Christos Tsatsanis1, Johannes Bobjer2, Hamideh Rastkhani3, Erini Dermitzaki4, Marianna Katrinaki4, Andrew N Margioris4, Yvonne Lundberg Giwercman3, Aleksander Giwercman2. 1. Molecular Reproductive Research Group, Department of Translational Medicine, Lund University, Malmö, Sweden Department of Clinical Chemistry, School of Medicine, University of Crete, Heraklion, Crete, Greece tsatsani@med.uoc.gr. 2. Molecular Reproductive Research Group, Department of Translational Medicine, Lund University, Malmö, Sweden Reproductive Medicine Centre, Skåne University Hospital Malmö, Malmö, Sweden. 3. Molecular Genetic Reproductive Medicine, Department of Translational Medicine, Lund University, Malmö, Sweden. 4. Department of Clinical Chemistry, School of Medicine, University of Crete, Heraklion, Crete, Greece.
Abstract
STUDY QUESTION: Are serum levels of micro-RNAs miR-155 and miR-146a associated with male fertility, low-grade systemic inflammation (LGSI) and androgens? SUMMARY ANSWER: miR-155 was associated with male subfertility independent of LGSI or androgens while miR-146a was only weakly associated with subfertility and LGSI. WHAT IS KNOWN ALREADY: Male subfertility has been associated with LGSI as well as with androgen deficiency. miR-155 and miR-146a are central regulators of inflammation and their level in cells and in the serum has been associated with several inflammatory conditions. STUDY DESIGN, SIZE, DURATION: In this case-control study, two independent groups of 60 subjects each (exploratory and confirmatory cohort) were randomly selected from an ongoing study on subfertile men (in total: hypogonadal; n = 40, eugonadal; n = 40 and control group n = 39) at a University Hospital Reproductive Medicine Centre. Individuals were matched for age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from cell-free serum. As internal control a synthetic miRNA, UniSp6, was added to each sample prior to extraction. miRNA expression levels were measured by real-time RT-PCR and presented as fold difference (arbitrary units, U) from control. Sera from these individuals had been previously analyzed for hormone and cytokine levels. MAIN RESULTS AND THE ROLE OF CHANCE: Serum levels of miR-155 were associated with levels of miR-146a (P < 0.0001), but only miR-146a was associated with inflammatory markers. miR-155 was strongly associated with subfertility (for subfertile group 1.88 U, 95% confidence interval (CI) 1.6-2.1 U versus 1.15, 95% CI 1.0-1.2 U in controls; P = 0.001). Receiver operating characteristic curve analysis indicated that miR-155 but not miR-146a can be used as a marker of subfertility. MiR-155 with a cutoff value of 1.77 had 47% sensitivity and 95% specificity for identifying subfertility and a positive predictive value (PPV) and negative predictive value (NPV) of 95 and 47%, respectively. When used in combination with FSH, sensitivity and specificity were 80 and 100%, respectively, while PPV and NPV were 100 and 71%, respectively, those values being higher than for the FSH alone. Repeating the results obtained in the exploratory cohort in an independent confirmatory cohort reduced the risk of a chance finding. LIMITATIONS, REASONS FOR CAUTION: Although the results from the exploratory cohort were confirmed in the confirmatory cohort, studies from other centers are needed to establish the role of miR-155 as a new biomarker of male fertility. Furthermore, the role of this marker in distinguishing between different groups of male subfertility is to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Association of the inflammatory miRNA miR-155 with male fertility contributes to our understanding of the pathophysiology of subfertility and suggests a novel biomarker. Serum miR-155 in combination with FSH has higher diagnostic specificity and sensitivity compared with FSH alone. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from Swedish Governmental Grant (ALF), Skane county council research and development foundation, Skane University Hospital Fonds and by the EU and Greek funds under the action 'Education and lifelong learning' program THALIS-FAT-VESSEL (No 379527). The authors have no competing interests to disclose.
STUDY QUESTION: Are serum levels of micro-RNAs miR-155 and miR-146a associated with male fertility, low-grade systemic inflammation (LGSI) and androgens? SUMMARY ANSWER: miR-155 was associated with male subfertility independent of LGSI or androgens while miR-146a was only weakly associated with subfertility and LGSI. WHAT IS KNOWN ALREADY: Male subfertility has been associated with LGSI as well as with androgen deficiency. miR-155 and miR-146a are central regulators of inflammation and their level in cells and in the serum has been associated with several inflammatory conditions. STUDY DESIGN, SIZE, DURATION: In this case-control study, two independent groups of 60 subjects each (exploratory and confirmatory cohort) were randomly selected from an ongoing study on subfertile men (in total: hypogonadal; n = 40, eugonadal; n = 40 and control group n = 39) at a University Hospital Reproductive Medicine Centre. Individuals were matched for age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from cell-free serum. As internal control a synthetic miRNA, UniSp6, was added to each sample prior to extraction. miRNA expression levels were measured by real-time RT-PCR and presented as fold difference (arbitrary units, U) from control. Sera from these individuals had been previously analyzed for hormone and cytokine levels. MAIN RESULTS AND THE ROLE OF CHANCE: Serum levels of miR-155 were associated with levels of miR-146a (P < 0.0001), but only miR-146a was associated with inflammatory markers. miR-155 was strongly associated with subfertility (for subfertile group 1.88 U, 95% confidence interval (CI) 1.6-2.1 U versus 1.15, 95% CI 1.0-1.2 U in controls; P = 0.001). Receiver operating characteristic curve analysis indicated that miR-155 but not miR-146a can be used as a marker of subfertility. MiR-155 with a cutoff value of 1.77 had 47% sensitivity and 95% specificity for identifying subfertility and a positive predictive value (PPV) and negative predictive value (NPV) of 95 and 47%, respectively. When used in combination with FSH, sensitivity and specificity were 80 and 100%, respectively, while PPV and NPV were 100 and 71%, respectively, those values being higher than for the FSH alone. Repeating the results obtained in the exploratory cohort in an independent confirmatory cohort reduced the risk of a chance finding. LIMITATIONS, REASONS FOR CAUTION: Although the results from the exploratory cohort were confirmed in the confirmatory cohort, studies from other centers are needed to establish the role of miR-155 as a new biomarker of male fertility. Furthermore, the role of this marker in distinguishing between different groups of male subfertility is to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Association of the inflammatory miRNA miR-155 with male fertility contributes to our understanding of the pathophysiology of subfertility and suggests a novel biomarker. Serum miR-155 in combination with FSH has higher diagnostic specificity and sensitivity compared with FSH alone. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from Swedish Governmental Grant (ALF), Skane county council research and development foundation, Skane University Hospital Fonds and by the EU and Greek funds under the action 'Education and lifelong learning' program THALIS-FAT-VESSEL (No 379527). The authors have no competing interests to disclose.
Authors: Dag Eckersten; Christos Tsatsanis; Aleksander Giwercman; Laila Bruun; Mats Pihlsgård; Anders Christensson Journal: Nephron Extra Date: 2017-04-07