based on feasibility as well as previous similar human studies conducted in different tissues. A total of 20 participants were recruited for the study. PARTICIPANTS/MATERIALS, SETTING, METHODS We conducted an exploratory study where blood samples were collected from 16 eumenorrheic females in the early follicular phase, the ovulation phase and the mid-luteal phase of the menstrual cycle. The levels of oestrogen, progesterone, LH and FSH were measured in serum by electrochemiluminescence. The levels of 174 plasma-enriched miRNAs were profiled using a PCR-based panel, including stringent internal and external controls to account for the potential differences in RNA extraction and reverse-transcription stemming from low-RNA input samples. MAIN RESULTS AND THE ROLE OF CHANCE This exploratory study suggests that cf-miRNAs may play an active role in the regulation of the female cycle by mediating the expression of genes during fluctuating hormonal changes. Linear mixed-models, adjusted for the relevant variables, showed associations between phases of the menstrual cycle, ovarian hormones and plasma cf-miRNA levels. Validated gene targets of the cf-miRNAs varying with the menstrual cycle were enriched within female reproductive tissues and are primarily involved in cell proliferation and apoptosis. LARGE SCALE DATA All relevant data are available from the Mendeley database: LEGER, Bertrand (2022), ‘MiRNA and menstrual cycle’, Mendeley Data, V1, doi: 10.17632/2br3zp79m3.1. LIMITATIONS, REASONS FOR CAUTION Our study was conducted on a small participant cohort. However, it was tightly controlled for endogenous and exogenous confounders, which is critical to ensure robust and reproducible cf-miRNA research. Both adjusted and non-adjusted P-values are presented throughout the article. WIDER IMPLICATIONS OF THE FINDINGS Measures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA levels in females and used as time-varying confounders. Our results reinforce the importance of accounting for female-specific biological processes in physiology research by implementing practical or statistical mitigation strategies during data collection and analysis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Clinique romande de réadaptation, Sion, Switzerland. S.L. was supported by an Australian Research Council (ARC) Future Fellowship (FT10100278). D.H. was supported by an Executive Dean’s Postdoctoral Research Fellowship from Deakin University. The authors declare no competing interests.