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Chagas disease (CD) is a multisystemic zoonotic infection whose causative agent is the protozoan parasite Trypanosoma cruzi. According to WHO, the CD is considered a neglected tropical disease that is endemic in Latin America. However, it has spread beyond its traditional geographical limits because of migration and congenital transmission, constituting a global public health problem. Congenital transmission depends on a complex network of host-pathogen interactions, where the placenta and virulence factors secreted in exovesicles play a fundamental role. Thus, to infect the developing fetus, the parasite must cross the placental barrier composed of a bistratified epithelium (trophoblast), fetal connective tissue (villous stroma) that contains fetal blood vessels, and the basal lamina that supports the different epithelia. To study the possible role of T. cruzi trypomastigote exovesicles (Tryp-TcEVs) in the susceptibility to infection and tissue damage caused by the parasite during ex vivo infection of human placental explants (HPE) we proposed two specific aims: 1) To determine the effect of Tryp-TcEVs during ex vivo infection of HPE by quantification of parasite DNA load; 2) To determine the role of Tryp-TcEVs in T. cruzi -induced tissue damage by histopathological and histochemical analysis of HPE. To do this, Tryp-TcEVs were obtained from trypomastigote cultures in MEM medium, followed by two ultra-centrifugations, filtration, and quality control in TEM, in collaboration with Dr. Antonio Osuna (University of Granada, Spain). Parasites were obtained by culture in VERO cells (ATTC ® CCL - 81™). Term placentas were obtained from the Obstetrics and Gynecology Department of the Hospital San José, Santiago de Chile. HPEs of 5 mm³ were obtained, followed by PBS washing and incubation in RPMI medium for 24 hours in the absence or presence of parasites (1x105 parasites/mL) and Tryp-TcEVs (5 μg/mL). Then, qPCR was performed to detect T. cruzi satellite DNA to evaluate parasite load (182-bp) (TCZ-F (5'-GCTCTCTTGCCCACAMAMGGGGGTGC-3'3') and TCZ-R (5'-CAAGCAGCGGATAGTTCAGG-3'3')) using the human GADPH gene as housekeeping gene (100-bp) (hGDHF (5'-TGATGCGTGTGTACAAGCGTTTTTT-3'3') and hGDH-R (5'-ACATGGTATTCACCACCACCCCAC TAT-3'3')). Data were analyzed using the double comparative control method (ΔΔCt). Routine histological processing was performed to assess the tissue damage, and samples were stained with Hematoxylin-Eosin, Masson's Trichrome, Picrosirious red, and Schiff's Periodic Acid. Our analysis shows that HPE incubated in the presence of Tryp-TcEVs (5 μg/mL) and T. cruzi trypomastigotes (1x105 parasites/mL) for 24 hours significantly increased their parasite load compared to those infected only with the parasite. In addition, it was evidenced that Tryp-TcEVs increase the damage caused by the parasite since increased trophoblast detachment, loss of continuity and organization of the basal lamina and villous stroma were observed. Finally, as a conclusion, we can confirm that Tryp-TcEVs contribute to the destruction of the placental barrier caused by the parasite, favoring infection. [ABSTRACT FROM AUTHOR]