Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring

{"Background": "Folate is essential for nucleotide synthesis and methylation reactions. Maternal folate deficiency causes neural tube and craniofacial defects, and methotrexate (MTX) is a known teratogenic folate antagonist. While maternal MTX use is contraindicated during pregnancy, the paternal influence of MTX on offspring was largely unknown. Recent evidence shows that sperm carry small non-coding RNAs (sncRNAs), particularly tRNA-derived small RNAs (tsRNAs) and miRNAs, which can transmit paternal experiences to offspring. This study investigated whether paternal MTX exposure affects offspring development through changes in sperm sncRNA abundance and methylation.", "Methods": "Adult male medaka fish were injected intraperitoneally with MTX at 10 mg/kg or 50 mg/kg body weight, or control solution (PBS/1% DMSO). After 7 days, sperm was collected for in vitro fertilization of wild-type oocytes and for small RNA extraction. Offspring were analyzed at 3 days post-hatching using alcian blue staining to measure cranial cartilage lengths and morphology. Small RNAs from sperm were sequenced (18-30 nt), and differential expression of sncRNAs, including 5' and 3' tRNA halves, was analyzed using DESeq2. RNA methylation status was assessed by UHPLC-MS-MS in 20-50 nt and 50-90 nt fractions. Expression of RNA methyltransferases (DNMT2, NSUN2, TRMT6) in testis was measured by RT-qPCR. Zygotic injection experiments were performed by injecting 20-50 nt, 50-90 nt, or combined 20-90 nt RNA fractions from MTX-treated and control sperm into fertilized wild-type eggs.", "Key Results": "Paternal MTX treatment did not affect sperm fecundity, hatching time, or overall embryo hatching. However, offspring from MTX-treated males showed significant craniofacial cartilage defects. The ceratohyal cartilage was reduced to almost half the length at both 10MTX (192.99 \u00b5m \u00b1 7.55, p < 0.0001) and 50MTX (185.42 \u00b5m \u00b1 8.71, p < 0.0001) compared with control (363.64 \u00b5m \u00b1 18.96). The anterior orbital cartilage was significantly reduced in the 50MTX group (115.02 \u00b5m \u00b1 9.03, p = 0.0164). Abnormal serpentine shape of the anterior orbital cartilage was significantly prevalent at 50MTX (p = 0.0059). Basihyal cartilage malformations (curved trowel or hook shape) were significantly increased at both 10MTX (p = 0.0329) and 50MTX (p = 0.0006). Sperm sncRNA sequencing revealed that tRNA fragments were the most affected population, with 5' tsRNA fragments of tRNA-Asp^GUC^, tRNA-Lys^CUU^, and tRNA-Glu^CUC^ significantly increased in MTX sperm without a concomitant increase in their 3' fragments. There was a shift towards longer tsRNA fragments in MTX-treated samples (median length 29 nt vs 24 nt in controls). UHPLC-MS-MS analysis of the 50-90 nt RNA fraction showed significantly increased m5C methylation (p = 0.0155), as well as m2G (p = 0.0027), m7G (p = 0.0063), and m2'2G (p = 0.0090) in MTX-treated sperm. DNMT2 expression was significantly upregulated in testis of MTX-treated males (p = 0.01), while NSUN2 and TRMT6 were not significantly changed. Zygotic injection of 20-50 nt, 50-90 nt, or combined 20-90 nt RNA fractions from MTX-treated sperm significantly reduced ceratohyal lengths (p < 0.0001) and increased basihyal cartilage abnormalities compared to controls.", "Clinical Implications": "This study provides evidence that paternal MTX exposure can cause craniofacial defects in offspring through changes in sperm small non-coding RNA content and modifications, independent of direct genetic mutations. The findings challenge the traditional view that paternal contribution to offspring health is limited to the haploid genome and suggest that men taking MTX (commonly used for rheumatoid arthritis, cancers, and dermatologic conditions) may need clearer preconception recommendations. The identification of DNMT2-mediated m5C methylation and specific tsRNA fragments as potential mediators of non-genetic inheritance opens avenues for understanding how paternal environmental exposures affect offspring development. The partial recapitulation of phenotypes through RNA injection supports a causal role for sperm RNAs. These results highlight the need for further research on paternal epigenetic contributions to congenital anomalies and may inform future clinical guidelines for men of reproductive age undergoing MTX therapy."}