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Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as
Michael Eikmans1, Jacqueline D H Anholts1, Laura Blijleven1
1Department of Immunohematology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
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.
Insights
Researchers identified specific microRNAs in seminal plasma linked to male subfertility. Optimized methods for microRNA extraction revealed miR-34b-5p levels correlate with semen quality, suggesting its potential as a diagnostic marker.
Area of Science:
- Reproductive biology
- Molecular diagnostics
- Biochemistry
Background:
- Male subfertility affects 10-15% of couples trying to conceive, with male factors contributing to 30% of cases.
- MicroRNAs (miRNAs) in seminal plasma are potential biomarkers for male reproductive health and spermatogenesis.
- Identifying reliable biomarkers is crucial for diagnosing and managing male subfertility.
Purpose of the Study:
- To optimize the protocol for extracting microRNAs from seminal plasma.
- To identify specific microRNAs in seminal plasma associated with semen motility and concentration.
- To evaluate the potential of identified microRNAs as diagnostic markers for male subfertility.
Main Methods:
- Optimized microRNA extraction from seminal plasma using the Biofluids RNA extraction kit with MS2 RNA carrier and proteinase K.
- Utilized quantitative polymerase chain reaction (qPCR) to profile 742 microRNAs in test and validation cohorts.
- Analyzed seminal plasma samples from normozoospermic, asthenozoospermic, and oligozoospermic individuals.
Main Results:
- The optimized protocol yielded high amounts of microRNA without exosome isolation.
- Out of 236 detected microRNAs, 54 showed significant differences between groups in the test cohort.
- In the validation cohort, miR-34b-5p levels were significantly higher in normozoospermic individuals compared to asthenozoospermic and oligozoospermic groups.
Conclusions:
- Optimized microRNA extraction from seminal plasma is feasible and effective.
- Seminal plasma miR-34b-5p levels are significantly associated with semen quality parameters.
- miR-34b-5p is a promising candidate biomarker for assessing male subfertility.
