Endocrine Abstracts

Background/Aims: Sex development at a cellular level involves complex interactions between chromosomal, hormonal, and cellular factors. Differences in sex development (DSD) provide a unique opportunity to explore how different factors influences metabolism. This study aims to investigate metabolic profiles of 46, XX fibroblasts, 46,XY fibroblasts and fibroblasts from DSD individuals. Extensive replication of the fibroblasts will lead to a senescent state of the cells that is characterized by permanent cell cycle arrest, altered morphology, and changes in metabolic activity. This will allow to investigate the effect of population doubling levels on metabolism.

Methods: Skin fibroblasts from 2 individuals with androgen insensitivity, and six 46, XX and six 46, XY fibroblasts are cultured under defined conditions. So far, cells have been harvested at a proliferating state. and intracellular metabolites were extracted using a chloroform/methanol protocols. Metabolomic profiles have been obtained using nuclear magnetic resonance spectroscopy and analysed using principal component analysis (PCA) to identify patterns associated with chromosomal state.

Results: Buffer preparation and metabolite extraction protocols were optimized to yield reproducible intracellular metabolite profiles, minimizing technical variability. Intracellular metabolomic profiling was performed so far on four fibroblast pellets. Principal component analysis (PCA) of these preliminary samples revealed metabolic variability among fibroblast lines at comparable proliferation stages. All cells were in a proliferative state, and none had reached senescence. Two fibroblast lines exhibited tighter clustering, whereas one showed greater intra-line variability, likely reflecting interindividual or donor-specific differences.

Conclusion: This project aims to investigate metabolic commonalities and differences in a human cellular model to better understand variations in sex development. Ongoing work will focus on expanding the NMR metabolomics analysis to additional fibroblast lines and biological replicates, including cells at later proliferation stages and replicative senescence. Furthermore, planned bulk RNA sequencing will allow integration of metabolomic and transcriptomic data to identify pathways associated with sex differences and DSD conditions. These next steps will help to better understand how chromosomal background and cellular aging influence metabolic regulation.