Sample recruitment for this study received ethical approval from Guizhou Provincial People's Hospital (Ethical Review Approval NO. [2019]61). All participants provided informed consent by signing forms that detailed the collection of semen specimens and the use of data for scientific research, publication, and public sharing. This ensured participants had full disclosure throughout the study. Strict inclusion and exclusion criteria were applied during recruitment. Males with asthenospermia were included and monitored continuously for 3–5 months. Exclusion criteria included a history of radiation exposure, prolonged sitting, familial infertility, relevant medication use, tumors, reproductive developmental anomalies, or organic lesions. Normal patients underwent semen vitality monitoring 2–3 times, with exclusion criteria including familial genetic history. Additionally, all participants abstained from ejaculation for 2–3 days before sample collection. Monitoring procedures adhered strictly to the guidelines outlined in the "WHO Laboratory Manual for the Examination and Processing of Human Semen (Fifth Edition)."
Following routine testing, sperm samples were transferred into a 15 ml centrifuge tube and combined with 10 ml of pre-chilled PBS at 4°C. The mixture was repeatedly pipetted and then centrifuged at 3000 rpm for 10 minutes. After centrifugation, the supernatant was carefully decanted. This washing and centrifugation process was repeated until the supernatant was clear, indicating the absence of sperm. If sperm cells were still present, the centrifugation force was increased until complete sedimentation was achieved.
Total RNA extraction was performed using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The purity and concentration of the extracted RNA were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The criteria for RNA quality assessment included an RNA Integrity Number (RIN) greater than 6 and a 28S/18S ratio greater than 0.7. Library preparation was then carried out using the VAHTS Universal V6 RNA-seq Library Prep Kit, following the manufacturer’s instructions. The library standards included a main peak band of 300-350bp, a library size range of 200-500bp, no primer dimers or small fragment residues, and a total amount above 10ng.
The libraries were sequenced on an Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. Approximately 47 million raw reads were generated per sample. The raw reads in fastq format underwent preprocessing using fastp12 to remove low-quality reads, resulting in the extraction of clean reads. Subsequently, around 47 million clean reads per sample were retained for further analysis. These clean reads were then mapped to the reference genome using HISAT213. FPKM (fragments per kilobase of transcript per million mapped reads)14 values for each gene were calculated, and read counts for each gene were obtained using HTSeq-count15.
Data Records
Eighty-three semen samples were collected from 28 males with asthenospermia (A) and 55 with normal sperm motility (N). Based on conventional RNA-seq quality standards (RIN and 28S/18S ratio), 37 semen samples were sequenced. Fifteen samples (Set 1: 8 A, 7 N) met these criteria (RIN > 6, 28S/18S > 0.7), while 22 additional samples (Set 2: 11 A, 11 N) approached the standards (RIN 5–6, 28S/18S > 0.7). Each sample's total RNA was labeled with a unique anonymized identifier to protect patient privacy. RNA sequencing data for Set 1 has been deposited in the GSA-Human database (accession HRA006250). Set 2 data is also accessible through GSA-Human (accession HRA006906).
Technical Validation
Table 1 summarizes the 37 sequenced semen samples (out of 83) based on RNA quality control criteria (pass/fail) and experimental group (A or N). Of these, 19 samples (68%) from group A (asthenospermia) and 18 (33%) from group N (normal motility) were sequenced. These data suggest that sperm cells from individuals with asthenospermia tend to have higher RNA content than those with normal sperm motility.
Table 1
| Group / QC* | Pass (%) | No Pass (%) | Total in the study |
| A* | 8(28.57) | 11(50.00) | 28 |
| N* | 7(12.73) | 11(20.00) | 55 |
| Total | 15 | 22 | 83 |
* The A group comprises individuals diagnosed with asthenospermia, while the N group comprises individuals with normal sperm parameters. QC: RNA quality control, including an RNA Integrity Number (RIN) greater than 6 and a 28S/18S ratio exceeding 0.7.
We conducted t-tests on various parameters, including age, monitored sperm count, volume, sperm concentration, total sperm count, total motility, and forward motility ratio, comparing the asthenospermia group (A) to the normal group (N) (see Table 2). These comparisons did not reveal significant differences in most variables between the two groups, except for total motility and forward motility parameters (P values < 0.000001). These results confirm that our sample collection effectively represents both asthenospermia and normal controls.
Table 2
Clinical information for 37 samples.
| group | A* | N* | p-value |
| Samples number | 19 | 18 | NA |
| Age (year) | 33.2 ± 5.8 | 30.8 ± 5.3 | 0.26 |
| Volume (mL) | 2.9 ± 1.2 | 3.0 ± 1.4 | 0.89 |
| total test sperm | 480.8 ± 232.4 | 526.2 ± 135.4 | 0.56 |
| Concentration (ng/µL) | 53.5 ± 26.6 | 64.8 ± 16.7 | 0.21 |
| total sperm | 147.8 ± 65.2 | 189.0 ± 101.7 | 0.26 |
| total motility (%) | 21.9 ± 7.3 | 52.0 ± 14.3 | 4.70E-07 |
| forward moving (%) | 19.2 ± 606 | 47.3 ± 13.3 | 3.82E-07 |
* The A group comprises individuals diagnosed with asthenospermia, while the N group comprises individuals with normal sperm parameters.
Usage Notes
Please register and log in at GSA-Human (https://ngdc.cncb.ac.cn/gsa-human), then search for data numbers HRA006250 and HRA006906. Click "Apply for Data" and complete the official Data Use Agreement online. Your application will be sent to the Data Access Committee (DAC) for review, and upon approval, you will receive a direct download link via email.