Introduction: Previous studies have demonstrated that heavy metal exposure can negatively impact male infertility and testicular carcinogenesis due to increased oxidative stress. This can lead to molecular dysfunction, part of which is mediated by small non-coding RNA known as miRNA, which may serve as potential biomarkers of disease. We present a miRNA analysis following thallium exposure to three-dimensional human testicular organoids (HTOs). Methods: 3D HTOs were created from 2D testicular cell cultures derived from three brain-dead adult patients. HTOs were exposed to either thallium or ultrapure water. The miRNA analysis was performed using the Nanostring nCounter Human v3b miRNA panel, representing 827 unique miRNA probes. Nanostring nSolver 4.0 was used to execute target normalization with the geometric mean of the top 100 miRNAs detected, followed by background subtraction of 57 counts, which was the average of exogenous negative controls in the panel plus two standard deviations. All miRNAs with average counts <5 within a sample group were excluded to ensure robust expression. Qiagen Ingenuity Pathway Analysis (IPA) miRNA target filter was used to select for experimentally observed mRNAs associated with reproductive pathology and testis cells. Results: 9 miRNAs were uniquely expressed within the thallium HTOs compared to 3 in the control. Seven of the 107 miRNAs expressed in both were significantly differentially expressed (miR-574-3p, miR-374a-5p, miR-423-5p, miR-106a-5p+miR-17-5p, miR-29b-3p, and miR-26b-5p were upregulated; miR-145-5p was downregulated). However, none survived the application of the false discovery rate. IPA miRNA target filter showed that these miRNAs regulated 161 mRNAs, including BCL2, MYC, CDKN1A, CCNA2, CCND1, and E2F1. Conclusions: Certain miRNAs have previously been linked to male infertility (miR-145-5p, miR-574-3p, miR-26b-5p), asthenozoospermia (miR-423-5p), and testicular diffuse large B-cell lymphoma (miR-17-5p). Previous studies have also demonstrated that oxidative stress from thallium-containing compounds can induce apoptosis by affecting Bcl-2 (BCL2) expression and activate nucleolar stress by altering c-Myc (MYC) and p21 (CDKN1A) expression. Thallium has also been shown to modify cell cycle progression through cyclin A (CCNA2), cyclin D1 (CCND1), and E2F-1 (E2F1). Although only 4 of these miRNAs have been implicated in male infertility and testicular cancer, further studies may elucidate other miRNAs and their downstream regulation of mRNA. SOURCE OF Funding: N/A
