Abstract
About 10–15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, a male factor plays a role. Levels of particular microRNAs in seminal plasma, including those involved in spermatogenesis, may serve as an indicative parameter for subfertility. We first optimized a protocol for acquiring microRNAs from seminal plasma. Next, using a test-validation strategy in a male cohort, we aimed to identify microRNAs of which the levels are related to semen motility and concentration. By qPCR, 742 microRNAs were profiled in three normozoospermic samples, three seminal samples with a low semen motility (asthenozoospermia), and three with a low semen concentration (oligozoospermia). MicroRNAs showing significant differences between groups were further validated in a second cohort consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (of which 74% also low motility). Highest microRNA yields were obtained with the Biofluids RNA extraction kit, with inclusion of MS2 RNA carrier and proteinase K treatment to the protocol, and when 50 µL of seminal plasma was used as input. Exosome isolation prior to RNA extraction did not lead to enhanced yields. In the test cohort, 236 microRNAs could be detected, of which 54 microRNAs showed a difference between groups. Five microRNAs were analyzed in the validation cohort. MiR-34b-5p levels in the control group were significantly higher compared to the asthenozoospermia group (p < 0.05) and compared to the oligozoospermia group (p < 0.001). We optimized microRNA acquirement from seminal plasma and identified microRNA levels in relation to semen concentration and motility. As recent human and mouse studies show that the miR-34 family is a marker of low semen concentration and is crucial in spermatogenesis, seminal plasma miR-34b-5p may represent a suitable candidate to study further as a marker of male subfertility.
Highlights
Spermatogenesis is the development of spermatozoa in males
We have optimized a protocol for microRNA assessment in seminal plasma, and we have shown in seminal plasma samples of a cohort of men that levels of microRNAs are related to semen quality
A previous study where microRNAs were extracted from blood plasma using various RNA extraction procedures showed that the usage of a Biofluids kit gave superior results compared to the other approaches [15]
Summary
Spermatogenesis is the development of spermatozoa in males. Semen consists of many biochemical molecules and is produced and released by specific organs of the male genital system, including seminiferous tubules, epididymis, and accessory glands (seminal vesicles, prostate, and urethral glands). Male subfertility is responsible for 30% of cases. Different forms of subfertility can be distinguished. The ejaculate contains a low concentration of spermatozoa, whereas azoospermia is a condition where the semen contain no spermatozoa. This latter condition can be distinguished in non-obstructive azoospermia (disturbed production of spermatozoa) and obstructive azoospermia (obstructed reproductive tract). Asthenozoospermia is a condition whereby the sperm has reduced motility
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