Effects of phthalate exposure on human ovarian extracellular matrix composition: insights from a 3D spheroid model

Phthalates, widely used as plasticizers in consumer products, have become a significant public health concern due to their toxic effects on reproductive and endocrine systems. However, the specific mechanisms by which phthalates affect ovarian function remain poorly understood. To address this knowledge gap, we employed 3D spheroids derived from human ovarian stromal cells, which mimic the ovarian tissue. Since remodeling the ovarian extracellular matrix (ECM) is centrally involved in follicle growth, ovulation, and ovarian aging, we decided to study the impact of environmentally relevant phthalate mixtures (PM) on ECM components. Spheroids were generated from both reproductive-aged and menopausal ovarian tissues, then treated with PM for four days. Collagen deposition was assessed using picrosirius red staining, while immunofluorescence was used to evaluate the proliferation and deposition of collagen type VI, elastin, fibrillin-1, and elastin microfibril interfacer 1 (EMILIN-1). Our results revealed that PM exposure significantly increased collagen deposition (p < 0.0001) in spheroids from reproductive-aged ovaries, while reducing collagen VI levels (p < 0.05), potentially compromising the structural and functional integrity of the ovarian ECM. In contrast, spheroids from menopausal ovaries exhibited a decrease in EMILIN-1 (p < 0.05) and fibrillin-1 (p < 0.001), both crucial for maintaining tissue elasticity. These findings underscore the detrimental effects of phthalates on ovarian ECM across different age groups, with a particular emphasis on ECM elastisity. Additionally, this study highlights the utility of 3D spheroids as a reliable in vitro model for mechanistic research, drug screening and toxicology testing.