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.

中文翻译：

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从精浆中获取microRNA的优化和精液microRNA-34b的减少可作为低精液浓度的指标

想要怀孕的夫妇中约有10–15％患有亚生育力，而在这些情况中，有30％的原因是男性因素。精浆中特定microRNA的水平，包括那些与精子发生有关的水平，可以作为亚生育力的指示性参数。我们首先优化了从精浆中获取microRNA的方案。接下来，我们在男性队列研究中使用测试验证策略，旨在鉴定其水平与精液运动性和浓度相关的microRNA。通过qPCR，在三个精子生精样本，三个精液活力低的精液样本（无精子症）和三个精液浓度低的精子样本（少精子症）中分析了742个microRNA。在第二组中进一步验证了显示组之间显着差异的MicroRNA，该组由40个具有正常精子症的样本（对照组），47个具有弱精子症的样本和19个具有少精子症的样本（其中74％的运动性也较低）组成。使用Biofluids RNA提取试剂盒可获得最高的microRNA产量，并在方案中加入了MS2 RNA载体和蛋白酶K处理，并且当使用50 µL精浆作为输入时。RNA提取之前的外来体分离未导致产量增加。在测试队列中，可以检测到236个microRNA，其中54个microRNA显示出组之间的差异。在验证队列中分析了五个microRNA。对照组的MiR-34b-5p水平显着高于弱精子症患者（p <0。05）并与少精症组比较（p <0.001）。我们优化了从精浆中获得的microRNA，并确定了与精液浓度和运动性相关的microRNA水平。正如最近的人类和小鼠研究表明，miR-34家族是低精液浓度的标志物，在精子发生中至关重要，精浆中的miR-34b-5p可能是进一步研究男性不育症的合适人选。