Abstract
Abstract
Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with adverse female reproductive outcomes. However, most prior studies examined individual PFAS, despite that humans are exposed to complex PFAS mixtures. Here, we investigated the ovarian effects of a mixture of five commonly detected long-chain PFAS, including PFOS, PFOA, PFNA, PFDA, and PFHxS, using complementary
in vivo
mouse and
in vitro
3D ovarian follicle culture models. Young adult female mice were exposed to a range of concentrations of the PFAS mixture (PFASmix) through daily drinking water for 8 weeks. PFASmix at high yet environmentally relevant concentrations (PFASmix-high) disrupted mouse estrous cyclicity, decreased circulating estradiol, increased testosterone, and promoted follicular atresia. Antral follicles from exposed mice showed decreased
Lhcgr
and increased
Amh
expression. Although the number of ovulated oocytes was unchanged following superovulation, PFASmix-high reduced oocyte size and polar body extrusion and altered oocyte cytoskeletal characteristics. In a 3D mouse ovarian follicle culture model, direct PFASmix exposure impaired follicle growth, ovulation, and oocyte maturation in a concentration-dependent manner. PFASmix-high suppressed the periovulatory expression of
Areg, Ereg, Tnfaip6,
and
Adamts1
and impaired corpus luteal (CL) development and function, as evidenced by reduced CL spheroid growth and cell survival, decreased expression of
Star, Cyp11a1, Hsd3b1,
and
Lhcgr
, and reduced progesterone secretion. PFASmix-high exposure additionally altered the expression of multiple PPARγ-associated genes in cultured ovarian follicles. Benchmark dose modeling identified estrous cyclicity and follicle rupture as sensitive
in vivo
and
in vitro
endpoints, respectively. Together, these findings demonstrate that exposure to the mixture of long-chain PFAS disrupts multiple stages of ovarian function, from follicular development, oocyte maturation to ovulation and luteal function, providing mechanistic and quantitative evidence for the reproductive toxicity of PFAS mixtures.