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The effect of the menstrual cycle on the circulating microRNA pool in human plasma: a pilot study

Study question Do ovarian hormones levels influence cf-miRNA expression across the menstrual cycle? Summary answer Measures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA expression in females and used as time-varying confounders. This exploratory study suggests that cf-miRNAs may play an active role in the regulation of the female cycle in various target tissues. What is known already Cell-free or “circulating” miRNAs (cf-miRNAs) are secreted from tissues into most physiological fluids, including plasma, where they play a role in cross-tissue communication. Endogenous and exogenous factors, including sex hormones, regulate cellular miRNA expression levels. Plasma cf-miRNA levels vary with numerous pathological and physiological conditions, including in females. Participants/materials, setting, methods We conducted an exploratory study where blood samples were collected from sixteen eumenorrheic females in the early follicular phase, the ovulation phase and the mid-luteal phase of the menstrual cycle. Ovarian hormones oestrogen, progesterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in serum by electrochemiluminescence. The expression levels of 179 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. Study design, size, duration This was a prospective monocentric study conducted between March and November 2021. 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 in various target tissues. Linear mixed-models adjusted for the relevant variables showed numerous 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 the female reproductive tissues and primarily involved in cell proliferation and apoptosis. Wider implications of the findings Measures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA expression in females and used as time-varying confounders. Limitations, reasons for caution Our study was conducted on a relatively small cohort of patients. However, it was tightly controlled for endogenous and exogenous confounders, which is critical to ensure robust and reproducible cf-miRNA research. Wider implications of the findings 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. Prof. Severine Lamon, is supported by an Australian Research Council Future Fellowship (FT10100278). The authors declare no competing interest Trial registration number N/A.