P-064 MicroRNAs as potential biomarkers for male infertility related to Testicular Germ Cell Tumors
Authors: Ferrara C , Battaglia R , Caponnetto A , Barbagallo C , Stella M , Vento M E , Borzì P , Scollo P , Lunelio F , Barbagallo D , Pernagallo S , Ragusa M , Di Pietro C 
Published: June 2023

Abstract

Study question
Could microRNAs represent efficient biomarkers to evaluate male infertility and the tendency of develop testicular germ cell tumors (TGCTs)?

Summary answer
The up-regulation of miR-193a-5p, miR-93-5p and let-7c-5p in TGCT patients with impaired spermiogram could represent a molecular signature of male infertility related to TGCTs.

What is known already
Testicular germ cell tumors (TGCTs) are the most recurrent tumors in young men with the highest incidence between the ages of 20 and 40 years and represent more than 60% of all cancers diagnosed in this age range. Several studies have shown a correlation between TGCTs and infertility, not only because infertility could be a consequence of testicular damage due to TGCTs but also because infertility may represent a risk factor for TGCTs. Therefore, it is important to find valid biomarkers that could be used to specifically identify infertility related to an increased risk of developing TGCTs.

Study design, size, duration
From March 2021 to January 2023, we collected 44 seminal plasma samples: 17 from TGCT patients who were undergoing sperm cryopreservation prior to chemotherapy, 14 with impaired spermiogram (IS) and 3 with normal spermiogram (NS) and 27 from control patients, 16 with IS and 11 with NS, undergoing assisted fertilization techniques. We evaluated the potential role of miRNAs as non-invasive biomarkers, analyzing the differential expression of 84 miRNAs in relation to cancer, infertility and both.

Participants/materials, setting, methods
Semen samples were placed 30’/37 °C, seminal plasma was purified using density-gradient centrifugation and stored at -80 °C. RNA was purified by Qiagen miRNeasy Serum/Plasma Kit and analyzed by miRCURY LNA miRNA SYBR®Green PCR_SerumPlasma, 96-well plate. We applied the 2−ΔΔCT method and statistical significance was evaluated by Significance Analysis of Microarrays, screened by p-values ≤ 0.05. We performed a Pearson correlation analysis applying two-sided p-values. miRTarBASE, MIENTURNET and Cytoscape were used for bioinformatics analysis.

Main results and the role of chance
In the four comparisons, we found 9 differentially expressed microRNAs. In particular, miR-221-3p, miR-222-3p, miR-204-5p and miR-205-5p down regulation would appear to be related to infertility, regardless to the cancer. Conversely, all TGCT patients shown an up-regulation of miR-376c-3p. Interestingly, up-regulation of miR-193a-5p, miR-93-5p and let-7c-5p specifically discriminates the infertile cancer patients vs other categories. Moreover, these three miRNAs showed a significant positive correlation both in cancer patients with impaired spermiogram and in infertile controls. This correlation demonstrates that their expression changes in the same way in the single samples and strongly suggests their role as early biomarkers of TGCT patients with impaired spermiogram. From the KEGG pathway analysis has emerged that DE miRNAs are involved in several signaling pathway, such as MAPK, mTOR, p53, PI3K-Akt, AMPK, FoxO, JAK-STAT, Ras, Estrogen signaling pathways, as well as cell cycle and cellular senescence. Moreover, from the Diseases Ontology analysis we found than those miRNAs are involved in germ cell tumor, male reproductive organ cancer, prostate cancer, male infertility and azoospermia.

Limitations, reasons for caution
The data must be validated by single assays in higher number of samples. These experiments are currently ongoing.

Wider implications of the findings
Three of the DE miRNAs are overexpressed in TGCT with impaired spermiogram patients. Specifically, they are over-expressed in TGCT with impaired spermiogram compared to infertile controls, so they may represent specific biomarkers able to discriminate, among the young men with infertility, those with higher risk of develop testicular cancer.

Trial registration number
Not applicable