Abstract
Disclosure: N.M. Pascuali: None. F.R. Tobias: None. A. Veiga-Lopez: None. The ovary is one of the first organs to undergo age-related decline, leading to reduced fertility and menopause. While ovarian aging hallmarks such as fibrosis and inflammation are well recognized, the role of lipids — key cellular components and hormone precursors — remains largely unexplored. To date, no studies have mapped ovarian lipids spatially in the context of aging. This study aimed to fill this gap by testing the hypotheses that ovarian lipid composition changes with aging and does so in a region-specific manner. Estrous cyclicity was recorded in young (2 mo), mature (6 mo), and reproductively old (10 mo) female mice (n=8/group), followed by assessment of ovarian fibrosis and spatial lipidomics using MALDI mass spectrometry imaging (MALDI-MSI). Ovarian sections were sprayed with matrices (9AA for negative mode, DHB for positive mode) and analyzed on a rapifleX MALDI Tissuetyper (20 µm resolution). Histological data was integrated with MSI to select regions of interest (follicles, corpora lutea, and stroma). Lipid abundance was assessed across ages by ANOVA. Spatial segmentation was performed to identify distinct lipid signatures. To enhance lipid identification in MALDI-MSI, an in-house ovarian lipid library was generated from 91 age- and strain-matched ovaries (21 - 220 days old) using LC-MS/MS. Ten-mo-old mice exhibited impaired estrous cyclicity and increased ovarian fibrosis, with a positive correlation between these features. Spatial lipidomics detected > 800 lipid features in negative mode and > 100 in positive mode. After manual curation and leveraging the in-house lipid library, ∼60% of those detected features were putatively assigned to specific lipids at the molecular species level. Over 50 lipid features exhibited significant age-dependent changes, including the phospholipid PE 16:0_18:0. However, most lipids were enriched in one ovarian compartment (either stroma, follicles or corpora lutea). Notably, most lipid abundances were dysregulated within specific regions in 10-mo-old mice. For instance, follicular levels of oleic acid, a highly-abundant fatty acid in the ovary, decreased in older mice, while phospholipids PC 33:0 and PC O-40:5 were downregulated in stroma and corpora lutea, respectively. Unbiased spatial segmentation aligned lipid clusters with ovarian structures and revealed age-dependent lipid shifts within distinct regions. In conclusion, these findings provide the first spatial lipidomics dataset of ovarian aging, demonstrating that lipid composition shifts both globally and in a region-specific manner in reproductively aged mice. Thus, the ovarian lipidome emerges as a dynamic and spatially organized feature of aging, with potential implications for reproductive longevity. Presentation: Sunday, July 13, 2025