Single-Cell Metallomics of ovarian granulosa cells enables assessment of Low-Level heavy metal exposure associated with impaired human oocyte development.

The oocyte microenvironment, comprising ovarian granulosa cells (GCs) and follicular fluid (FF), sustains oocyte development with nutrients and regulatory signals but may also transfer heavy metals to the oocyte. While high concentrations of heavy metals in FF and blood have been associated with impaired oocyte development, the effect of low-level exposure on the oocyte remains unclear, partly due to the analytical challenges in measuring low-level heavy metals in biological fluids. Here, we investigated the associations between low-level heavy metal exposure and oocyte development. Using a highly sensitive single-cell metallomic technique, we analyzed ovarian GCs collected from women undergoing in vitro fertilization and embryo transfer (IVF-ET) to serve as a biomarker of intracellular heavy metal burden in the peri-oocyte microenvironment. The associations between heavy metals in GCs and reproductive outcomes across multiple key reproductive stages were analyzed. Our results showed that the heavy metals content in GCs were generally below 0.5 fg/cell. Higher mean intensities and prevalence of several metals in GCs were significantly associated with reduced oocyte maturation rate, normal fertilization rate, and good-quality embryo rate (all adjusted P < 0.05). Elevated intracellular levels of lead and mercury were also associated with adverse reproductive outcomes and increased risk of early miscarriage. In contrast, higher heterogeneity of Hg within GCs was associated with reduced miscarriage risk (OR = 0.37 [0.04, 0.92], P = 0.034). Notably, these associations are significantly contributed by intercellular heterogeneity of metal accumulation within the GC population. Our study demonstrates the feasibility of applying single-cell metallomic technique to ovarian GCs to assess the reproductive risk associated with low-level intracellular heavy metal burden in ovarian follicles, and underscores the need for further reducing environmental heavy metal exposure to protect female reproductive health.