Dear Editor,Metformin, the first-line therapy for type 2 diabetes1,2, is increasingly used not only in diabetic patients but also being actively explored for cardiovascular risk reduction, anti-neoplastic effects, with real-world adoptions in healthy individuals for metabolic risk management and healthy aging3,4,5. While the Paternal Origins of Health and Disease (POHaD) paradigm posits that preconceptional environmental exposures in fathers can reprogram offspring health through epigenetic modifications in sperm6,7,8,9, the intergenerational consequences of paternal metformin use remain elusive. Recent clinical debates have focused on whether paternal metformin exposure increases the risk of genital malformations in male offspring; however, findings remain conflicting, with a Danish registry study reporting a positive association10 while larger multinational cohorts found no such link11. Crucially, against the backdrop of the POHaD paradigm, beyond the risk of congenital malformations, the intergenerational impact of paternal metformin use on offspring metabolism, particularly via epigenetic mechanisms, remains unknown.To investigate the transgenerational impact of paternal metformin treatment, we administered metformin (Met, 200 mg/kg) or vehicle (Veh, equivalent volume of saline) to 8-week-old male Sprague-Dawley rats for 21 days and mated them with untreated females to generate offspring (Fig. 1a). We observed no differences in litter size, sex ratio, or birth weight between the Met-F1 and Veh-F1 groups (Supplementary Fig. S1a–c). However, Met-F1 offspring of both sexes exhibited significantly higher body weight since week 8, compared to Veh-F1 offspring (Fig. 1b, left). By week 15, Met‑F1 offspring exhibited a significant right shift in body weight Z‑score distribution. The incidence of overweight (Z‑score ≥ 2) reached 50.0% in males and 45.8% in females, compared to only 4.2% and 0.0% in Veh‑F1 males and females, respectively (Fig. 1b, right). MRI-based body composition analysis confirmed that this increased body weight in both sexes was due to a specific increase in fat mass, with no change in lean mass (Fig. 1c).Fig. 1: Paternal metformin induces intergenerational metabolic dysfunction in F1 offspring.Full size imagea Schematic of the experimental design for paternal metformin treatment, drug washout, and transgenerational transmission. b Left, body weight of F1 male and female offspring (Veh-F1, n = 24 from 12 litters; Met-F1, n = 24 from 12 litters). Right, distribution of body weight Z‑scores at 15 weeks, with density curves colored by Z‑score values. c MRI-based quantification of fat mass (left) and lean mass (right) in F1 male and female offspring (Veh-F1, n = 5 from 5 litters; Met-F1, n = 5 from 5 litters). d IPGTT curves and corresponding AUC for F1 male and female offspring (Veh-F1, n = 12 from 12 litters; Met-F1, n = 12 from 12 litters). e Fasting insulin levels and HOMA-IR in F1 male and female offspring (Veh-F1, n = 6 from 6 litters; Met-F1, n = 6 from 6 litters). f Left, body weight of F1 male and female offspring generated after 3-week drug withdrawal (Veh-WD-F1, n = 12 from 6 litters; Met-WD-F1, n = 12 from 6 litters). Right, distribution of body weight at 15 weeks, with density curves shaded by Z-scores. g IPGTT curves and AUC for F1 male and female offspring generated after 3-week drug withdrawal (Veh-WD-F1, n = 6 from 6 litters; Met-WD-F1, n = 6 from 6 litters). h Left, body weight of F2 male and female offspring (Veh-F2, n = 10 from 5 litters; Met-F2, n = 12 from 6 litters). Right, distribution of body weight at 12 weeks, with density curves shaded by Z-scores. i IPGTT curves and AUC for F2 male and female offspring (males: Veh-F2, n = 10 from 10 litters, Met-F2, n = 11 from 11 litters; females: Veh-F2, n = 7 from 7 litters, Met-F2, n = 10 from 10 litters). j Circular heatmap showing correlations between F0 sperm DNA methylation and body weight (BW) or glucose tolerance (GTT AUC) in male and female F1 offspring. k Upper, heatmap of metformin-induced differentially methylated regions (DMRs) in F0 sperm. Lower, ranking plot of methylation changes at metformin-associated DMRs across two comparisons: Met-F0 vs Veh-F0 (metformin effect), Met-WD-F0 vs Met-F0 (withdrawal effect). l Chromosomal distribution of metformin-induced DMRs. Regions associated with rat imprinted genes (green) or the tryptophan pathway (red) are highlighted. m KEGG pathway enrichment analysis of genes overlapping metformin-induced DMRs. n Heatmap of 53 significantly altered metabolites identified by untargeted plasma metabolomics in F1 offspring at 17 weeks (males: Veh-F1, n = 4 from 4 litters, Met-F1, n = 4 from 4 litters; females: Veh-F1, n = 4 from 4 litters, Met-F1, n = 4 from 4 litters). Selection criteria: VIP > 1, P 1.2 or