"The testis remembers”: lifestyle-based weight loss partially rescues fertility despite testicular metabolic memory of early-life obesity

Childhood obesity is a risk factor for male infertility, creating the need for non-pharmacological therapeutic strategies. Thus, we aimed to assess whether lifestyle-based strategies mitigate the negative metabolic and reproductive effects of early-life obesity. We developed a Mus musculus model, divided into five cohorts: control (CTRL), control diet and baseline activity (200 days); diet-induced obesity (DIO), lifelong high-fat diet (HFD) and baseline activity (200 days); DIO with dietary correction (DIOc), HFD (60 days) followed by a switch to control diet (140 days); DIO with aerobic exercise (DIO_EX), lifelong HFD (200 days), treadmill exercise during the final 140 days; DIO with dietary correction and aerobic exercise (DIOc_EX), HFD (60 days) followed by a switch to control diet and treadmill exercise (140 days). Body and organ weight were recorded. Intraperitoneal glucose and insulin tolerance tests (ipGTT and ipITT) were performed. Estradiol (E₂) and testosterone (T) were measured through ELISA. Hematoxylin-eosin-stained sections were examined for testicular histological characterization. Testicular metabolomes were characterized through untargeted ¹H-NMR metabolomics. DIO established a phenotype of metabolic syndrome (glucose intolerance) and reproductive decline (elevated E₂/T and poor sperm quality). While all interventions normalized the E₂/T ratios, diet-based interventions (DIOc and DIOc_EX) were superior in reducing weight, restoring glucose homeostasis, and testicular histoarchitecture. Sperm parameters showed the greatest rescue in the exercise-based groups (DIO_EX and DIOc_EX). Metabolomic profiling revealed a "testicular metabolic memory", characterized by a depletion of energy and antioxidant metabolites that persisted despite weight loss. Our data demonstrates that functional reproductive recovery can occur despite the altered testicular metabolome, suggesting that sperm quality may improve without complete metabolic recovery. Our results also support a "Two-Hit" recovery model, in which diet improves systemic damage and exercise improves sperm quality. This establishes multimodal lifestyle-based therapies as a mechanistically superior framework for addressing obesity-related subfertility.