Environmentally Relevant Effects of Benzyl Butyl Phthalate (Bbp) on Folliculogenesis, Follicular Growth, and Embryo Development

Abstract

The chemical benzyl butyl phthalate (BBP) has been studied for its endocrine-disrupting capabilities (Begum et al. 2020; Du et al. 2019), in human reproduction. Human exposure to BBP ranges from 2-300 µg/kg/day (Kohn et al. 2000) with approximately 2 ng/ml detected in follicular fluid (Sacha et al. 2021). Despite sharing a metabolite with DBP (Sicińska et al. 2022), a well-studied phthalate compound linked to several effects in females (Li et al. 2022), BBP has received very little attention. Consequently, an investigation on the effects of BBP on folliculogenesis, antral follicle survival, and pre-implanted embryos was executed to bridge existing knowledge gaps. First, the ability of BBP to influence folliculogenesis and early embryo development was evaluated. To evaluate folliculogenesis in vivo, archived histological ovarian samples from mice treated for 10 days with tocopherol-stripped corn oil (vehicle control) or three doses of BBP (3, 300, 1000 μg/kg/day) were used to classify and count ovarian follicles according to their developmental and health status. In vitro late folliculogenesis was evaluated using archived data from isolated mouse antral follicles (250 - 450 μm) cultured for 72 h in the presence of dimethylsulfoxide (DMSO; vehicle control) or increasing concentrations of BBP (0.001, 0.01, 0.1, 1, 10, 100, 500 μg/ml), and subjected to luminescence-based growth measurements every 24 h. Lastly, commercially acquired zygotes (one-celled embryos) were allowed to develop in vitro over 96 h in the presence of DMSO vehicle control or increasing concentrations of BBP (0.001, 0.01, 0.1, 1, 10 μg/ml), and their cleavage and blastocyst rate, developmental timelines, and morphological characteristics compared between treatment groups. Compared to control treatment, oral exposure to BBP did not alter the ovarian reserve as BBP treated mice had similar total follicle number and follicular health status as the vehicle-treated controls. In vitro antral follicle growth was mostly unaffected by BBP exposure except for a transient loss of fast-growing follicles at 48 h in the groups treated with BBP at 1, 100, and 500 µg/ml concentrations. Finally, BBP treatment in vitro did not alter cleavage or blastocyst rate, developmental timing, or blastocyst size and composition. These findings indicate that a short term exposure to human relevant levels of BBP does not affect in vivo folliculogenesis, antral follicle survival, and embryo development. However, transient growth pattern effects observed in vitro, suggest the existence of molecular effects on the fast-growing antral follicles. Even though BBP does not seem to increase reproductive toxicity by itself, a reduction in the use of plastic is still advised to prevent the negative impacts of other known phthalates on female reproduction.