Exploratory Assessment of Preconception Phthalate Exposure on Pregnancy and Offspring Health Outcomes in Mice

Abstract Understanding how endocrine-disrupting chemicals (EDCs) influence reproductive success requires attention to sensitive windows beyond gestation, including the understudied preconception period. In this exploratory pilot study, female CD-1 mice were exposed to a human-relevant phthalate mixture (200 µg/kg/day) for 30 days prior to mating. Although implantation and litter size were unaffected, exposed dams exhibited non-significant shifts in estrus cyclicity, spending more time in proestrus and less in metestrus. Maternal liver transcriptomics revealed persistent changes more than a month after exposure ceased, with differential expression of genes involved in mitochondrial metabolism, oxidative phosphorylation, and xenobiotic processing, suggesting long-term metabolic reprogramming in the absence of overt toxicity. Maternal effects coincided with developmental alterations at mid-gestation. At E14.5, fetuses from exposed dams were heavier, and placentas displayed expansion of the junctional zone, a region critical for endocrine function. This early growth enhancement reversed later in life, as exposed male offspring exhibited reduced adult body weight, consistent with altered developmental programming. Transcriptomic profiling revealed pronounced sex-specific placental responses: female placentas exhibited extensive reprogramming across immune, metabolic, and extracellular matrix pathways (518 DEGs), whereas male placentas showed minimal differential expression (9 DEGs), despite enrichment for RNA processing and mitochondrial pathways. Adult offspring livers also displayed sex-specific transcriptional signatures, with exposed females downregulating metabolic and immune-regulatory genes and exposed males upregulating inflammatory and signaling pathways. Collectively, these hypothesis-generating findings provide early evidence that preconception exposures can shape maternal physiology, placental development, and long-term offspring health, highlighting the preconception period as a critical yet understudied window of susceptibility.