Gut microbiome influences uterine development in mice

Diisononyl phthalate (DiNP), a plasticizer increasingly replacing di(2-ethylhexyl) phthalate, is an endocrine-disrupting chemical linked to female reproductive harm. Ingestion is the most common route of DiNP exposure, making the gastrointestinal tract and gut microbiome a direct target for endocrine-disrupting chemical exposure. This study examined the effects of acute DiNP exposure either in the absence or presence of a gut microbiome on uterine development. Female C57Bl/6 germ-free (-microbiome) 40-day-old mice were orally dosed, over 3 days, with either sterile phosphate-buffered (n = 8) to remain germ-free (GF, -microbiome) or with colon contents (n = 10) to develop a gut-microbiome (+microbiome). This was followed by a 10-day period where half of the -microbiome and +microbiome mice were orally dosed with corn oil while half were orally dosed with 200 μg/kg/day DiNP. The control group were specific pathogen-free conventionally housed mice born with a microbiome. Mice were euthanized in diestrus at the end of the 10 days. Uteri were collected for histological analyses. Uterine development was significantly delayed in GF mice, regardless of later microbiome reintroduction or DiNP exposure. Key findings included reduced uterine diameter, stroma area, and gland number, and thinner myometrial layers. Endometrial stromal cell proliferation was also lower in GF mice. DiNP exposure alone showed no significant effects. Estradiol levels and ovarian follicle counts were similar across groups, but GF mice had fewer, smaller litters in fertility tests. The study highlights that the gut microbiome critically influences postnatal uterine development, with its absence leading to persistent structural deficits. DiNP, at the tested dose, did not exacerbate these effects.