BACKGROUND: Adenomyosis is a uterine disorder closely linked to infertility and adverse pregnancy outcomes. Despite its clinical significance, the mechanisms by which adenomyosis impairs embryo implantation and perinatal outcomes remain incompletely defined. Previous studies have indicated that alterations in the uterine microenvironment may contribute to these reproductive challenges. AIM: To investigate the effects of adenomyosis on fertility and perinatal outcomes using a novel murine model and to identify molecular pathways involved in implantation failure and pregnancy loss. STUDY DESIGN: A mechanically induced adenomyosis model was established in female BALB/c mice through endometrial-myometrial interface disruption (EMID) to closely simulate the clinical condition observed in humans. Mice were randomly assigned to either the adenomyosis group or a sham-operated control group. Reproductive outcomes were systematically assessed at multiple gestational time points, focusing on embryo implantation site distribution, implantation rates, pregnancy loss, fetal growth parameters, and postnatal uterine recovery. In parallel, uterine tissues collected from implantation sites and inter-implantation regions at 4.5 days post coitus were subjected to RNA sequencing. Differential gene expression analyses were performed, and enriched pathways were identified using Gene Ontology and KEGG enrichment tools. RESULTS: Mice in the adenomyosis group demonstrated significant disruptions in the uterine microenvironment compared to controls. Notably, the adenomyosis group exhibited irregular distribution of implantation sites with reduced inter-embryo distances, leading to compromised spatial organization of the developing conceptuses. Although the total number of embryos at early gestation did not differ significantly between groups, a marked increase in pregnancy loss was observed during mid-gestation, accompanied by a reduction in the size of surviving embryos. Histological evaluation revealed extensive architectural disruptions in the uterine muscle layers and increased local inflammatory responses in adenomyotic uteri. RNA sequencing further revealed that adenomyosis was associated with the dysregulation of multiple genes involved in immune modulation, apoptotic regulation, and metabolic processes. In particular, significant enrichment of the PI3K-Akt, MAPK, and TNF signaling pathways was observed, suggesting that variation in these cascades may underlie the impaired uterine receptivity and embryo development seen in adenomyosis. CONCLUSION: Our findings indicate that adenomyosis exerts a adverse effect on fertility and perinatal outcomes by disrupting the uterine environment and interfering with critical molecular pathways essential for proper embryo implantation and development. The results of this study not only enhance our understanding of adenomyosis pathophysiology but also pinpoint potential molecular targets-such as the PI3K-Akt, MAPK, and TNF pathways-for therapeutic intervention. These insights offer promising targets for developing treatments aimed at reversing the adverse reproductive impacts associated with adenomyosis.